Primary cervical cancer screening with HPV detection has higher longitudinal sensitivity than conventional cytology to detect cervical intraepithelial neoplasia grade 2.

Evidence regarding human papillomavirus testing in secondary prevention of cervical cancer.

More than ever, clinicians need regularly updated reviews given the continuously increasing amount of new information regarding innovative cervical cancer prevention methods. A summary is given from recent meta-analyses and systematic reviews on 3 possible clinical applications of human papillomavirus (HPV) testing: triage of women with equivocal or low-grade cytologic abnormalities; prediction of the therapeutic outcome after treatment of cervical intraepithelial neoplasia (CIN) lesions, and last not but not least, primary screening for cervical cancer and pre-cancer. Consistent evidence is available indicating that HPV-triage with the Hybrid Capture(®) 2 assay (Qiagen Gaithersburg, Inc., MD, USA [previously Digene Corp.] (HC2) is more accurate (higher sensitivity, similar specificity) than repeat cytology to triage women with equivocal Pap smear results. Several other tests show at least similar accuracy but mRNA testing with the APTIMA(®) (Gen-Probe Inc., San Diego, CA, USA) test is similarly sensitive but more specific compared to HC2. In triage of low-grade squamous intraepithelial lesions (LSIL), HC2 is more sensitive but its specificity is substantially lower compared to repeat cytology. The APTIMA(®) test is more specific than HC2 without showing a loss in sensitivity. Identification of DNA of HPV types 16 and/or 18, or RNA from the five most carcinogenic HPV types allow selecting women at highest risk for CIN3+ but the sensitivity and negative predictive value of these markers are lower than full-range high-risk HPV (hrHPV) testing. After conservative treatment of cervical pre-cancer, HPV testing picks up more quickly, with higher sensitivity and not lower specificity, residual or recurrent high-grade CIN than follow-up cytology. Primary screening for hrHPV generally detects more CIN2, CIN3 or cancer compared to cytology at cut-off atypical squamous cells of undetermined significance (ASC-US) or LSIL, but is less specific. Combined HPV and cytology screening provides a further small gain in sensitivity at the expense of a considerable loss in specificity if positive by either test is referred to colposcopy, in comparison with HPV testing only. Randomised trials and follow-up of cohort studies consistently demonstrate a significantly lower cumulative incidence of CIN3+ and even of cancer, in women aged 30 years or older, who were at enrollment hrHPV DNA negative compared to those who were cytologically negative. The difference in cumulative risk of CIN3+ or cancer for double negative (cytology & HPV) versus only HPV-negative women is small. HC2, GP5+/6+ PCR (polymerase chain reaction), cobas(®) 4800 PCR (Roche Molecular Systems Inc., Alameda, CA, USA) and Real Time PCR (Abbott Molecular, Des Plaines, IL, USA) can be considered as clinically validated for use in primary screening. The loss in specificity associated with primary HPV-based screening can be compensated by appropriate algorithms involving reflex cytology and/or HPV genotyping for HPV16 or 18. There exists a substantial evidence base to support that HPV testing is advantageous both in triage of women with equivocal abnormal cytology, in surveillance after treatment of CIN lesions and in primary screening of women aged 30 years or older. However, the possible advantages offered by HPV-based screening require a well organised program with good compliance with screening and triage policies. This article forms part of a special supplement entitled "Comprehensive Control of HPV Infections and Related Diseases" Vaccine Volume 30, Supplement 5, 2012.

Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial.

BACKGROUND Human papillomavirus (HPV) testing is more sensitive for the detection of high-grade cervical lesions than is cytology, but detection of HPV by DNA screening in two screening rounds 5 years apart has not been assessed. The aim of this study was to assess whether HPV DNA testing in the first screen decreases detection of cervical intraepithelial neoplasia (CIN) grade 3 or worse, CIN grade 2 or worse, and cervical cancer in the second screening. METHODS In this randomised trial, women aged 29-56 years participating in the cervical screening programme in the Netherlands were randomly assigned to receive HPV DNA (GP5+/6+-PCR method) and cytology co-testing or cytology testing alone, from January, 1999, to September, 2002. Randomisation (in a 1:1 ratio) was done with computer-generated random numbers after the cervical specimen had been taken. At the second screening 5 years later, HPV DNA and cytology co-testing was done in both groups; researchers were masked to the patient's assignment. The primary endpoint was the number of CIN grade 3 or worse detected. Analysis was done by intention to screen. The trial is now finished and is registered, number ISRCTN20781131. FINDINGS 22,420 women were randomly assigned to the intervention group and 22 518 to the control group; 19 999 in the intervention group and 20,106 in the control group were eligible for analysis at the first screen. At the second screen, 19 579 women in the intervention group and 19,731 in the control group were eligible, of whom 16,750 and 16,743, respectively, attended the second screen. In the second round, CIN grade 3 or worse was less common in the intervention group than in the control group (88 of 19 579 in the intervention group vs 122 of 19,731 in the control group; relative risk 0·73, 95% CI 0·55-0·96; p=0·023). Cervical cancer was also less common in the intervention group than in the control group (four of 19 579 in the intervention group vs 14 of 19,731; 0·29, 0·10-0·87; p=0·031). In the baseline round, detection of CIN grade 3 or worse did not differ significantly between groups (171 of 19 999 vs 150 of 20,106; 1·15, 0·92-1·43; p=0·239) but was significantly more common in women with normal cytology (34 of 19,286 vs 12 of 19,373; 2·85, 1·47-5·49; p=0·001). Furthermore, significantly more cases of CIN grade 2 or worse were detected in the intervention group than in the control group (267 of 19 999 vs 215 of 20,106; 1·25, 1·05-1·50; p=0·015). In the second screen, fewer HPV16-positive CIN grade 3 or worse were detected in the intervention group than in the control group (17 of 9481 vs 35 of 9354; 0·48, 0·27-0·85; p=0·012); detection of non-HPV16-positive CIN grade 3 or worse did not differ between groups (25 of 9481 vs 25 of 9354; 0·99, 0·57-1·72; p=1·00). The cumulative detection of CIN grade 3 or worse and CIN grade 2 or worse did not differ significantly between study arms, neither for the whole study group (CIN grade 3 or worse: 259 of 19 999 vs 272 of 20,106; 0·96, 0·81-1·14, p=0·631; CIN grade 2 or worse: 427 of 19 999 vs 399 of 20,106; 1·08, 0·94-1·24; p=0·292), nor for subgroups of women invited for the first time (CIN grade 3 or worse in women aged 29-33 years: 102 of 3139 vs 105 of 3128; 0·97, 0·74-1·27; CIN grade 2 or worse in women aged 29-33 years: 153 of 3139 vs 151 of 3128; 1·01, 0·81-1·26; CIN grade 3 or worse in women aged 34-56 years: 157 of 16,860 vs 167 of 16 978; 0·95, 0·76-1·18; CIN grade 2 or worse in women aged 34-56 years: 274 of 16,860 vs 248 of 16 978; 1·11, 0·94-1·32). INTERPRETATION Implementation of HPV DNA testing in cervical screening leads to earlier detection of clinically relevant CIN grade 2 or worse, which when adequately treated, improves protection against CIN grade 3 or worse and cervical cancer. Early detection of high-grade cervical legions caused by HPV16 was a major component of this benefit. Our results lend support to the use of HPV DNA testing for all women aged 29 years and older. FUNDING Zorg Onderzoek Nederland (Netherlands Organisation for Health Research and Development).

Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial.

BACKGROUND Human papillomavirus (HPV) testing is known to be more sensitive, but less specific than cytology for detecting cervical intraepithelial neoplasia (CIN). We assessed the efficacy of cervical-cancer screening policies that are based on HPV testing. METHODS Between March, 2004, and December, 2004, in two separate recruitment phases, women aged 25-60 years were randomly assigned to conventional cytology or to HPV testing in combination with liquid-based cytology (first phase) or alone (second phase). Randomisation was done by computer in two screening centres and by sequential opening of numbered sealed envelopes in the remaining seven centres. During phase one, women who were HPV-positive and aged 35-60 years were referred to colposcopy, whereas women aged 25-34 years were referred to colposcopy only if cytology was also abnormal or HPV testing was persistently positive. During phase two, women in the HPV group were referred for colposcopy if the HPV test was positive. Two rounds of screening occurred in each phase, and all women had cytology testing only at the second round. The primary endpoint was the detection of grade 2 and 3 CIN, and of invasive cervical cancers during the first and second screening rounds. Analysis was done by intention to screen. This trial is registered, number ISRCTN81678807. FINDINGS In total for both phases, 47,001 women were randomly assigned to the cytology group and 47,369 to HPV testing. 33,851 women from the cytology group and 32,998 from the HPV-testing group had a second round of screening. We also retrieved the histological diagnoses from screening done elsewhere. The detection of invasive cervical cancers was similar for the two groups in the first round of screening (nine in the cytology group vs seven in the HPV group, p=0.62); no cases were detected in the HPV group during round two, compared with nine in the cytology group (p=0.004). Overall, in the two rounds of screening, 18 invasive cancers were detected in the cytology group versus seven in the HPV group (p=0.028). Among women aged 35-60 years, at round one the relative detection (HPV vs cytology) was 2.00 (95% CI 1.44-2.77) for CIN2, 2.08 (1.47-2.95) for CIN3, and 2.03 (1.60-2.57) for CIN2 and 3 together. At round two the relative detection was 0.54 (0.23-1.28) for CIN2, 0.48 (0.21-1.11) for CIN3, and 0.51 (0.28-0.93) for CIN2 and 3 together. Among women aged 25-34 years, there was significant heterogeneity between phases in the relative detection of CIN3. At round one the relative detection was 0.93 (0.52-1.64) in phase one and 3.91 (2.02-7.57) in phase two. At round two the relative detection was 1.34 (0.46-3.84) in phase one and 0.20 (0.04-0.93) in phase two. Pooling both phases, the detection ratio of CIN2 for women aged 25-34 years was 4.09 (2.24-7.48) at round one and 0.64 (0.23-1.27) at round two. INTERPRETATION HPV-based screening is more effective than cytology in preventing invasive cervical cancer, by detecting persistent high-grade lesions earlier and providing a longer low-risk period. However, in younger women, HPV screening leads to over-diagnosis of regressive CIN2. FUNDING European Union, Italian Ministry of Health, Regional Health Administrations of Piemonte, Tuscany, Veneto and Emilia-Romagna, and Public Health Agency of Lazio.

Human papillomavirus and Papanicolaou tests to screen for cervical cancer.

BACKGROUND Screening for cervical cancer based on testing for human papillomavirus (HPV) increases the sensitivity of detection of high-grade (grade 2 or 3) cervical intraepithelial neoplasia, but whether this gain represents overdiagnosis or protection against future high-grade cervical epithelial neoplasia or cervical cancer is unknown. METHODS In a population-based screening program in Sweden, 12,527 women 32 to 38 years of age were randomly assigned at a 1:1 ratio to have an HPV test plus a Papanicolaou (Pap) test (intervention group) or a Pap test alone (control group). Women with a positive HPV test and a normal Pap test result were offered a second HPV test at least 1 year later, and those who were found to be persistently infected with the same high-risk type of HPV were then offered colposcopy with cervical biopsy. A similar number of double-blinded Pap smears and colposcopies with biopsy were performed in randomly selected women in the control group. Comprehensive registry data were used to follow the women for a mean of 4.1 years. The relative rates of grade 2 or 3 cervical intraepithelial neoplasia or cancer detected at enrollment and at subsequent screening examinations were calculated. RESULTS At enrollment, the proportion of women in the intervention group who were found to have lesions of grade 2 or 3 cervical intraepithelial neoplasia or cancer was 51% greater (95% confidence interval [CI], 13 to 102) than the proportion of women in the control group who were found to have such lesions. At subsequent screening examinations, the proportion of women in the intervention group who were found to have grade 2 or 3 lesions or cancer was 42% less (95% CI, 4 to 64) and the proportion with grade 3 lesions or cancer was 47% less (95% CI, 2 to 71) than the proportions of control women who were found to have such lesions. Women with persistent HPV infection remained at high risk for grade 2 or 3 lesions or cancer after referral for colposcopy. CONCLUSIONS The addition of an HPV test to the Pap test to screen women in their mid-30s for cervical cancer reduces the incidence of grade 2 or 3 cervical intraepithelial neoplasia or cancer detected by subsequent screening examinations. (ClinicalTrials.gov number, NCT00479375 [ClinicalTrials.gov].).

Evaluation of human papillomavirus testing in primary screening for cervical abnormalities: comparison of sensitivity, specificity, and frequency of referral.

CONTEXT Human papillomavirus (HPV) DNA testing of women having Papanicolaou (Pap) smears showing atypical squamous cells of undetermined significance (ASCUS) has clinical usefulness. Whether HPV DNA testing alone is useful in primary screening remains to be determined. OBJECTIVE To determine the accuracy of HPV DNA testing for detecting cervical intraepithelial neoplasia (CIN) grade 3 or cancer (the criterion standard). DESIGN, SETTING, AND PARTICIPANTS Between December 1997 and October 2000, 4075 women who attended Planned Parenthood clinics in Washington State were screened simultaneously using thin-layer Pap and HPV DNA testing by a polymerase chain reaction (PCR)-based method and by a liquid-based RNA-DNA hybridization capture with signal amplification assay (signal amplification). Women who were positive for high-risk HPV types, or had Pap results of ASCUS or higher, were considered to have positive screening test results and were referred for colposcopy and biopsy. Additionally, a random sample of women with negative screening test results was referred for colposcopy. Based on individual and combined thin-layer Pap, HPV PCR, and HPV signal amplification test results from the screening and the colposcopy visits, 7 colposcopy triage strategies were defined and evaluated. MAIN OUTCOME MEASURE Sensitivity and specificity for detecting cervical lesions graded CIN 3 or higher for each of the 7 triage strategies. RESULTS The estimated prevalence of CIN 3 or higher was 3.2%. The sensitivity (95% confidence interval) of thin-layer Pap (with a result of > or = ASCUS) for identifying women with CIN 3 or higher was only 61.3% (48.5%-70.9%) compared with 88.2% (78.9%-93.8%) for HPV testing by PCR and 90.8% (83.1%-95.8%) by signal amplification. Differences in specificities were also observed: 82.4% (81.8%-83.1%) for thin-layer Pap (with a result of > or = ASCUS), 78.8% (77.9%-79.7%) for PCR, and 72.6% (69.4%-75.0%) for signal amplification. Compared with referral for colposcopy of all women with ASCUS or higher, signal amplification testing of women with ASCUS and referral of those with a positive result was about as sensitive (61.3% vs 60.3%, respectively) and significantly more specific (82.4% vs 88.9%, respectively). The strategy requiring repeat positive PCR tests on 2 visits had a sensitivity of 84.2% (75.3%-91.0%) and a specificity of 86.2% (85.1%-87.3%). All tests were more specific and less sensitive in older (> or = 30 years) vs younger women. CONCLUSIONS Testing for HPV has higher sensitivity but lower specificity than thin-layer Pap screening. In some settings, particularly where screening intervals are long or haphazard, screening for HPV DNA may be a reasonable alternative to cytology-based screening of reproductive-age women.

A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection.

BACKGROUND Human papillomavirus (HPV) has been associated with cervical intraepithelial neoplasia, but the temporal relation between the infection and the neoplasia remains unclear, as does the relative importance of the specific type of HPV, other sexually transmitted diseases, and other risk factors. METHODS We studied prospectively a cohort of 241 women who presented for evaluation of sexually transmitted disease and had negative cervical cytologic tests. The women were followed every four months with cytologic and colposcopic examinations of the uterine cervix and tests for HPV DNA and other sexually transmitted diseases. RESULTS Cervical intraepithelial neoplasia grade 2 or 3 was confirmed by biopsy in 28 women. On the basis of survival analysis, the cumulative incidence of cervical intraepithelial neoplasia at two years was 28 percent among women with a positive test for HPV and 3 percent among those without detectable HPV DNA: The risk was highest among those with HPV type 16 or 18 infection (adjusted relative risk as compared with that in women without HPV infection, 11; 95 percent confidence interval, 4.6 to 26; attributable risk, 52 percent). All 24 cases of cervical intraepithelial neoplasia grade 2 or 3 among HPV-positive women were detected within 24 months after the first positive test for HPV. After adjustment for the presence of HPV infection, the development of cervical intraepithelial neoplasia was also associated with younger age at first intercourse, the presence of serum antibodies to Chlamydia trachomatis, the presence of serum antibodies to cytomegalovirus, and cervical infection with Neisseria gonorrhoeae. CONCLUSIONS Cervical intraepithelial neoplasia is a common and apparently early manifestation of cervical infection by HPV, particularly types 16 and 18.

Efficacy of HPV DNA testing with cytology triage and/or repeat HPV DNA testing in primary cervical cancer screening.

BACKGROUND Primary cervical screening with both human papillomavirus (HPV) DNA testing and cytological examination of cervical cells with a Pap test (cytology) has been evaluated in randomized clinical trials. Because the vast majority of women with positive cytology are also HPV DNA positive, screening strategies that use HPV DNA testing as the primary screening test may be more effective. METHODS We used the database from the intervention arm (n = 6,257 women) of a population-based randomized trial of double screening with cytology and HPV DNA testing to evaluate the efficacy of 11 possible cervical screening strategies that are based on HPV DNA testing alone, cytology alone, and HPV DNA testing combined with cytology among women aged 32-38 years. The main outcome measures were sensitivity for detection of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) within 6 months of enrollment or at colposcopy for women with a persistent type-specific HPV infection and the number of screening tests and positive predictive value (PPV) for each screening strategy. All statistical tests were two-sided. RESULTS Compared with screening by cytology alone, double testing with cytology and for type-specific HPV persistence resulted in a 35% (95% confidence interval [CI] = 15% to 60%) increase in sensitivity to detect CIN3+, without a statistically significant reduction in the PPV (relative PPV = 0.76, 95% CI = 0.52 to 1.10), but with more than twice as many screening tests needed. Several strategies that incorporated screening for high-risk HPV subtypes were explored, but they resulted in reduced PPV compared with cytology. Compared with cytology, primary screening with HPV DNA testing followed by cytological triage and repeat HPV DNA testing of HPV DNA-positive women with normal cytology increased the CIN3+ sensitivity by 30% (95% CI = 9% to 54%), maintained a high PPV (relative PPV = 0.87, 95% CI = 0.60 to 1.26), and resulted in a mere 12% increase in the number of screening tests (from 6,257 to 7,019 tests). CONCLUSIONS Primary HPV DNA-based screening with cytology triage and repeat HPV DNA testing of cytology-negative women appears to be the most feasible cervical screening strategy.

Short term persistence of human papillomavirus and risk of cervical precancer and cancer: population based cohort study

OBJECTIVE To evaluate the cumulative incidence of cervical intraepithelial neoplasia II or worse (grade II+) or cervical intraepithelial neoplasia grade III+ after short term persistence of prevalently detected carcinogenic human papillomavirus (HPV). DESIGN Population based cohort study. SETTING Guanacaste, Costa Rica. PARTICIPANTS 2282 sexually active women actively followed after enrolment. MAIN OUTCOME MEASURES Primary end points: three year and five year cumulative incidence of histologically confirmed cervical intraepithelial neoplasia grade II+ (n=70). Cervical specimens collected at each visit tested for more than 40 HPV genotypes. HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82 were considered the primary carcinogenic genotypes. RESULTS Women who tested positive for a carcinogenic HPV at enrolment and after about one year (9-21 months) (positive/positive) had a three year cumulative incidence of cervical intraepithelial neoplasia grade II+ of 17.0% (95% confidence interval 12.1% to 22.0%). Those who tested negative/positive (3.4%, 0.1% to 6.8%), positive/negative (1.2%, -0.2% to 2.5%), and negative/negative (0.5%, 0.1% to 0.9%) were at a significantly lower risk. There was little difference in the cumulative incidence of cervical intraepithelial neoplasia grade II+ between testing positive twice for any carcinogenic HPV genotype (same genotype or different genotypes) v testing positive twice for the same carcinogenic genotype (17.0% v 21.3%, respectively). Short term persistence of HPV 16 strongly predicted cervical intraepithelial neoplasia grade II+, with a three year cumulative incidence of 40.8% (26.4% to 55.1%). Similar patterns were observed for the five year cumulative incidence of grade II+ and for three year and five year cumulative incidence of grade III+. CONCLUSIONS Short term persistence of a prevalently detected carcinogenic HPV infection, especially HPV 16, strongly predicts a subsequent diagnosis of cervical intraepithelial neoplasia II+ over the next few years.

Rate of cervical cancer, severe intraepithelial neoplasia, and adenocarcinoma in situ in primary HPV DNA screening with cytology triage: randomised study within organised screening programme.

OBJECTIVE To assess the performance and impact of primary human papillomavirus (HPV) DNA screening with cytology triage compared with conventional cytology on cervical cancer and severe pre-cancerous lesions. DESIGN Randomised trial. SETTING Population based screening programme for cervical cancer in southern Finland in 2003-5. PARTICIPANTS 58 076 women, aged 30-60, invited to the routine population based screening programme for cervical cancer. INTERVENTIONS Primary HPV DNA test (hybrid capture II) with cytology triage if the result was positive or conventional cytological screening (reference). MAIN OUTCOME MEASURES Rate of cervical cancer, cervical intraepithelial neoplasia (CIN) grade III, and adenocarcinoma in situ (as a composite outcome referred to as CIN III+) during 2003-7 through record linkage between files from the screening registry and the national cancer registry. RESULTS In the HPV and conventional arms there were 95 600 and 95 700 woman years of follow-up and 76 and 53 cases of CIN III+, respectively (of which six and eight were cervical cancers). The relative rate of CIN III+ in the HPV arm versus the conventional arm was 1.44 (95% confidence interval 1.01 to 2.05) among all women invited for screening and 1.77 (1.16 to 2.74) among those who attended. Among women with a normal or negative test result, the relative rate of subsequent CIN III+ was 0.28 (0.04 to 1.17). The rate of cervical cancer between arms was 0.75 (0.25 to 2.16) among women invited for screening and 1.98 (0.52 to 9.38) among those who attended. CONCLUSIONS When incorporated into a well established organised screening programme, primary HPV screening with cytology triage was more sensitive than conventional cytology in detecting CIN III+ lesions. The number of cases of cervical cancer was small, but considering the high probability of progression of CIN III the findings are of importance regarding cancer prevention. TRIAL REGISTRATION Current Controlled Trials ISRCTN23885553.

Findings to date from the ASCUS-LSIL Triage Study (ALTS).

Controversy exists in the United States regarding the proper evaluation and management of low-grade squamous intraepithelial lesion (LSIL) and equivocal (atypical squamous cells of undetermined significance [ASCUS, now ASC-US]) cervical cytologic interpretations. To address this issue, the National Cancer Institute initiated the ASCUS-LSIL Triage Study (ALTS). ALTS is a multicenter, randomized clinical trial designed to evaluate 3 alternative methods of management, namely, immediate colposcopy, cytologic follow-up, and triage by human papillomavirus (HPV) DNA testing. This article summarizes the major findings of ALTS that have been published to date. Patients with ASCUS (n = 3488) or LSIL (n = 1572) were randomly assigned to research arms between November 1996 and December 1998, and were monitored for 2 years. The disease outcome was histologic cervical intraepithelial neoplasia (CIN) 3/cancer. The prevalence of oncogenic HPV was too high to permit effective triage of LSIL using HPV DNA testing by Hybrid Capture 2. However, for the women referred with a cytologic interpretation of ASCUS, HPV triage proved useful, with sensitivity equivalent to immediate colposcopy and a halving of colposcopic referrals. Among older women with ASCUS, HPV testing remained sensitive for detecting CIN 3 and cancer, but the referral percentage was dramatically lower compared to younger women. ALTS yielded insight into the performance of cytology and histopathology; experienced pathologists differed significantly in their interpretations of cervical abnormalities, especially histologic CIN 1 and cytologic ASCUS. Nonetheless, it was possible to distinguish a relatively uncommon type of ASCUS, equivocal for high-grade squamous intraepithelial lesion, that has a high positive predictive value for identifying women with underlying high-grade CIN. Many additional analyses are underway.

Baseline cytology, human papillomavirus testing, and risk for cervical neoplasia: A 10-year cohort analysis

As more is learned about the development of cervical cancer, the value of annual Pap smear screening for all women is being questioned. This study was conducted to investigate whether women at higher risk for the development of cervical cancer could be identified by testing for the presence of human papillomavirus (HPV) in the cervical smear. These women could be followed annually, and the interval between screening Pap smears for women at lower risk could be increased. Study participants were women enrolled in the Kaiser Permanente healthcare plan in Portland, Oregon, who underwent annual Pap smear screening between April 1989, and November 1990. More than 20,000 women (20,810 of 23,702) had satisfactory cervical smears with sufficient samples for HPV testing, which was conducted using a polymerase chain reaction-based method with MYO9/11 primers. Most women (83.6%) had at least one follow-up smear during the study period; however, women with atypical squamous cells (ASC) or worse had more smears than women with normal results (mean, 4.4 vs. 3.3). Follow-up was conducted more or less annually for a total period of 122 months. HPV results were not used in deciding patient management. Ninety-six percent of the 20,810 baseline Pap smears were diagnosed as negative (N = 20,156). Thirteen percent of these patients tested positive for HPV. The baseline smears of 654 of the 20,810 women (3.1%) were classified as ASC or worse. Of these 654 smears, 417 (63.8%) were positive for HPV. One hundred seventy-eight women had a cytologic diagnosis of a low-grade squamous intraepithelial lesion or worse; of these, 143 (80.3%) tested positive for HPV. During the 10 years of follow-up, 171 patients developed cervical intraepithelial neoplasia (CIN) 3 or cervical cancer. The baseline smear was normal in 112 of these women and ASC or worse in 59 (34.5%). Only half (49.2%) of the 58 patients diagnosed within the first 45 months of follow-up had an abnormal baseline smear. During this first 45 months, 7.85% of the women whose initial Pap test was at least ASC were diagnosed with CIN 3 or cancer. The cumulative incidence at 10 years of follow-up was 10.2%. Sixty of the 171 women with CIN 3 or cervical cancer had a negative baseline HPV test. Of the 118 women who were diagnosed during the first 45 months of follow-up, 89 (79.4%) were HPV positive initially. The cumulative incidence of CIN 3 or cancer among the group with a positive baseline HPV test was 6.92% over 10 years but only 1.73% at 45 months. The risk of developing CIN 3 or cancer remained elevated throughout the study in those women with a positive baseline HPV test. The predictive ability of the baseline Pap smear diminished as the follow-up interval increased. Fifteen percent of the patients (N = 3216) had a positive Pap smear, a positive HPV test, or both at the initial examination. One hundred twenty-three (71.9%) were among the 171 women who developed CIN 3 or cancer. Eighty-six percent (102 of 123) of the patients who were diagnosed within the first 45 months were positive with at least one of the screening studies. The cumulative incidence over 45 months for women who had positive HPV testing and/or abnormal Pap smear results was 4.54%. Women with negative results in both screening tests had a cumulative risk of 0.16% for the same period. At 10 years the cumulative risk incidence for these two groups was 6.83% and 0.79%, respectively, yielding a negative predictive value of 99.1% for combined testing.

Clinical human papillomavirus detection forecasts cervical cancer risk in women over 18 years of follow-up.

PURPOSE To describe the long-term (≥ 10 years) benefits of clinical human papillomavirus (HPV) DNA testing for cervical precancer and cancer risk prediction. METHODS Cervicovaginal lavages collected from 19,512 women attending a health maintenance program were retrospectively tested for HPV using a clinical test. HPV positives were tested for HPV16 and HPV18 individually using a research test. A Papanicolaou (Pap) result classified as atypical squamous cells of undetermined significance (ASC-US) or more severe was considered abnormal. Women underwent follow-up prospectively with routine annual Pap testing up to 18 years. Cumulative incidence rates (CIRs) of ≥ grade 3 cervical intraepithelial neoplasia (CIN3+) or cancer for enrollment test results were calculated. RESULTS A baseline negative HPV test provided greater reassurance against CIN3+ over the 18-year follow-up than a normal Pap (CIR, 0.90% v 1.27%). Although both baseline Pap and HPV tests predicted who would develop CIN3+ within the first 2 years of follow-up, only HPV testing predicted who would develop CIN3+ 10 to 18 years later (P = .004). HPV16- and HPV18-positive women with normal Pap were at elevated risk of CIN3+ compared with other HPV-positive women with normal Pap and were at similar risk of CIN3+ compared with women with a low-grade squamous intraepithelial Pap. CONCLUSION HPV testing to rule out cervical disease followed by Pap testing and possibly combined with the detection of HPV16 and HPV18 among HPV positives to identify those at immediate risk of CIN3+ would be an efficient algorithm for cervical cancer screening, especially in women age 30 years or older.

2001. Comparison of three management strategies for patients with atypical squamous cells of undetermined significance. Baseline results from a randomized trial

BACKGROUND More than 2 million U.S. women receive an equivocal cervical cytologic diagnosis (atypical squamous cells of undetermined significance [ASCUS]) each year. Effective colposcopy triage strategies are needed to identify the minority of women who have clinically significant disease while avoiding excessive follow-up evaluation for others. METHODS The ASCUS/LSIL (i.e., low-grade squamous intraepithelial lesion) Triage Study (ALTS) is a multicenter, randomized trial comparing the sensitivity and specificity of the following three management strategies to detect cervical intraepithelial neoplasia grade 3 (CIN3): 1) immediate colposcopy (considered to be the reference standard), 2) triage to colposcopy based on human papillomavirus (HPV) results from Hybrid Capture 2(TM) (HC 2) and thin-layer cytology results, or 3) triage based on cytology results alone. This article summarizes the cross-sectional enrollment results for 3488 women with a referral diagnosis of ASCUS. All statistical tests are two-sided. RESULTS Among participants with ASCUS, the underlying prevalence of histologically confirmed CIN3 was 5.1%. Sensitivity to detect CIN3 or above by testing for cancer-associated HPV DNA was 96.3% (95% confidence interval [CI] = 91.6% to 98.8%), with 56.1% of women referred to colposcopy. Sensitivity of a single repeat cytology specimen with a triage threshold of HSIL or above was 44.1% (95% CI = 35.6% to 52.9%), with 6.9% referred. Sensitivity of a lower cytology triage threshold of ASCUS or above was 85.3% (95% CI = 78.2% to 90.8%), with 58.6% referred. CONCLUSIONS HC 2 testing for cancer-associated HPV DNA is a viable option in the management of women with ASCUS. It has greater sensitivity to detect CIN3 or above and specificity comparable to a single additional cytologic test indicating ASCUS or above.

Basal keratinocyte tetrasomy in low-grade squamous intra-epithelial lesions of the cervix is restricted to high and intermediate risk HPV infection but is not type-specific

Human papillomavirus (HPV) infection appears to be an early event in cervical carcinogenesis with additional abnormalities being required for biological transformation. We have analysed 179 low-grade cervical squamous intra-epithelial lesions (SILs) and 15 normal cervices for the presence of HPV using both in situ hybridization and polymerase chain reaction (PCR). PCR was performed with GP5+/GP6+ primers followed by hybridization using probes for low (HPV 6, 11, 40, 42, 43, 44), intermediate (HPV 31, 33, 35, 39, 51, 52, 58, 59, 66 and 68) and high-risk HPVs (HPV 16, 18, 45 and 56). Interphase cytogenetic analysis using pericentromeric probes for chromosomes 1, 3, 4, 6, 10, 11, 17, 18 and X was also performed to identify numerical chromosomal abnormalities. Tetrasomy of all nine chromosomes was identified within basal keratinocytes, was restricted to epithelia infected with high risk (17 of 46) or intermediate risk (23 of 83) HPVs but was not HPV type-specific. Tetrasomy was not identified in any of the epithelia infected with low risk HPVs (n = 62). These numbers include multiple infection. These findings indicate that the induction of tetrasomy is a property restricted to high and intermediate-risk HPV types but that it is not type-specific. The factors governing which lesions will develop this abnormality are as yet unclear. © 2000 Cancer Research Campaign

Long-term follow-up of cervical disease in women screened by cytology and HPV testing: results from the HART study

BACKGROUND Several studies have shown that testing for high-risk human papillomavirus (HPV) types results in an improved sensitivity for CIN2+, compared with cytology, although with a somewhat lower specificity. METHODS We obtained follow-up results, with at least one smear after participation in the HART study, which compared HPV testing (HC-II) with cytology as a primary screening modality. RESULTS With a median follow-up of 6 years, 42 additional cases of CIN2+ were identified; women who were HPV positive at baseline were more likely to develop CIN2+ than those who were HPV negative (hazard ratio (HR) 17.2; 95% confidence interval (CI) (9.3-31.6)) and the risk increased with increasing viral load. Compared with HPV-negative women (relative light unit (RLU) <1), the HR (95% CI) was 5.4 (1.6, 18.2) for 1-10 RLU and 25.5 (13.6, 47.9) for RLU > or = 10. Positive cytology (borderline or worse compared with negative) was also predictive of developing CIN2, although to a lesser extent (HR 8.7; 95% CI (4.5-17.1)). Only one case of CIN3 and three cases of CIN2 were found in women who showed a positive cytology result but were HPV negative at baseline. CONCLUSION After 5 years of follow-up, CIN2+ occurred in 0.23% of women who were HPV negative at baseline compared with 0.48% of women who showed a negative cytology result, indicating a much longer low-risk interval for CIN2+ after HPV testing.

Persistent human papillomavirus infection and cervical neoplasia: a systematic review and meta-analysis.

Detection of persistent cervical carcinogenic human papillomavirus (HPV) DNA is used as a marker for cervical cancer risk in clinical trials. The authors performed a systematic review and meta-analysis of the association between persistent HPV DNA and high-grade cervical intraepithelial neoplasia (CIN2-3), high-grade squamous intraepithelial lesions (HSIL), and invasive cervical cancer (together designated CIN2-3/HSIL+) to evaluate the robustness of HPV persistence for clinical use. MEDLINE and Current Contents were searched through January 30, 2006. Relative risks (RRs) were stratified by HPV comparison group. Of 2,035 abstracts, 41 studies were eligible for inclusion in the meta-analysis. Over 22,500 women were included in calculation of RRs for persistent HPV DNA detection and cervical neoplasia. RRs ranged from 1.3 (95% confidence interval: 1.1, 1.5) to 813.0 (95% confidence interval: 168.2, 3,229.2) for CIN2-3/HSIL+ versus 12 months), wider testing intervals, CIN2-3/HSIL+, and use of an HPV-negative reference group were consistently associated with higher RRs. Thus, HPV persistence was consistently and strongly associated with CIN2-3/HSIL+, despite wide variation in definitions and study methods. The magnitude of association varied by duration of persistence and testing interval. Precise definition and standardization of HPV testing, sampling procedure, and test interval are needed for reliable clinical testing. These findings validate HPV persistence as a clinical marker and endpoint.

Mass screening programmes and trends in cervical cancer in Finland and the Netherlands.

With respect to cervical cancer management, Finland and the Netherlands are comparable in relevant characteristics, e.g., fertility rate, age-of-mother at first birth and a national screening programme for several years. The aim of this study is to compare trends in incidence of and mortality from cervical cancer in Finland and the Netherlands in relation to the introduction and intensity of the screening programmes. Therefore, incidence and mortality rates were calculated using the Cancer Registries of Finland and the Netherlands. Data on screening intensity were obtained from the Finnish Cancer Registry and the Dutch evaluation centre at ErasmusMC-Rotterdam. Women aged 30-60 have been screened every 5 years, in Finland since 1992 and in the Netherlands since 1996. Screening protocols for smear taking and referral to the gynaecologist are comparable. Incidence and mortality rates have declined more in Finland. In 2003, age-adjusted incidence and mortality in Finland were 4.0 and 0.9 and in the Netherlands 4.9 and 1.4 per 100,000 woman-years, respectively. Excess smear use in the Netherlands was estimated to be 24 per 1,000 women during a 5-year interval compared to 121 in Finland. The decline in mortality in Finland seems to be almost completely related to the screening programme whereas in the Netherlands it was initially considered to be a natural decline. Differences in risk factors might also play a role: the Netherlands has higher population density and higher percentages of immigrants and (female) smokers. The greater excess smear use in Finland might also have affected incidence.

Rapid clearance of human papillomavirus and implications for clinical focus on persistent infections.

Health professionals and the public need to understand the natural history of human papillomavirus (HPV) infections of the cervix to best use the information provided by new molecular screening tests. We investigated outcomes of 800 carcinogenic HPV infections detected in 599 women at enrollment into a population-based cohort (Guanacaste, Costa Rica). For individual infections, we calculated cumulative proportions of three outcomes (viral clearance, persistence without cervical intraepithelial neoplasia grade 2 or worse [CIN2+], or persistence with new diagnosis of CIN2+) at successive 6-month time points for the first 30 months of follow-up. Cervical specimens were tested for carcinogenic HPV genotypes using an L1 degenerate-primer polymerase chain reaction method. Infections typically cleared rapidly, with 67% (95% confidence interval [CI] = 63% to 70%) clearing by 12 months. However, among infections that persisted at least 12 months, the risk of CIN2+ diagnosis by 30 months was 21% (95% CI = 15% to 28%). The risk of CIN2+ diagnosis was highest among women younger than 30 years with HPV-16 infections that persisted for at least 12 months (53%; 95% CI = 29% to 76%). These findings suggest that the medical community should emphasize persistence of cervical HPV infection, not single-time detection of HPV, in management strategies and health messages.

Interlaboratory reproducibility and proficiency testing within the human papillomavirus cervical cancer screening program in Catalonia, Spain.

In Catalonia, a screening protocol for cervical cancer, including human papillomavirus (HPV) DNA testing using the Digene Hybrid Capture 2 (HC2) assay, was implemented in 2006. In order to monitor interlaboratory reproducibility, a proficiency testing (PT) survey of the HPV samples was launched in 2008. The aim of this study was to explore the repeatability of the HC2 assay's performance. Participating laboratories provided 20 samples annually, 5 randomly chosen samples from each of the following relative light unit (RLU) intervals: <0.5, 0.5 to 0.99, 1 to 9.99, and ≥10. Kappa statistics were used to determine the agreement levels between the original and the PT readings. The nature and origin of the discrepant results were calculated by bootstrapping. A total of 946 specimens were retested. The kappa values were 0.91 for positive/negative categorical classification and 0.79 for the four RLU intervals studied. Sample retesting yielded systematically lower RLU values than the original test (P<0.005), independently of the time elapsed between the two determinations (median, 53 days), possibly due to freeze-thaw cycles. The probability for a sample to show clinically discrepant results upon retesting was a function of the RLU value; samples with RLU values in the 0.5 to 5 interval showed 10.80% probability to yield discrepant results (95% confidence interval [CI], 7.86 to 14.33) compared to 0.85% probability for samples outside this interval (95% CI, 0.17 to 1.69). Globally, the HC2 assay shows high interlaboratory concordance. We have identified differential confidence thresholds and suggested the guidelines for interlaboratory PT in the future, as analytical quality assessment of HPV DNA detection remains a central component of the screening program for cervical cancer prevention.

The effect of mass screening on incidence and mortality of squamous and adenocarcinoma of cervix uteri.

OBJECTIVE To describe the efficacy of the Finnish mass screening program for cervical squamous carcinoma and adenocarcinoma, as reflected by changes of incidence and mortality rate. METHODS Cervical cancer incidence and mortality data were obtained from the Finnish Cancer Registry. Data were available from the year 1953, when the registry was established. The nationwide mass screening program in Finland was started in the mid-1960s. A centralized organization administers this program. Women age 30-60 years are notified for screening every 5 years. RESULTS The mean incidence of cervical carcinoma in the early 1960s was 15.4 per 10(5) woman-years. In 1991, it was only 2.7 per 10(5) woman-years. The mortality rate has decreased in the same proportion since the mass screening program. In the early 1960s, the mortality was 6.6 and in 1991 1.4 per 10(5) woman-years. However, the decrease of the incidence is seen almost exclusively in squamous cell carcinomas. The mortality caused by adenocarcinoma has decreased in screened birth cohorts, but the incidence rate has remained the same. CONCLUSIONS The Finnish mass screening program has been effective and its continuation is of utmost importance. In the future more attention should be given to glandular cell atypias in cervical smears. Thus, it might be possible to decrease the incidence of cervical adenocarcinoma.

Apoptosis in breast cancer: relationship with other pathological parameters.

Apoptosis is a frequent phenomenon in breast cancer and it can be detected by light microscopy in conventional histopathological sections or by special staining techniques. The number of apoptotic cells as a percentage of cells present, or the number of apoptotic cells per square millimetre of neoplastic tissue, is usually described as the apoptotic index (AI). In breast cancer, the AI is not related to tumour size, axillary lymph node metastasis or distant metastasis at diagnosis. It is greater in invasive ductal carcinomas than in other histological types. High AI is also related to high histological grade, high nuclear grade, comedo-type necrosis, lack of tubule formation, and dense infiltration of the tumour by lymphocytes. Sex steroid receptor-negative tumours have greater AIs than the sex steroid receptor-positive ones. Aneuploid breast cancers with high S-phase fractions (SPFs) also have high AI values compared with diploid tumours with low SPFs. p53-Positive breast cancers have high AIs, whereas tumours that are Bcl-2 positive have low AIs. The AI shows a strong positive correlation to all direct or indirect indicators of cell proliferation, such as mitotic index and Ki67 immunolabelling. Univariate survival analyses show that a high AI is linked with unfavourable disease outcome in axillary lymph node-negative and -positive breast cancer, but multivariate analyses indicate that AI is not an independent prognostic factor. In conclusion, a high AI is related to malignant cellular features and indicators of invasiveness and cell proliferation in breast cancer.

Cost-effectiveness of human papillomavirus vaccination and screening in Spain.

BACKGROUND In Spain, prophylactic vaccination against human papillomavirus (HPV) types 16 and 18 is being offered free-of-charge to one birth cohort of girls aged 11-14. Screening is opportunistic (annual/biannual) contributing to social and geographical disparities. METHODS A multi-HPV-type microsimulation model was calibrated to epidemiologic data from Spain utilising likelihood-based methods to assess the health and economic impact of adding HPV vaccination to cervical cancer screening. Strategies included (1) screening alone of women over age 25, varying frequency (every 1-5 years) and test (cytology, HPV DNA testing); (2) HPV vaccination of 11-year-old girls combined with screening. Outcomes included lifetime cancer risk, life expectancy, lifetime costs, number of clinical procedures and incremental cost-effectiveness ratios. RESULTS After the introduction of HPV vaccination, screening will need to continue, and strategies that incorporated HPV testing are more effective and cost-effective than those with cytology alone. For vaccinated girls, 5-year organised cytology with HPV testing as triage from ages 30 to 65 costs 24,350€ per year of life saved (YLS), assuming life-long vaccine immunity against HPV-16/18 by 3 doses with 90% coverage. Unvaccinated girls would benefit from organised cytology screening with HPV testing as triage; 5-year screening from ages 30 to 65 costs 16,060€/YLS and 4-year screening from ages 30 to 85 costs 38,250€/YLS. Interventions would be cost-effective depending on the cost-effectiveness threshold and the vaccine price. CONCLUSIONS In Spain, inequitable coverage and overuse of cytology make screening programmes inefficient. If high vaccination coverage among pre-adolescent girls is achieved, organised cytology screening with HPV triage starting at ages 30 to at least 65 every 4-5 years represents the best balance between costs and benefits.

A cluster randomized, controlled trial of breast and cervix cancer screening in Mumbai, India: methodology and interim results after three rounds of screening.

Cervix and Breast cancers are the most common cancers among women worldwide and extract a large toll in developing countries. In May 1998, supported by a grant from the NCI (US), the Tata Memorial Hospital, Mumbai, India, started a cluster-randomized, controlled, screening-trial for cervix and breast cancer using trained primary health workers to provide health-education, visual-inspection of cervix (with 4% acetic acid-VIA) and clinical breast examination (CBE) in the screening arm, and only health education in the control arm. Four rounds of screening at 2-year intervals will be followed by 8 years of monitoring for incidence and mortality from cervix and breast cancers. The methodology and interim results after three rounds of screening are presented here. Good randomization was achieved between the screening (n = 75360) and control arms (n = 76178). In the screening arm we see: High screening participation rates; Low attrition; Good compliance to diagnostic confirmation; Significant downstaging; Excellent treatment completion rate; Improving case fatality ratios. The ever-screened and never-screened participants in the screening arm show significant differences with reference to the variables religion, language, age, education, occupation, income and health-seeking behavior for gynecological and breast-related complaints. During the same period, in the control arm we see excellent participation rate for health education; Low attrition and a good number of symptomatic referrals for both cervix and breast.

Knowledge, attitudes, and practice on second primary cancer screening among cancer survivors: a qualitative study.

OBJECTIVE The goal of this study was to examine the knowledge, attitude, and practice on second cancer screening among cancer survivors. METHODS Three focus group interviews were conducted with thirteen disease-free stomach, colorectal, breast and thyroid cancer survivors. Recurrent issues were identified and then placed into thematic categories. RESULTS None of the study participants had heard SPC, and they could not differentiate SPC from 'recurrence' or 'metastasis.' Survivors believed that they had been cured, and they were not aware of their increased risk of SPC. Although they had high awareness of cancer screening, they could not make a distinction between 'cancer screening' and 'routine surveillance test' after cancer treatment. Survivors said that they would have had the screening for SPC if they had known about it. They preferred physicians as the most reliable source for screening information. CONCLUSION Cancer survivors had limited knowledge about SPC, and lack of information was the main barrier for SPC screening. PRACTICE IMPLICATIONS An educational intervention would help cancer survivors to understand their risk of SPC and the needs of screening after the first cancer.

Mammographic density and the risk and detection of breast cancer.

BACKGROUND Extensive mammographic density is associated with an increased risk of breast cancer and makes the detection of cancer by mammography difficult, but the influence of density on risk according to method of cancer detection is unknown. METHODS We carried out three nested case-control studies in screened populations with 1112 matched case-control pairs. We examined the association of the measured percentage of density in the baseline mammogram with risk of breast cancer, according to method of cancer detection, time since the initiation of screening, and age. RESULTS As compared with women with density in less than 10% of the mammogram, women with density in 75% or more had an increased risk of breast cancer (odds ratio, 4.7; 95% confidence interval [CI], 3.0 to 7.4), whether detected by screening (odds ratio, 3.5; 95% CI, 2.0 to 6.2) or less than 12 months after a negative screening examination (odds ratio, 17.8; 95% CI, 4.8 to 65.9). Increased risk of breast cancer, whether detected by screening or other means, persisted for at least 8 years after study entry and was greater in younger than in older women. For women younger than the median age of 56 years, 26% of all breast cancers and 50% of cancers detected less than 12 months after a negative screening test were attributable to density in 50% or more of the mammogram. CONCLUSIONS Extensive mammographic density is strongly associated with the risk of breast cancer detected by screening or between screening tests. A substantial fraction of breast cancers can be attributed to this risk factor.

Clinicopathological and prognostic significance of Bmi-1 expression in human cervical cancer.

OBJECTIVE To investigate the clinical significance of Bmi-1 expression as a prognostic marker for cervical cancer. Design. Retrospectively collected data from a population-based cohort. SETTING Jiangsu Province Hospital. Population. Eighty-eight women diagnosed with cervical carcinoma between 2000 and 2003. METHODS RT-PCR assay was performed to determine Bmi-1 mRNA expression in 18 cervical cancer and noncancerous tissue samples and immunohistochemistry to detect Bmi-1 protein expression in 88 cervical cancer samples. The correlation between Bmi-1 expression and clinicopathological factors was analyzed. Additionally, statistical analyses were applied to test for prognostic associations. RNA interference was used to downregulate Bmi-1 expression in a cervical cancer cell line (HeLa). In vitro cytotoxicity was measured by the methylthiazoletetrazolium and colony formation assays. Effects of Bmi-1 inhibition on in vivo growth of cancer cells was detected by the tumorigenicity assay. Cell cycle distribution and cell apoptosis were measured by flow cytometry. MAIN OUTCOME MEASURES The levels of Bmi-1 mRNA and protein expression in tissues were evaluated by RT-PCR and Western Blot assays. RESULTS The level of Bmi-1 mRNA expression in cervical cancer tissues was significantly higher than that in corresponding noncancerous tissues. High Bmi-1 expression was significantly correlated with poor tumor differentiation, advanced International Federation of Gynecology and Obstetrics stage and positive lymph node metastasis. Patients with high Bmi-1 expression showed shorter overall survival than those with low expression. Univariate and multivariate analyses showed that high Bmi-1 expression was an independent prognostic factor. CONCLUSIONS RNA interference-mediated Bmi-1 inhibition could inhibit in vitro and in vivo growth, enhance apoptosis and induce cell cycle arrest of cervical cancer cells. Bmi-1 might be an independent prognostic marker for cervical cancer patients.

The assessment of the prognostic value of tumor markers and cytokines as SCCAg, CYFRA 21.1, IL-6, VEGF and sTNF receptors in patients with squamous cell cervical cancer, particularly with early stage of the disease

The aim of this study is to determine the prognostic value of tumor markers, as squamous cell carcinoma antigen (SCCAg) and cytokeratin-19 fragment (CYFRA 21.1) and interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), soluble tumor necrosis factor receptor I (sTNF RI), and sTNF RII in patients with squamous cell carcinoma of the cervix. The subjects of analysis were 138 patients with stage I-IVA according to the International Federation of Gynecology and Obstetrics (FIGO) classification. The collected research material comes from one oncology center. During the 10 years of follow-up, 56 relapses and 53 deaths were observed, and recurrent disease in early stage was confirmed in 45 % of patients. The pretreatment serum levels of SCCAg and CYFRA 21.1, and cytokines IL-6, VEGF, sTNF RI, and sTNF RII were determined in all patients. The probability of disease-free survival (DFS) and overall survival (OS) was evaluated using the log-rank test and the Cox regression model. Based on the ROC curve analysis for patients with recurrence, the largest area under the curve was demonstrated for SCCAg and IL-6 and for patients who died, for SCCAg and VEGF. Cox analysis demonstrated that independent prognostic factor for DFS was only SCCAg and for OS cytokine IL-6 and SCCAg, but in patients with early stage the prognostic value for DFS was VEGF, whereas IL-6 and CYFRA 21.1 for OS. Serum level of VEGF, CYFRA 21.1 and IL-6 before treatment in patients with early stage cervical cancer appears to be an important prognostic factor.

Expression of basal cell keratin 15 and keratin 19 in oral squamous neoplasms represents diverse pathophysiologies.

Human epithelium contains keratin, which is expressed during differentiation. Depending on the target cell type, different types of keratin are expressed, and their alterations seem to represent changes in cell properties. The basal cells of oral epithelium express keratin 5 (K5), K14, K15 and K19, but their alterations in tumors are unclear. To address this issue and to seek possible diagnostic application, we examined the expression of these keratins in oral squamous cell carcinoma (OSCC) and squamous intraepithelial neoplasm (SIN). cDNA microarray analysis of 43 OSCC revealed slight upregulation of KRT14, downregulation of KRT15 and KRT19, and unaltered KRT5 expression. There were great variations in KRT15 and KRT19 expression across each cancer. Well-differentiated OSCC tended to express more KRT15 and less KRT19 compared to moderately- or poorly-differentiated OSCC. KRT15 was positively correlated with differentiation-related keratin, KRT13. These observations were further investigated by immunohistochemical examination. K5 and K14 were ubiquitously expressed in all 50 OSCC and 50 SIN examined. K15 and K19 were generally downregulated, but were considerably retained in about half of the cases and showed diverse expression patterns. K15-positive cancers tended to show a well-differentiated phenotype, and K19-positive cancers tended to show more invasive tumor fronts. Most K19-positive cancers appeared to develop with little associating SIN. K19 was consistently downregulated in SIN, while K15 was downregulated mainly in high grade SIN. In summary, K15 and K19, unlike K5 or K14, are expressed variably in both SIN and OSCC, which reflects the differences in their pathogenesis and biological behaviors, suggesting their prospective applications as markers for subclassifying OSCC and SIN.

The prognostic impact of the platelet distribution width-to-platelet count ratio in patients with breast cancer

Activated platelets promote tumor cell growth, angiogenesis, and invasion. Platelet activity can be inferred by platelet volume indices (PVIs), which include platelet distribution width (PDW), mean platelet volume (MPV), platelet distribution width-to-platelet count ratio (PDW/P), and mean platelet volume-to-platelet count ratio. Platelets and platelet-related markers, such as the platelet-to-lymphocyte ratio, have been found to be significant prognostic factors in patients with breast cancer. However, the role of PVIs for predicting survival in breast cancer remains unknown; hence, we performed this retrospective analysis of 275 patients with breast cancer. PVIs were compared with clinicopathological variables, and were assessed to identify independent indicators associated with disease-free survival (DFS) using the Cox proportional hazards model. An elevated PDW/P significantly correlated with age and HER2 status. Univariate analysis revealed that elevated PDW, MPV, and PDW/P as well as tumor size, nuclear grade, and lymph node involvement were significantly associated with inferior DFS rates (tumor size: p<0.01; nuclear grade, lymph node involvement, PDW, MPV, and PDW/P: p<0.05). On multivariate analysis, a large tumor size and elevated PDW/P were significant prognostic factors for DFS, with hazard ratios of 3.24 (95% confidence interval [CI]: 1.24-8.47) and 2.99 (95% CI: 1.18-7.57), respectively (p<0.05). Our study is the first to reveal that an elevated PDW/P significantly reduces DFS in patients with breast carcinoma. Measuring the PDW/P is simple, relatively inexpensive, and almost universally available using routine blood counts; this makes it an attractive biomarker for improved risk assessment.

A comparison of cancer screening practices in cancer survivors and in the general population: the Korean national health and nutrition examination survey (KNHANES) 2001–2007

This study aimed to describe cancer screening rates for second primary cancer among cancer survivors in Korea, and to compare these rates with those of two control groups: individuals without a history of cancer but with other chronic diseases, and individuals without a history of cancer and without other chronic diseases. The study is a cross-sectional analysis of 15,556 adults ≥30 years old who participated in the 2001, 2005, and 2007 Korean National Health and Nutrition Examination Surveys (KNHANES). The prevalence of breast, cervical, gastric, and colorectal cancer screening examinations according to national guidelines was assessed and compared to two control groups. Screening rates among cancer survivors were 48.5, 54.7, 34.7, and 28.6% for breast, cervical, gastric, and colorectal cancer screening, respectively. Cancer survivors showed higher screening rates for all four cancer sites compared with both control groups, but breast cancer screening was only statistically significant after adjusting gender, age, marital status, education, income, working status, health insurance, smoking and drinking status, and self-reported health status. Cancer survivors were more likely than individuals without a cancer history to obtain screening examinations according to recommended guidelines. Still, screening rates even among survivors were suboptimal, emphasizing the need for a more systematic approach to second primary cancer screening and prevention.

Geovisual analytics to enhance spatial scan statistic interpretation: an analysis of U.S. cervical cancer mortality

BACKGROUND Kulldorff's spatial scan statistic and its software implementation - SaTScan - are widely used for detecting and evaluating geographic clusters. However, two issues make using the method and interpreting its results non-trivial: (1) the method lacks cartographic support for understanding the clusters in geographic context and (2) results from the method are sensitive to parameter choices related to cluster scaling (abbreviated as scaling parameters), but the system provides no direct support for making these choices. We employ both established and novel geovisual analytics methods to address these issues and to enhance the interpretation of SaTScan results. We demonstrate our geovisual analytics approach in a case study analysis of cervical cancer mortality in the U.S.   RESULTS We address the first issue by providing an interactive visual interface to support the interpretation of SaTScan results. Our research to address the second issue prompted a broader discussion about the sensitivity of SaTScan results to parameter choices. Sensitivity has two components: (1) the method can identify clusters that, while being statistically significant, have heterogeneous contents comprised of both high-risk and low-risk locations and (2) the method can identify clusters that are unstable in location and size as the spatial scan scaling parameter is varied. To investigate cluster result stability, we conducted multiple SaTScan runs with systematically selected parameters. The results, when scanning a large spatial dataset (e.g., U.S. data aggregated by county), demonstrate that no single spatial scan scaling value is known to be optimal to identify clusters that exist at different scales; instead, multiple scans that vary the parameters are necessary. We introduce a novel method of measuring and visualizing reliability that facilitates identification of homogeneous clusters that are stable across analysis scales. Finally, we propose a logical approach to proceed through the analysis of SaTScan results. CONCLUSION The geovisual analytics approach described in this manuscript facilitates the interpretation of spatial cluster detection methods by providing cartographic representation of SaTScan results and by providing visualization methods and tools that support selection of SaTScan parameters. Our methods distinguish between heterogeneous and homogeneous clusters and assess the stability of clusters across analytic scales. METHOD We analyzed the cervical cancer mortality data for the United States aggregated by county between 2000 and 2004. We ran SaTScan on the dataset fifty times with different parameter choices. Our geovisual analytics approach couples SaTScan with our visual analytic platform, allowing users to interactively explore and compare SaTScan results produced by different parameter choices. The Standardized Mortality Ratio and reliability scores are visualized for all the counties to identify stable, homogeneous clusters. We evaluated our analysis result by comparing it to that produced by other independent techniques including the Empirical Bayes Smoothing and Kafadar spatial smoother methods. The geovisual analytics approach introduced here is developed and implemented in our Java-based Visual Inquiry Toolkit.

BMI-1 Autoantibody as a New Potential Biomarker for Cervical Carcinoma

BMI-1 is overexpressed in a variety of cancers, which can elicit an immune response leading to the induction of autoantibodies. However, BMI-1 autoantibody as a biomarker has seldom been studied with the exception of nasopharyngeal carcinoma. Whether BMI-1 autoantibodies can be used as a biomarker for cervical carcinoma is unclear. In this study,BMI-1 proteins were isolated by screening of a T7 phage cDNA library from mixed cervical carcinoma tissues. We analyzed BMI-1 autoantibody levels in serum samples from 67 patients with cervical carcinoma and 65 controls using ELISA and immunoblot. BMI-1 mRNA or protein levels were over-expressed in cervical carcinoma cell lines. Immunoblot results exhibited increased BMI-1 autoantibody levels in patient sera compared to normal sera. Additionally, the results for antibody affinity assay showed that there was no difference between cervical polyps and normal sera of BMI-1 autoantibody levels, but it was significantly greater in patient sera than that in normal controls (patient 0.827±0.043 and normal 0.445±0.023; P<0.001). What's more, the levels of BMI-1 autoantibody increased significantly at stage I (0.672±0.019) compared to normal sera (P<0.001), and levels of BMI-1 autoantibodies were increased gradually during the tumor progression (stage I 0.672±0.019; stage II 0.775 ±0.019; stage III 0.890 ±0.027; stage IV 1.043±0.041), which were significantly correlated with disease progression of cervical cancer (P<0.001). Statistical analyses using logistic regression and receiver operating characteristics (ROC) curves indicated that the BMI-1 autoantibody level can be used as a biomarker for cervical carcinoma (sensitivity 0.78 and specificity 0.76; AUC = 0.922). In conclusion, measuring BMI-1 autoantibody levels of patients with cervical cancer could have clinical prognostic value as well as a non-tissue specific biomarker for neoplasms expressing BMI-1.

(99m)Tc-HisoDGR as a Potential SPECT Probe for Orthotopic Glioma Detection via Targeting of Integrin α5β1.

Integrins, a large family of cell adhesion receptors, have been shown to play an important role for glioma proliferation and invasion. Several integrin receptors, including αvβ3, αvβ5, and α5β1, have generated clinical interest for glioma diagnosis and antitumor therapy. Integrin α5β1 has been highlighted as a prognostic and diagnostic marker in glioma, and its expression is correlated with a worse prognosis in high-grade glioma. However, unlike extensively studied integrins αvβ3 and αvβ5, very few integrin α5β1-specific radiotracers have been reported. Developing α5β1-specific radiotracers may provide alternative diagnosis and evaluation options in addition to well-studied αvβ3/αvβ5-specific tracers, and they may add new documents for profiling tumor progression. Here, a novel integrin α5β1-specific probe (99m)Tc-HisoDGR was fabricated for SPECT (single-photon emission computed tomography) imaging of glioma. To confirm its selective targeting of integrin α5β1 in vivo, the mouse models of α5β1-positive U87MG human glioma were subjected to SPECT/CT scans, and biodistribution experiments and blocking studies were performed. Small-animal SPECT/CT imaging experiments demonstrated that the tumors were clearly visualized in both subcutaneous and orthotopic glioma tumor models with clear background at 0.5, 1, and 2 h p.i. The tumor accumulation of (99m)Tc-HisoDGR showed significant reduction when excess cold isoDGR peptide was coinjected, suggesting that the tumor uptake was specifically mediated. Our work revealed that (99m)Tc-HisoDGR represented a powerful molecular probe for integrin α5β1-positive cancer imaging; moreover, it might be a promising tool for evaluating malignancy, predicting prognosis, selecting subpopulations of patients who might be sensitive to integrin α5β1-targeted drugs, and assessing and monitoring the response to integrin α5β1-targeted drugs in clinical trials.

Multipoint Kras oncogene mutations potentially indicate mucinous carcinoma on the entire spectrum of mucinous ovarian neoplasms

Kras mutation is a common phenomenon in many human neoplasms. We aimed to assess the Kras mutational status along the histological continuum from normal ovaries to the development of benign, borderline and malignant ovarian mucinous neoplasms. We analyzed 41 cases of malignant, 10 cases of borderline, 7 cases of benign mucinous ovarian tumors and 7 cases of normal ovarian tissue. The prevalence of Kras mutations in the normal ovary was 0.00% (n=0/7), while the prevalence in benign, borderline and malignant mucinous neoplasms was 57.14% (n=4/7), 90.00% (n=9/10) and 75.61% (n=31/41), respectively. Multiple Kras mutations were detected in 6 cases of mucinous carcinoma, including 5 double mutations with G13D/V14I (n=1), G12V/G13S (n=1), G12D/G13S (n=3) and one triple mutation with A11V/G13N/V14I (n=1). We identified six cases with 3 novel Kras mutations not previously described in the COSMIC database, which included A11V (n=3) and V14I (n=2) in mucinous carcinomas, and A11T (n=1) in a mucinous borderline tumor. In conclusion, Kras mutation appears to be one of the imperative events in the ovarian mucinous adenoma-borderline tumor-carcinoma sequence, as increased numbers of Kras mutations have been shown to be the strongest predictor of unequivocal malignancy in ovarian mucinous neoplasms.

Comparisons of patient and physician expectations for cancer survivorship care.

PURPOSE To compare expectations for cancer survivorship care between patients and their physicians and between primary care providers (PCPs) and oncologists. METHODS Survivors and their physicians were surveyed to evaluate for expectations regarding physician participation in primary cancer follow-up, screening for other cancers, general preventive health, and management of comorbidities. RESULTS Of 992 eligible survivors and 607 physicians surveyed, 535 (54%) and 378 (62%) were assessable, respectively. Among physician respondents, 255 (67%) were PCPs and 123 (33%) were oncologists. Comparing patients with their oncologists, expectations were highly discrepant for screening for cancers other than the index one (agreement rate, 29%), with patients anticipating significantly more oncologist involvement. Between patients and their PCPs, expectations were most incongruent for primary cancer follow-up (agreement rate, 35%), with PCPs indicating they should contribute a much greater part to this aspect of care. Expectations between patients and their PCPs were generally more concordant than between patients and their oncologists. PCPs and oncologists showed high discordances in perceptions of their own roles for primary cancer follow-up, cancer screening, and general preventive health (agreement rates of 3%, 44%, and 51%, respectively). In the case of primary cancer follow-up, both PCPs and oncologists indicated they should carry substantial responsibility for this task. CONCLUSION Patients and physicians have discordant expectations with respect to the roles of PCPs and oncologists in cancer survivorship care. Uncertainties around physician roles and responsibilities can lead to deficiencies in care, supporting the need to make survivorship care planning a standard component in cancer management.

Birth weight and mammographic density among postmenopausal women in Sweden.

Birth weight is a significant predictor of breast cancer risk in adult life and mammary gland mass could be an intermediate stage in this long process. We have studied the association of birth size measurements with mammographic density, a marker of mammary gland mass. For a population-based sample of 893 postmenopausal women without previous cancer in Sweden, we retrieved information on birth size from birth records and their most recent mammography. Film mammograms of the medio-lateral oblique view were digitized and the Cumulus software was used for computer-assisted semi-automated thresholding of mammographic density. Results were analyzed using generalized linear models controlling for possible confounders. Mean percent mammographic density increased when comparing the extreme categories of birth weight (from 15.6% to 18.6%) and head circumference (from 15.5% to 20.4%), and the corresponding linear trends were statistically significant (p values 0.02 and 0.007, respectively). The associations were particularly strong when the cutoff for high versus low mammographic density was set at the relatively high value of 50%. Compared to women weighing 3001-3500 grams at birth, women with birth weights >4000g were at almost 3-fold risk of developing high mammographic density (odds ratio: 2.9, 95% confidence interval 1.1 to 7.9). No association with mammographic density was evident with respect to birth length which, however, is known to be less accurately measured. These results indicate that adult breast density, a powerful predictor of breast cancer risk, has intrauterine roots, as reflected in birth size.

Knowledge, attitudes, risk perception, and cancer screening behaviors among cancer survivors.

BACKGROUND Knowledge, attitudes, and risk perception in relation to second primary cancer (SPC) screening and their impact on screening practices in cancer survivors are largely unknown. METHODS A total of 326 cancer survivors who had completed primary treatment for cancer >1 year previously were recruited from 6 oncology care outpatient clinics in the Republic of Korea. Survivors' knowledge, attitudes, perceived risk, and screening practices were assessed along with sociodemographic, behavioral, and clinical characteristics. Multivariate logistic regression was used to examine behavioral factors associated with the completion of all appropriate SPC screening according to national guidelines. RESULTS Approximately 37.7% of survivors had undergone all appropriate SPC screening tests. Survivors were found to have a high perceived risk of SPC, high perceived benefits of screening, and positive attitudes toward cancer screening. However, they had limited knowledge regarding SPC screening tests and few had received a recommendation from a physician to undergo SPC screening. Although there was no association found between perceived risk and positive attitudes with screening behavior, higher knowledge was noted to be significantly associated with the completion of all appropriate SPC screening (adjusted odds ratio, 1.81; 95% confidence interval, 1.03-3.33). CONCLUSIONS In the current study, cancer survivors were found to have limited knowledge regarding second cancer screening tests, which may have resulted in lower rates of completion of screening practices in this population.

Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development

BACKGROUND Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development. METHODS AND FINDINGS Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression. CONCLUSIONS HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies. Please see later in the article for the Editors' Summary.

Risk for breast cancer development determined by mammographic parenchymal pattern.

A classification of risk for developing breast cancer has been devised based solely on the appearance of the breast parenchyma by mammography. Four groups of patients were isolated. The study encompassed a five-year period and was done by reviewing the mammograms of all women over the age of 30 who had been examined at Hutzel Hospital, Detroit. The average time of followup would be approximately 2 1/2 years. Four groups had an incidence of developing breast cancer of 0.1, 0.4, 1.7, and 2.2. These parenchymal patterns are described and criteria for their identification are given.

Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase.

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase-immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16(INK4A) locus and downregulation of RASSF1A expression. The deletion of the p16(INK4A) locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array-based CGH revealed a gain of a 17q21-q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16(INK4A) locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF-kappaB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development.

KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer

BACKGROUND Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas. METHODS We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally. RESULTS Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity. CONCLUSION These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.

Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx.

A long-term cell culture epithelioid cell line was established from a recurrent squamous carcinoma of the nasopharynx of a Chinese male 17 1/2 years after radiation therapy. The cell line, designated NPC/HK1, has been passed 72 times over a period 1 year. The cells have been shown by light and electron microscopies to be of the squamous epithelial type. When they were transplanted subcutaneously into the back of athymic nude BALB/c (nu/nu) mice, tumors developed at the sites of inoculation, which on histological examination were shown to be well-differentiated squamous carcinomas, similar in morphology to the recurrent human tumor from which they were derived. Karyotypic analysis of cells from the cell line demonstrates an aneuploid human type with a modal chromosome number of 74 with both numerical and structural aberrations. Viral particles or Epstein-Barr viral nuclear antigen (EBNA) has not been demonstrated in the cells from the primary culture or several of the subcultures tested. The presence of EBNA in touch smears prepared from the biopsy tissue was inconclusive. Infection of the subcultured cells with EBV from P3HR1 and B95-8 cells was unsuccessful.

Reproducibility of the diagnosis of atypical endometrial hyperplasia: a Gynecologic Oncology Group study.

BACKGROUND Most gynecologists determine therapy based on current International Society of Gynecologic Pathologists (ISGP)/World Health Organization classification of endometrial hyperplasia, the reproducibility of which has been questioned. The Gynecologic Oncology Group (GOG) initiated a protocol to assess the efficacy of hormonal therapy of atypical endometrial hyperplasia (AEH). Primary goals of the first phase (Part A) were to prospectively determine reproducibility of referring institution's pathologist's diagnosis of AEH by a panel of 3 gynecologic pathologists and to determine reproducibility of diagnoses by panel members. METHODS Three hundred six women were entered on this protocol with a referring institution's pathologist diagnosis of AEH based on biopsy or curettage. Available slides were assessed independently and interpreted by each of a panel of 3 gynecologic pathologists who used International Society of Gynecologic Pathologists (ISGP)/World Health Organization criteria. The majority diagnosis was based on diagnostic concordance by at least 2 of the 3 panelists. RESULTS The referring institution's pathologist's diagnosis of AEH was supported by the majority of the panel in only 38% of cases. Overall kappa value for the panel diagnosis of AEH was 0.28. The majority diagnosis was adenocarcinoma in 29%, cycling endometrium in 7%, and nonatypical hyperplasia in 18% of cases. Unanimous agreement for any diagnosis was reached among all 3 of the panel in 40% of cases. For the panel, paired kappa values for any diagnosis ranged 0.34-0.43, with an overall kappa value of 0.40. CONCLUSION Reproducibility of referring institution's pathologists' diagnosis of AEH by a panel of gynecologic pathologists is poor. Both underestimation and overestimation of the severity of the lesion are very common. The level of reproducibility among subspecialist panel members for diagnosis of AEH in these specimens also is poor. Better criteria and better sampling are needed to improve reproducibility of this diagnosis, particularly if it is to be used for clinical decisions.

The PneuCarriage Project: A Multi-Centre Comparative Study to Identify the Best Serotyping Methods for Examining Pneumococcal Carriage in Vaccine Evaluation Studies

BACKGROUND The pneumococcus is a diverse pathogen whose primary niche is the nasopharynx. Over 90 different serotypes exist, and nasopharyngeal carriage of multiple serotypes is common. Understanding pneumococcal carriage is essential for evaluating the impact of pneumococcal vaccines. Traditional serotyping methods are cumbersome and insufficient for detecting multiple serotype carriage, and there are few data comparing the new methods that have been developed over the past decade. We established the PneuCarriage project, a large, international multi-centre study dedicated to the identification of the best pneumococcal serotyping methods for carriage studies. METHODS AND FINDINGS Reference sample sets were distributed to 15 research groups for blinded testing. Twenty pneumococcal serotyping methods were used to test 81 laboratory-prepared (spiked) samples. The five top-performing methods were used to test 260 nasopharyngeal (field) samples collected from children in six high-burden countries. Sensitivity and positive predictive value (PPV) were determined for the test methods and the reference method (traditional serotyping of >100 colonies from each sample). For the alternate serotyping methods, the overall sensitivity ranged from 1% to 99% (reference method 98%), and PPV from 8% to 100% (reference method 100%), when testing the spiked samples. Fifteen methods had ≥70% sensitivity to detect the dominant (major) serotype, whilst only eight methods had ≥70% sensitivity to detect minor serotypes. For the field samples, the overall sensitivity ranged from 74.2% to 95.8% (reference method 93.8%), and PPV from 82.2% to 96.4% (reference method 99.6%). The microarray had the highest sensitivity (95.8%) and high PPV (93.7%). The major limitation of this study is that not all of the available alternative serotyping methods were included. CONCLUSIONS Most methods were able to detect the dominant serotype in a sample, but many performed poorly in detecting the minor serotype populations. Microarray with a culture amplification step was the top-performing method. Results from this comprehensive evaluation will inform future vaccine evaluation and impact studies, particularly in low-income settings, where pneumococcal disease burden remains high.

Relation between Medicare screening reimbursement and stage at diagnosis for older patients with colon cancer.

CONTEXT Medicare's reimbursement policy was changed in 1998 to provide coverage for screening colonoscopies for patients with increased colon cancer risk, and expanded further in 2001 to cover screening colonoscopies for all individuals. OBJECTIVE To determine whether the Medicare reimbursement policy changes were associated with an increase in either colonoscopy use or early stage colon cancer diagnosis. DESIGN, SETTING, AND PARTICIPANTS Patients in the Surveillance, Epidemiology, and End Results Medicare linked database who were 67 years of age and older and had a primary diagnosis of colon cancer during 1992-2002, as well as a group of Medicare beneficiaries who resided in Surveillance, Epidemiology, and End Results areas but who were not diagnosed with cancer. MAIN OUTCOME MEASURES Trends in colonoscopy and sigmoidoscopy use among Medicare beneficiaries without cancer were assessed using multivariate Poisson regression. Among the patients with cancer, stage was classified as early (stage I) vs all other (stages II-IV). Time was categorized as period 1 (no screening coverage, 1992-1997), period 2 (limited coverage, January 1998-June 2001), and period 3 (universal coverage, July 2001-December 2002). A multivariate logistic regression (outcome = early stage) was used to assess temporal trends in stage at diagnosis; an interaction term between tumor site and time was included. RESULTS Colonoscopy use increased from an average rate of 285/100,000 per quarter in period 1 to 889 and 1919/100,000 per quarter in periods 2 (P<.001) and 3 (P vs 2<.001), respectively. During the study period, 44,924 eligible patients were diagnosed with colorectal cancer. The proportion of patients diagnosed at an early stage increased from 22.5% in period 1 to 25.5% in period 2 and 26.3% in period 3 (P<.001 for each pairwise comparison). The changes in Medicare coverage were strongly associated with early stage at diagnosis for patients with proximal colon lesions (adjusted relative risk period 2 vs 1, 1.19; 95% confidence interval, 1.13-1.26; adjusted relative risk period 3 vs 2, 1.10; 95% confidence interval, 1.02-1.17) but weakly associated, if at all, for patients with distal colon lesions (adjusted relative risk period 2 vs 1, 1.07; 95% confidence interval, 1.01-1.13; adjusted relative risk period 3 vs 2, 0.97; 95% confidence interval, 0.90-1.05). CONCLUSIONS Expansion of Medicare reimbursement to cover colon cancer screening was associated with an increased use of colonoscopy for Medicare beneficiaries, and for those who were diagnosed with colon cancer, an increased probability of being diagnosed at an early stage. The selective effect of the coverage change on proximal colon lesions suggests that increased use of whole-colon screening modalities such as colonoscopy may have played a pivotal role.

Occurrence of chromosome 9 and p53 alterations in multifocal dysplasia and carcinoma in situ of human urinary bladder

To define the genetic changes of flat urothelial lesions, carcinoma in situ (CIS) and moderate dysplasias (DII) were investigated for alterations in the two chromosomal regions most frequently involved in bladder cancer. Overall, 33 CIS and 16 DII from 21 patients were used to microdissect urothelium. Dual color fluorescence in situ hybridization (FISH) using gene locus probes of 9q22 (FACC), 9p21 (CDK), 17p13 (p53), and related centromeric probes was applied on interphase nuclei. In parallel, preamplified DNA of these samples was used for loss of heterozygosity (LOH) analyses with eight microsatellite markers on chromosomes 9p, 9q and 17p, and for sequencing of exons 5-9 of p53. Data indicated nearly identical deletion frequencies for chromosomes 9 and 17 for CIS (chromosome 9, 86%; p53, 84%). DII showed a lower deletion rate in comparison with CIS (chromosome 9, 75%; p53, 53%). A very high correlation between the results of FISH and LOH analyses was found. p53 mutations were detected in 12 of 15 patients (CIS, 72%; DII, 67%). In three of 16 patients with multifocal tumors, oligoclonal lesions were identified by LOH analyses, a finding further supported by sequencing of p53, by which two different p53 deletions were detected in two cases. In conclusion, data from microdissected flat urothelial lesions indicate that chromosome 9 deletions cannot be regarded as indicators of papillary growth, because they are found frequently in both types of flat lesions of the urothelium: those associated with papillary tumors and those that are not. The similar distribution and lower amount of genetic changes in DII render DII a possible precursor lesion of CIS.

Altered PTEN expression; a diagnostic marker for differentiating normal, hyperplastic and neoplastic endometrium

BACKGROUND Different molecular alterations have been described in endometrioid endometrial carcinoma (EECA). Among them the most frequently altered is loss of the PTEN protein, a tumor suppressor gene. The purpose of this study was to evaluate the expression pattern of PTEN gene in normal, hyperplastic and neoplastic endometrium. METHODS In a study in a referral gynecologic hospital in Tehran, Iran, immunohistochemical (IHC) evaluation of PTEN was performed on 87 consecutive specimens to the following three groups; group A- normal proliferative endometrium(n = 29); group B- hyperplastic endometrium [including simple hyperplasia without atypia(n = 21) and complex hyperplasia with atypia (n = 8)] and group C- EECA(n = 29). Immunostaining of cells was analyzed by arbitrary quantitative methods according to both slide's area staining and intensity of color reaction. RESULTS PTEN immunoreactivity was present in all normal proliferative endometrium, all simple hyperplasia, 75% of atypical complex hyperplasia and in 48% of EECA (P < 0.001). The intensity of PTEN reaction was significantly higher in group with proliferative endometrium than hyperplastic endometrium and EECA (P < 0.001). CONCLUSION PTEN expression was significantly higher in cyclical endometrium than in atypical hyperplasia and endometrioid carcinoma.

Effect of screening on cervical cancer mortality in England and Wales: analysis of trends with an age period cohort model.

The number of women dying from cervical cancer in 1997 was 7% lower than in 1996 and has fallen by over 25% since 1992.1 Such rapid change must be at least partly due to cervical screening, although strong cohort effects have caused large fluctuations in cervical mortality in the past.2 We modelled mortality data, taking into account the effects of age and year of birth and looking for trends in time within four age groups to estimate the beneficial effects of cervical screening. We obtained mortality data, in 5 year age bands, from death registrations in England and Wales and calculated rates using mid-year population estimates. Mortality since 1993 was adjusted upwards by 4% because of changes in classification of cause of death.3  We modelled the data assuming that the age specific mortality is the product of a smoothly varying age effect, birth cohort effect, and age dependent …

Epithelial to Mesenchymal Transition by TGFβ-1 Induction Increases Stemness Characteristics in Primary Non Small Cell Lung Cancer Cell Line

BACKGROUND Cancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line. MATERIALS AND METHODS A549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed. RESULTS TGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable. CONCLUSIONS The induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers.

Glioblastoma Subclasses Can Be Defined by Activity among Signal Transduction Pathways and Associated Genomic Alterations

BACKGROUND Glioblastoma multiforme (GBM) is an umbrella designation that includes a heterogeneous group of primary brain tumors. Several classification strategies of GBM have been reported, some by clinical course and others by resemblance to cell types either in the adult or during development. From a practical and therapeutic standpoint, classifying GBMs by signal transduction pathway activation and by mutation in pathway member genes may be particularly valuable for the development of targeted therapies. METHODOLOGY/PRINCIPAL FINDINGS We performed targeted proteomic analysis of 27 surgical glioma samples to identify patterns of coordinate activation among glioma-relevant signal transduction pathways, then compared these results with integrated analysis of genomic and expression data of 243 GBM samples from The Cancer Genome Atlas (TCGA). In the pattern of signaling, three subclasses of GBM emerge which appear to be associated with predominance of EGFR activation, PDGFR activation, or loss of the RAS regulator NF1. The EGFR signaling class has prominent Notch pathway activation measured by elevated expression of Notch ligands, cleaved Notch receptor, and downstream target Hes1. The PDGF class showed high levels of PDGFB ligand and phosphorylation of PDGFRbeta and NFKB. NF1-loss was associated with lower overall MAPK and PI3K activation and relative overexpression of the mesenchymal marker YKL40. These three signaling classes appear to correspond with distinct transcriptomal subclasses of primary GBM samples from TCGA for which copy number aberration and mutation of EGFR, PDGFRA, and NF1 are signature events. CONCLUSIONS/SIGNIFICANCE Proteomic analysis of GBM samples revealed three patterns of expression and activation of proteins in glioma-relevant signaling pathways. These three classes are comprised of roughly equal numbers showing either EGFR activation associated with amplification and mutation of the receptor, PDGF-pathway activation that is primarily ligand-driven, or loss of NF1 expression. The associated signaling activities correlating with these sentinel alterations provide insight into glioma biology and therapeutic strategies.

The epidemiology of sexually transmitted co-infections in HIV-positive and HIV-negative African-Caribbean women in Toronto

BACKGROUND HIV disproportionately affects African-Caribbean women in Canada but the frequency and distribution of sexually transmitted infections in this community have not been previously studied. METHODS We recruited women based on HIV status through a Toronto community health centre. Participants completed a socio-behavioural questionnaire using Audio Computer Assisted Self-Interview (ACASI) and provided blood for syphilis, HIV, hepatitis B and C, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and human cytomegalovirus (CMV) serology, urine for chlamydia and gonorrhea molecular testing and vaginal secretions for bacterial vaginosis (BV) and human papillomavirus (HPV). Differences in prevalence were assessed for statistical significance using chi-square. RESULTS We recruited 126 HIV-positive and 291 HIV-negative women, with a median age of 40 and 31 years, respectively (p < 0.001). Active HBV infection and lifetime exposure to HBV infection were more common in HIV-positive women (4.8% vs. 0.34%, p = 0.004; and 47.6% vs. 21.2%, p < 0.0001), as was a self-reported history of HBV vaccination (66.1% vs. 44.0%, p = 0.0001). Classical STIs were rare in both groups; BV prevalence was low and did not vary by HIV status. HSV-2 infection was markedly more frequent in HIV-positive (86.3%) than HIV-negative (46.6%) women (p < 0.0001). Vaginal HPV infection was also more common in HIV-positive than in HIV-negative women (50.8% vs. 22.6%, p < 0.0001) as was infection with high-risk oncogenic HPV types (48.4% vs. 17.3%, p < 0.0001). CONCLUSIONS Classical STIs were infrequent in this clinic-based population of African-Caribbean women in Toronto. However, HSV-2 prevalence was higher than that reported in previous studies in the general Canadian population and was strongly associated with HIV infection, as was infection with hepatitis B and HPV.

Aberrant expression and altered cellular localization of desmosomal and hemidesmosomal proteins are associated with aggressive clinicopathological features of oral squamous cell carcinoma

Disruption of cell adhesion plays a central role in dedifferentiation, invasion, and metastasis of various cancers. The desmosome and hemidesmosome are anchoring junctions that control cell-cell and cell-matrix adhesion, respectively. To clarify their contributions in mediating the biological properties of oral cancer, we immunohistochemically examined the expression of desmoglein 1 (DSG1), DSG2, DSG3, desmocollin 2 (DSC2), integrin beta 4 (ITGB4), laminin gamma chain 2 (LAMC2), and collagen type 17 alpha 1 (COL17A1) in 51 cases of oral squamous cell carcinoma. On normal oral epithelial cells, DSG1, DSG3, DSC2, and COL17A1 were expressed on the plasma membrane, while ITGB4 and mature LAMC2 were present at the basement membrane. In cancer, the expression of DSG1, DSG3, DSC2, and COL17A1 decreased and internalized to the cytoplasm. Cytoplasmic expression of DSG2, ITGB4, and LAMC2 was induced in the cancer cells facing to the stroma. We scored immunohistochemical expression and correlated this to clinicopathological parameters including histologic differentiation, pattern of invasion, and presence of lymph node metastasis. Decrease of DSG3 and DSC2 expression correlated with a more aggressive cancer phenotype: less differentiated and more invasive histologic features and a higher incidence of nodal metastasis. Lower COL17A1 and higher LAMC2 expression were also associated with a more aggressive phenotype. The present study demonstrates that aberrant expression and altered cellular localization of desmosomal and hemidesmosomal proteins are associated with aggressive clinicopathological features of oral cancer. This reinforces the notion that disturbance of the keratin-associated anchoring junctions confers aggressive features to cancer cells.

COX-2 and survivin are overexpressed and positively correlated in endometrial carcinoma.

OBJECTIVES To investigate the expressions of survivin and Cyclooxygenase-2 (COX-2), and their possible correlations in the development of endometrial adenocarcinoma (EC). We also looked at their association with classical prognostic factors in EC. To our knowledge, this is the first time survivin expression is investigated in terms of its relation to COX-2 in the developmental pathway of EC. METHODS Archived tissue samples of 50 EC, 30 endometrial hyperplasia and 20 proliferative endometrium were selected and immunohistochemically analyzed for survivin and COX-2 expression. RESULTS Both survivin and COX-2 were overexpressed in hyperplasia and endometrial adenocarcinoma cases compared to proliferative endometrium, which was statistically significant (p=0.01, p=0.02, respectively). Among EC cases, survivin and COX-2 were strongly positive in 38 (76%) and 30 (60%) patients, respectively. Furthermore, we found survivin and COX-2 to be positively correlated, which was also statistically significant (p=0.0001, r=0.46). Neither survivin nor COX-2 expression was correlated with classical prognostic factors of endometrial carcinoma such as myometrial invasion, grade or lymph node metastasis (p>0.05). Neither COX-2 nor survivin had an impact on overall survival (p>0.05). CONCLUSIONS Both survivin and COX-2 are overexpressed, and they seem to be early events in the occurrence of EC. Moreover, protein products of these two genes are positively correlated. COX-2 and survivin might share a common molecular pathway or enhance each other's actions in the developmental pathway of EC. Molecular basis of such a relationship should be further investigated in endometrial carcinogenesis.

Predictors of mammographic density: insights gained from a novel regression analysis of a twin study.

Understanding which factors influence mammographically dense and nondense areas is important because percent mammographic density adjusted for age is a strong, continuously distributed risk factor for breast cancer, especially when adjusted for weight or body mass index. Using computer-assisted methods, we measured mammographically dense areas for 571 monozygotic and 380 dizygotic Australian and North American twin pairs ages 40 to 70 years. We used a novel regression modeling approach in which each twin's measure of dense and nondense area was regressed against one or both of the twin's and co-twin's covariates. The nature of changes to regression estimates with the inclusion of the twin and/or co-twin's covariates can be evaluated for consistency with causal and/or other models. By causal, we mean that if it were possible to vary a covariate experimentally then the expected value of the outcome measure would change. After adjusting for the individual's weight, the co-twin associations with weight were attenuated, consistent with a causal effect of weight on mammographic measures, which in absolute log cm(2)/kg was thrice stronger for nondense than dense area. After adjusting for weight, later age at menarche, and greater height were associated with greater dense and lesser nondense areas in a manner inconsistent with causality. The associations of dense and nondense areas with parity are consistent with a causal effect and/or within-person confounding. The associations between mammographic density measures and height are consistent with shared early life environmental factors that predispose to both height and percent mammographic density and possibly breast cancer risk.

High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma.

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) can be inactivated by multiple genetic mechanisms. We analyzed 29 invasive primary head and neck squamous cell carcinomas (HNSCC) for p16 inactivation with immunohistochemistry utilizing a new monoclonal antibody (mAb), DCS-50. p16 staining of the primary lesions was correlated with genetic analysis including: (a) detailed microsatellite analysis of markers at the p16 locus to detect homozygous deletion; (b) sequence analysis of p16; and (c) Southern blot analysis to determine the methylation status of the 5' CpG island of p16. Twenty-four of 29 (83%) head and neck squamous cell carcinoma tumors displayed an absence of p16 nuclear staining using immunohistochemistry. Of these 24 tumors, we found that 16 (67%) harbored homozygous deletions, 5 (21%) were methylated, 1 displayed a rearrangement at the p16 locus, and 1 displayed a frameshift mutation in exon 1. These data suggest that: (a) inactivation of the p16 tumor suppressor gene is a frequent event in squamous cell carcinomas of the head and neck; (b) p16 is inactivated by several distinct and exclusive events including homozygous deletion, point mutation, and promoter methylation; and (c) immunohistochemical analysis for expression of the p16 gene product is an accurate and relatively simple method for evaluating p16 gene inactivation.

Results of screening for intracranial aneurysms in patients with coarctation of the aorta.

BACKGROUND AND PURPOSE IAs are found in 2.3% of adults; the mean age at detection is 52 years. Prevalence is <0.5% in young adults. Early studies suggest that 10%-50% of patients with aortic coarctation have IAs. Screening recommendations are variable. We sought to examine the prevalence of IAs through screening with MRA. MATERIALS AND METHODS Consecutive patients older than 16 years of age with coarctation undergoing brain MRA between May 1999 and October 2007 were included. MRA was performed by using a 1.5T scanner with a 3D time-of-flight protocol; simultaneous MR imaging was performed of the heart and aorta. Cerebral MRAs were double-reported by a neuroradiologist. Statistics are described as mean ± SD and median ± range. Continuous variables were compared by using Student t tests and Mann-Whitney U tests (categoric variables, by using the Fisher exact test). RESULTS One hundred seventeen MRAs were double-reported. The median age was 29 ± 11 years (range, 16-59 years). IAs were found in 12 patients (10.3%). The mean diameter of IAs was 3.9 mm (range, 2.0-8.0 mm). Patients with aneurysms were older (median, 37 years; range, 16-50 years) than those without (median, 23 years; range, 16-59 years; Z = -2.01, P = .04). Hypertension was more common in those with IAs (IA 83% versus no IA 43%, P = .01). There was no association between ascending aortopathy, bicuspid aortic valves, and IAs. CONCLUSIONS Patients with coarctation have a higher prevalence of IAs, occurring at an earlier age than in population studies. Whether routine screening is appropriate for this group of patients is unclear. Hypertension is likely to be an important pathophysiologic factor.

RNA is favourable for analysing EGFR mutations in malignant pleural effusion of lung cancer.

Malignant pleural effusion (MPE) is a useful specimen allowing for the evaluation of EGFR status in nonsmall cell lung cancer (NSCLC). However, direct sequencing of genomic DNA from MPE samples was found not to be sensitive for EGFR mutation detection. To test whether EGFR analysis from RNA is less prone to interference from nontumour cells that have no or lower EGFR expression, we compared three methods (sequencing from cell-derived RNA versus sequencing and mass-spectrometric analysis from genomic DNA), in parallel, for EGFR mutation detection from MPE samples in 150 lung adenocarcinoma patients receiving first-line tyrosine kinase inhibitors (TKIs). Among these MPE samples, EGFR mutations were much more frequently identified by sequencing using RNA than by sequencing and mass-spectrometric analysis from genomic DNA (for all mutations, 67.3 versus 44.7 and 46.7%; for L858R or exon 19 deletions, 61.3 versus 41.3 and 46.7%, respectively). The better mutation detection yield of sequencing from RNA was coupled with the superior prediction of clinical efficacy of first-line TKIs. In patients with acquired resistance, EGFR sequencing from RNA provided satisfactory detection of T790M (54.2%). These results demonstrated that EGFR sequencing using RNA as template greatly improves sensitivity for EGFR mutation detection from samples of MPE, highlighting RNA as the favourable source for analysing EGFR mutations from heterogeneous MPE specimens in NSCLC.

Periodic screening for breast cancer: the HIP Randomized Controlled Trial. Health Insurance Plan.

This paper summarizes the findings of the first breast cancer screening trial, which was initiated in December 1963 to explore the efficacy of screening. Women aged 40-64 years were selected from enrollees in the Health Insurance Plan (HIP) of Greater New York and were randomly assigned to study and control groups. Study group women were invited for screening, an initial examination, and three annual reexaminations. Screening consisted of film mammography (cephalocaudal and lateral views of each breast) and clinical examination of breasts. Breast cancer and mortality from breast cancer were examined by treatment group (study vs. control) and by entry-age subgroup. By the end of 18 years from entry, the study group had about a 25% lower breast cancer mortality among women aged 40-49 and 50-59 at time of entry than did the control group. However, to a large extent the difference among the 40-49-year-olds occurred in the subgroup with breast cancer diagnosed after these women had passed their 50th birthday, and utility of screening women in their forties is questionable.

Endometrial carcinoma risk among women diagnosed with endometrial hyperplasia: the 34-year experience in a large health plan

Classifying endometrial hyperplasia (EH) according to the severity of glandular crowding (simple hyperplasia (SH) vs complex hyperplasia (CH)) and nuclear atypia (simple atypical hyperplasia (SAH) vs complex atypical hyperplasia (CAH)) should predict subsequent endometrial carcinoma risk, but data on progression are lacking. Our nested case–control study of EH progression included 138 cases, who were diagnosed with EH and then with carcinoma (1970–2003) at least 1 year (median, 6.5 years) later, and 241 controls, who were individually matched on age, date, and follow-up duration and counter-matched on EH classification. After centralised pathology panel and medical record review, we generated rate ratios (RRs) and 95% confidence intervals (CIs), adjusted for treatment and repeat biopsies. With disordered proliferative endometrium (DPEM) as the referent, AH significantly increased carcinoma risk (RR=14, 95% CI, 5–38). Risk was highest 1–5 years after AH (RR=48, 95% CI, 8–294), but remained elevated 5 or more years after AH (RR=3.5, 95% CI, 1.0–9.6). Progression risks for SH (RR=2.0, 95% CI, 0.9–4.5) and CH (RR=2.8, 95% CI, 1.0–7.9) were substantially lower and only slightly higher than the progression risk for DPEM. The higher progression risks for AH could foster management guidelines based on markedly different progression risks for atypical vs non-atypical EH.

Stromal p16 expression differentiates endometrial polyp from endometrial hyperplasia

Endometrial polyps are very common benign endometrial lesions, but their pathogenesis is poorly understood, except for a few studies indicating the possibility of benign stromal neoplasm. Although the histopathological diagnosis of endometrial polyp on a surgical specimen is straightforward, it is often difficult to differentiate endometrial polyp from endometrial hyperplasia on a biopsy or curettage specimen. Presently, there is no immunohistochemical marker helpful in this differential diagnosis. In this study, we examined p16 expression in 35 endometrial polyps and 33 cases of endometrial hyperplasia that included 16 simple hyperplasias, 14 complex atypical hyperplasias, and 3 complex hyperplasias without atypia. Stromal p16 expression differed significantly between the two groups; it was seen in 31 (89 %) endometrial polyps, but in only 1 (3 %) endometrial hyperplasia. The percentage of p16-positive stromal cells ranged from 10 to 90 % (mean, 47 %) and the positive cells tended to be distributed around glands. Six cases of endometrial hyperplasia within an endometrial polyp were also examined and all cases showed stromal p16 expression. There was no difference in glandular p16 expression between endometrial polyps 33 (94 %) and hyperplasia 27 (82 %). The p16-immunoreactivity was mostly confined to metaplastic epithelial cells in both groups. Stromal p16 expression might be a peculiar characteristic of endometrial polyp and constitute a useful marker for the diagnosis, especially in fragmented specimens from biopsy or curettage. Stromal p16 expression might be a reflection of p16-induced cellular senescence, which has been documented in several benign mesenchymal neoplasms.

Diverse tumorigenic pathways in ovarian serous carcinoma.

This study was undertaken to analyze genetic alterations in 108 sporadic serous ovarian neoplasms to elucidate ovarian serous carcinogenesis. Our results demonstrate that K-ras mutations occur in approximately 50% of serous borderline tumors (SBTs), non-invasive micropapillary serous carcinomas (MPSCs), and invasive micropapillary serous carcinomas, which represent a morphological continuum of tumor progression. Moreover, progressive increase in the degree of allelic imbalance of chromosomes 1p, 5q, 8p, 18q, 22q, and Xp was observed comparing serous borderline tumors to noninvasive and invasive micropapillary serous carcinomas. In contrast, high-grade (conventional serous carcinoma) tumors contained wild-type K-ras in all 23 cases studied and a high frequency of allelic imbalance even in small (early) primary tumors similar to that found in advanced stage tumors. Based on these findings, we propose a dualistic model for ovarian serous carcinogenesis. One pathway involves a stepwise progression from SBT to noninvasive and then invasive MPSC. The other pathway is characterized by rapid progression from the ovarian surface epithelium or inclusion cysts to a conventional (high-grade) serous carcinoma.

New pathologic classification of lung cancer: relevance for clinical practice and clinical trials.

We summarize significant changes in pathologic classification of lung cancer resulting from the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) lung adenocarcinoma classification. The classification was developed by an international core panel of experts representing IASLC, ATS, and ERS with oncologists/pulmonologists, pathologists, radiologists, molecular biologists, and thoracic surgeons. Because 70% of patients with lung cancer present with advanced stages, a new approach to small biopsies and cytology with specific terminology and criteria focused on the need for distinguishing squamous cell carcinoma from adenocarcinoma and on molecular testing for EGFR mutations and ALK rearrangement. Tumors previously classified as non-small-cell carcinoma, not otherwise specified, because of the lack of clear squamous or adenocarcinoma morphology should be classified further by using a limited immunohistochemical workup to preserve tissue for molecular testing. The terms "bronchioloalveolar carcinoma" and "mixed subtype adenocarcinoma" have been discontinued. For resected adenocarcinomas, new concepts of adenocarcinoma in situ and minimally invasive adenocarcinoma define patients who, if they undergo complete resection, will have 100% disease-free survival. Invasive adenocarcinomas are now classified by predominant pattern after using comprehensive histologic subtyping with lepidic, acinar, papillary, and solid patterns; micropapillary is added as a new histologic subtype with poor prognosis. Former mucinous bronchioloalveolar carcinomas are now called "invasive mucinous adenocarcinoma. " Because the lung cancer field is now rapidly evolving with new advances occurring on a frequent basis, particularly in the molecular arena, this classification provides a much needed standard for pathologic diagnosis not only for patient care but also for clinical trials and TNM classification.

Reduced Cytokine Release in Ex Vivo Response to Cilengitide and Cetuximab Is a Marker for Improved Survival of Head and Neck Cancer Patients

Targeting of αVβ3 and αVβ5 integrins by cilengitide may reduce growth of solid tumors including head and neck squamous cell carcinoma (HNSCC). Preclinical investigations suggest increased activity of cilengitide in combination with other treatment modalities. The only published trial in HNSCC (ADVANTAGE) investigated cisplatin, 5-fluorouracil, and cetuximab (PFE) without or with once (PFE+CIL1W) or twice weekly cilengitide (PFE+CIL2W) in recurrent/metastatic HNSCC. ADVANTAGE showed good tolerability of the cilengitide arms and even lower adverse events (AEs) compared to PFE but not the benefit in overall survival expected based on preclinical data. As we found in the FLAVINO assay, a short-time ex vivo assay for prediction of chemosensitivity, only a subgroup of HNSCC had an increased suppressive effect of cilengitide containing combination therapies on colony formation of epithelial cells (CFec) and release of pro-angiogenetic and pro-inflammatory cytokines, whereas other HNSCC failed to respond. Response to αVβ3 and αVβ5 integrin targeting by cilengitide classifies HNSCC regarding outcome. We present FLAVINO data arguing for further development of cilengitide plus cetuximab in treatment of a subgroup of HNSCC potentially identified by the FLAVINO assay using a set of biomarkers for response evaluation.

Evaluation of HER-2/neu amplification and other biological markers as predictors of response to neoadjuvant anthracycline-based chemotherapy in primary breast cancer: the role of anthracycline dose intensity.

OBJECTIVES The value of HER-2/neu status as a predictor of response to anthracycline-based chemotherapy is still a matter of debate. We evaluated the contribution of HER-2/neu gene amplification and other biologic markers in predicting response to different doses of neoadjuvant anthracycline-based chemotherapy. METHODS Clinical and pathologic records of 115 primary breast cancer patients were reviewed. Forty-eight and 67 patients received high (doxorubicin > or =20 mg/m2/wk; epirubicin > or =30 mg/m2/wk) and moderate-low anthracycline dose intensity regimens, respectively. Pathologic diagnosis, hormonal receptor status (HR), Ki67, and HER-2/neu status were assessed on tumor samples before neoadjuvant chemotherapy. HER-2/neu was determined by fluorescence in situ hybridization (FISH). RESULTS HER-2/neu amplification was observed in 29/115 (25%) tumors, 18 from moderate-low-dose and 11 from high-dose group. In the univariate analysis, a high Ki67 index (> or =20%) and positive clinical axillary nodes were predictive of an objective tumor response (P = 0.033 and 0.001, respectively). In the multivariate analysis, Ki67 was the only factor predictive of response (OR = 3.08, 95% CI = 1.1-8.5, P = 0.03). HER-2/neu status was not a factor in predicting objective response to different anthracycline dose intensities. The same finding was observed with regards to HR and Ki67. CONCLUSIONS In our series, no significant dose-response relationship was found according to HER-2/neu status.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus type 8-positive solid lymphomas: a tissue-based variant of primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus type 8, is consistently identified in Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. Here we report four cases of KSHV-bearing solid lymphomas that occurred in AIDS patients (cases 1 to 3) and in a human immunodeficiency virus (HIV)-seronegative person (case 4). The patients presented extranodal masses in the abdomen (cases 1, 3, and 4) or skin (case 2), and nodal involvement, together with Kaposi's sarcoma (case 3). The gastrointestinal tract was involved in two patients (cases 1 and 3). The patients did not develop a lymphomatous effusion. KSHV was detected in the tumor cells of all cases by immunohistochemistry and by polymerase chain reaction. Epstein-Barr virus was detected in two of the HIV-related cases. All KSHV-positive solid lymphomas exhibited PEL-like cell morphology. To investigate the relationship of these disorders to PEL and to other AIDS-associated diffuse large cell lymphomas, KSHV-positive solid lymphomas were tested for the expression of a set of genes that were previously shown by gene profiling analysis to define PEL tumor cells. The results showed that expression of this set of genes in KSHV-positive lymphomas is similar to that of PEL but distinct from KSHV-negative AIDS-associated diffuse large cell lymphomas. Because pathobiological features of KSHV-positive solid lymphomas closely mimic those of PEL, our results suggest that KSHV-positive solid lymphomas should be considered as a tissue-based variant of classical PEL, irrespective of HIV status.

Cancer risks attributable to low doses of ionizing radiation: assessing what we really know.

High doses of ionizing radiation clearly produce deleterious consequences in humans, including, but not exclusively, cancer induction. At very low radiation doses the situation is much less clear, but the risks of low-dose radiation are of societal importance in relation to issues as varied as screening tests for cancer, the future of nuclear power, occupational radiation exposure, frequent-flyer risks, manned space exploration, and radiological terrorism. We review the difficulties involved in quantifying the risks of low-dose radiation and address two specific questions. First, what is the lowest dose of x- or gamma-radiation for which good evidence exists of increased cancer risks in humans? The epidemiological data suggest that it is approximately 10-50 mSv for an acute exposure and approximately 50-100 mSv for a protracted exposure. Second, what is the most appropriate way to extrapolate such cancer risk estimates to still lower doses? Given that it is supported by experimentally grounded, quantifiable, biophysical arguments, a linear extrapolation of cancer risks from intermediate to very low doses currently appears to be the most appropriate methodology. This linearity assumption is not necessarily the most conservative approach, and it is likely that it will result in an underestimate of some radiation-induced cancer risks and an overestimate of others.

Heterogenic Loss of the Wild-Type BRCA Allele in Human Breast Tumorigenesis

For individuals genetically predisposed to breast and ovarian cancer through inheritance of a mutant BRCA allele, somatic loss of heterozygosity affecting the wild-type allele is considered obligatory for cancer initiation and/or progression. However, several lines of evidence suggest that phenotypic effects may result from BRCA haploinsufficiency. Archival fixed and embedded tissue specimens from women with germ line deleterious mutations in BRCA1 or BRCA2 were identified. After pathologic review, focal areas of normal breast epithelium, atypical ductal hyperplasia, ductal carcinoma-in-situ, and invasive ductal carcinoma were identified from 14 BRCA1-linked and 9 BRCA2-linked breast cancers. Ten BRCA-linked prophylactic mastectomy specimens and 12 BRCA-linked invasive ovarian carcinomas were also studied. Laser catapult microdissection was used to isolate cells from the various pathologic lesions and corresponding normal tissues. After DNA isolation, real-time polymerase chain reaction assays were used to quantitate the proportion of wild-type to mutant BRCA alleles in each tissue sample. Quantitative allelotyping of microdissected cells revealed a high level of heterogeneity in loss of heterozygosity within and between preinvasive lesions and invasive cancers from BRCA1 and BRCA2 heterozygotes with breast cancer. In contrast, all BRCA-associated ovarian cancers displayed complete loss of the wild-type BRCA allele. These data suggest that loss of the wild-type BRCA allele is not required for BRCA-linked breast tumorigenesis, which would have important implications for the genetic mechanism of BRCA tumor suppression and for the clinical management of this patient population.

Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis.

Store-operated Ca(2+) entry (SOCE) is the principal Ca(2+) entry mechanism in nonexcitable cells. Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) sensor that triggers SOCE activation. However, the role of STIM1 in regulating cancer progression remains controversial and its clinical relevance is unclear. Here we show that STIM1-dependent signaling is important for cervical cancer cell proliferation, migration, and angiogenesis. STIM1 overexpression in tumor tissue is noted in 71% cases of early-stage cervical cancer. In tumor tissues, the level of STIM1 expression is significantly associated with the risk of metastasis and survival. EGF-stimulated cancer cell migration requires STIM1 expression and EGF increases the interaction between STIM1 and Orai1 in juxta-membrane areas, and thus induces Ca(2+) influx. STIM1 involves the activation of Ca(2+)-regulated protease calpain, as well as Ca(2+)-regulated cytoplasmic kinase Pyk2, which regulate the focal-adhesion dynamics of migratory cervical cancer cells. Because of an increase of p21 protein levels and a decrease of Cdc25C protein levels, STIM1-silencing in cervical cancer cells significantly inhibits cell proliferation by arresting the cell cycle at the S and G2/M phases. STIM1 also regulates the production of VEGF in cervical cancer cells. Interference with STIM1 expression or blockade of SOCE activity inhibits tumor angiogenesis and growth in animal models, confirming the crucial role of STIM1-mediated Ca(2+) influx in aggravating tumor development in vivo. These results make STIM1-dependent signaling an attractive target for therapeutic intervention.

Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types.

Cell-free tumor DNA (ctDNA) has the potential to enable non-invasive diagnostic tests for personalized medicine in providing similar molecular information as that derived from invasive tumor biopsies. The histology-independent phase II SHIVA trial matches patients with targeted therapeutics based on previous screening of multiple somatic mutations using metastatic biopsies. To evaluate the utility of ctDNA in this trial, as an ancillary study we performed de novo detection of somatic mutations using plasma DNA compared to metastasis biopsies in 34 patients covering 18 different tumor types, scanning 46 genes and more than 6800 COSMIC mutations with a multiplexed next-generation sequencing panel. In 27 patients, 28 of 29 mutations identified in metastasis biopsies (97%) were detected in matched ctDNA. Among these 27 patients, one additional mutation was found in ctDNA only. In the seven other patients, mutation detection from metastasis biopsy failed due to inadequate biopsy material, but was successful in all plasma DNA samples providing three more potential actionable mutations. These results suggest that ctDNA analysis is a potential alternative and/or replacement to analyses using costly, harmful and lengthy tissue biopsies of metastasis, irrespective of cancer type and metastatic site, for multiplexed mutation detection in selecting personalized therapies based on the patient's tumor genetic content.

Time from finding abnormality on mass-screening to final diagnosis of lung cancer.

Mass-screening for lung cancer is rather a unique system in Japan. This study illustrates time from finding abnormality on mass-screening to final diagnosis of lung cancer. Among the 517 patients with lung cancer who were admitted to our hospital over a 10-year period up to December 2001, 83 (16.1%) were detected by mass-screening. We reviewed medical records of the 83 patients and determined the intervals from the mass-screening to the pathological diagnosis with clinical staging. Time from the mass-screening to the date of hospital visit was <2 months in 62 (74.7%) cases. Five (6.0%) patients visited hospital more than 6 months after the mass-screening. With respect to the interval, there was no statistical difference in gender (p=0.0680) and age (p=0.1532). Among 60 patients who were referred from outside, on average, patients visited our hospital 0.5 month after they first sought medical attention at nearby clinic, and at our hospital 0.5 month was required to make a pathological diagnosis of lung cancer with TNM staging. There was a statistical difference in survival between the patients who were diagnosed <4 months and the patients who were diagnosed >4 months from the screening (p=0.0487). The interval in most cases was acceptable. However, further improvements are still needed to minimize the delay and to maximize the benefits of early cancer detection.

Population density profiles of nasopharyngeal carriage of 5 bacterial species in pre-school children measured using quantitative PCR offer potential insights into the dynamics of transmission.

Bacterial vaccines can reduce carriage rates. Colonization is usually a binary endpoint. Real time quantitative PCR (qPCR) can quantify bacterial DNA in mucosal samples over a wide range. Using culture and single-gene species-specific qPCRs for Streptococcus pneumoniae (lytA), Streptococcus pyogenes (ntpC), Moraxella catarrhalis (ompJ), Haemophilus influenzae (hdp) and Staphylococcus aureus (nuc) and standard curves against log-phase reference strain broth cultures we described frequency and peak density distributions of carriage in nasopharyngeal swabs from 161 healthy 2-4 y old children collected into STGG broth. In general, detection by qPCR and culture was consistent. Discordance mostly occurred at lower detection thresholds of both methods, although PCR assays for S. pyogenes and S. aureus were less sensitive. Density varied across 5-7 orders of magnitude for the 5 species with the abundant species skewed toward high values (modes: S. pneumoniae log3-4, M. catarrhalis & H. influenzae log4-5 CFU/ml broth). Wide ranges of bacterial DNA concentrations in healthy children carrying these bacteria could mean that different individuals at different times vary greatly in infectiousness. Understanding the host, microbial and environmental determinants of colonization density will permit more accurate prediction of vaccine effectiveness.

Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells

BACKGROUND microRNAs (miRNAs) stably exist in circulating blood and are encapsulated in extracellular vesicles such as exosomes. The aims of this study were to identify which exosomal miRNAs are highly produced from epithelial ovarian cancer (EOC) cells, to analyze whether serum miRNA can be used to discriminate patients with EOC from healthy volunteers, and to investigate the functional role of exosomal miRNAs in ovarian cancer progression. METHODS Exosomes were collected from the culture media of serous ovarian cancer cell lines, namely TYK-nu and HeyA8 cells. An exosomal miRNA microarray revealed that several miRNAs including miR-99a-5p were specifically elevated in EOC-derived exosomes. Expression levels of serum miR-99a-5p in 62 patients with EOC, 26 patients with benign ovarian tumors, and 20 healthy volunteers were determined by miRNA quantitative reverse transcription-polymerase chain reaction. To investigate the role of exosomal miR-99a-5p in peritoneal dissemination, neighboring human peritoneal mesothelial cells (HPMCs) were treated with EOC-derived exosomes and then expression levels of miR-99a-5p were examined. Furthermore, mimics of miR-99a-5p were transfected into HPMCs and the effect of miR-99a-5p on cancer invasion was analyzed using a 3D culture model. Proteomic analysis with the tandem mass tag method was performed on HPMCs transfected with miR-99a-5p and then potential target genes of miR-99a-5p were examined. RESULTS The serum miR-99a-5p levels were significantly increased in patients with EOC, compared with those in benign tumor patients and healthy volunteers (1.7-fold and 2.8-fold, respectively). A receiver operating characteristic curve analysis showed with a cut-off of 1.41 showed sensitivity and specificity of 0.85 and 0.75, respectively, for detecting EOC (area under the curve = 0.88). Serum miR-99a-5p expression levels were significantly decreased after EOC surgeries (1.8 to 1.3, p = 0.002), indicating that miR-99a-5p reflects tumor burden. Treatment with EOC-derived exosomes significantly increased miR-99a-5p expression in HPMCs. HPMCs transfected with miR-99a-5p promoted ovarian cancer invasion and exhibited increased expression levels of fibronectin and vitronectin. CONCLUSIONS Serum miR-99a-5p is significantly elevated in ovarian cancer patients. Exosomal miR-99a-5p from EOC cells promotes cell invasion by affecting HPMCs through fibronectin and vitronectin upregulation and may serve as a target for inhibiting ovarian cancer progression.

Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation.

Although 75% of endometrial cancers are treated at an early stage, 15% to 20% of these recur. We performed an integrated analysis of genome-wide expression and copy-number data for primary endometrial carcinomas with extensive clinical and histopathological data to detect features predictive of recurrent disease. Unsupervised analysis of the expression data distinguished 2 major clusters with strikingly different phenotypes, including significant differences in disease-free survival. To identify possible mechanisms for these differences, we performed a global genomic survey of amplifications, deletions, and loss of heterozygosity, which identified 11 significantly amplified and 13 significantly deleted regions. Amplifications of 3q26.32 harboring the oncogene PIK3CA were associated with poor prognosis and segregated with the aggressive transcriptional cluster. Moreover, samples with PIK3CA amplification carried signatures associated with in vitro activation of PI3 kinase (PI3K), a signature that was shared by aggressive tumors without PIK3CA amplification. Tumors with loss of PTEN expression or PIK3CA overexpression that did not have PIK3CA amplification also shared the PI3K activation signature, high protein expression of the PI3K pathway member STMN1, and an aggressive phenotype in test and validation datasets. However, mutations of PTEN or PIK3CA were not associated with the same expression profile or aggressive phenotype. STMN1 expression had independent prognostic value. The results affirm the utility of systematic characterization of the cancer genome in clinically annotated specimens and suggest the particular importance of the PI3K pathway in patients who have aggressive endometrial cancer.

Risk of breast cancer in relation to blood lipids: a prospective study of 31,209 Norwegian women

In this prospective study, the relationship between blood lipids and breast cancer risk was examined. Between 1977 and 1983, 31,209 Norwegian women, 20 to 54 years of age, attended a health screening carried out by the Norwegian National Health Screening Services. The screening consisted of a questionnaire, anthropometric measurements, and nonfasting blood drawn for analysis of total serum cholesterol (TC), triglyceride (TG), and high density lipoprotein (HDL) cholesterol. Low density lipoprotein (LDL) cholesterol was calculated by the Friedewald's formula. During the seven to 13 years of follow-up, 302 breast cancer cases were identified by linkage to the Norwegian Cancer Registry. After adjustment for some of the known risk factors of breast cancer, the relative risk of women in the highest quartile of TC compared with women in the lowest quartile was 0.87 (95 percent confidence interval [CI]=0.61–1.23). The corresponding relative risks and CIs were 0.82 (CI=0.58–1.16) for TG, 1.02 (CI=0.73–1.42) for HDL, and 0.93 (CI=0.67–1.29) for LDL. No association between breast cancer risk and blood lipids was found in the total population, nor when the data were divided into those diagnosed before or after the age of 50 as a dividing line between pre- and postmenopausal diagnosis.

Risk of malignant neoplasms in patients with pulmonary sarcoidosis.

BACKGROUND For over 20 years the association between sarcoidosis and malignancy, particularly lymphoma and lung cancer, has been disputed with misclassification being the major concern. The aim of the present study was to analyse the incidence of malignancies in a cohort of patients with sarcoidosis by linkage to a nationwide population based cancer register. METHODS The cohort comprised 254 patients followed for a median of 25 years until death, emigration, or 31 December 1992, whichever came first. The expected number of cancer cases was calculated using the annual age and sex specific cancer rates from the Danish Cancer Registry. RESULTS Thirty six cancers were registered, three of which were misclassified as sarcoidosis, leaving 33 cancers compared with 23 expected (standardised incidence ratio (SIR) = 1.4; 95% CI 0.99 to 2.0). Five lung cancers were observed compared with 2.5 expected, yielding an SIR of 2.0 (95% CI 0.7 to 4.7). There was no incidence of lymphoma and only one case of leukaemia. There was a significant excess number of pharyngeal cancers based on two cases (SIR = 15.4; 95% CI 1.7 to 56). CONCLUSIONS This study does not support the theory of an association between sarcoidosis and malignancy, and the main reason other studies have shown such an association is most likely to have been due to selection bias and misclassification.

Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor

The activation of T cells is the fundamental on switch for the adaptive immune system. Ca2+ signaling is essential for T cell activation and starts as initial, short-lived, localized Ca2+ signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca2+ signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca2+ indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca2+ signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca2+ signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1−/− mice, either these early Ca2+ signals were not detected or the number of signals was markedly reduced. Local Ca2+ signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca2+ signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca2+ release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.

An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.

The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

Oncologists' perceptions of the effects of cancer patients' companions on physician-patient interactions

To determine physicians' perceptions of the effects that the companions of cancer patients have on physician-patient communication, semistructured interviews were conducted with 12 oncologists (6 medical, 4 surgical, and 2 radiation) from a total population of 21 oncologists. The physicians estimated that threefourths of their patients brought companions with them to consultations and said that these consultations were more complex for the physician. The behaviors of the companions varied from domination to passive note taking, and the companions who were young professional men or older women who accompanied their husbands were the most assertive and asked the most questions. All possible coalitions were observed during medical visits. The physicians perceived that companions and patients often had different agendas and noted differences in the companions' behaviors according to their gender and whether they lived in rural or urban areas.

painDETECT: a new screening questionnaire to identify neuropathic components in patients with back pain.

OBJECTIVE Nociceptive and neuropathic components both contribute to pain. Since these components require different pain management strategies, correct pain diagnosis before and during treatment is highly desirable. As low back pain (LBP) patients constitute an important subgroup of chronic pain patients, we addressed the following issues: (i) to establish a simple, validated screening tool to detect neuropathic pain (NeP) components in chronic LBP patients, (ii) to determine the prevalence of neuropathic pain components in LBP in a large-scale survey, and (iii) to determine whether LBP patients with an NeP component suffer from worse, or different, co-morbidities. METHODS In co-operation with the German Research Network on Neuropathic Pain we developed and validated the painDETECT questionnaire (PD-Q) in a prospective, multicentre study and subsequently applied it to approximately 8000 LBP patients. RESULTS The PD-Q is a reliable screening tool with high sensitivity, specificity and positive predictive accuracy; these were 84% in a palm-top computerised version and 85%, 80% and 83%, respectively, in a corresponding pencil-and-paper questionnaire. In an unselected cohort of chronic LBP patients, 37% were found to have predominantly neuropathic pain. Patients with NeP showed higher ratings of pain intensity, with more (and more severe) co-morbidities such as depression, panic/anxiety and sleep disorders. This also affected functionality and use of health-care resources. On the basis of given prevalence of LBP in the general population, we calculated that 14.5% of all female and 11.4% of all male Germans suffer from LBP with a predominant neuropathic pain component. CONCLUSION Simple, patient-based, easy-to-use screening questionnaires can determine the prevalence of neuropathic pain components both in individual LBP patients and in heterogeneous cohorts of such patients. Since NeP correlates with more intense pain, more severe co-morbidity and poorer quality of life, accurate diagnosis is a milestone in choosing appropriate therapy.

Venous thrombosis in cancer patients: insights from the FRONTLINE survey.

BACKGROUND Venous thromboembolism (VTE) is a common complication in cancer patients and a significant cause of morbidity and mortality. However, little information is available on oncologists' perceptions of the risk of VTE and its management. The Fundamental Research in Oncology and Thrombosis (FRONTLINE) study is the first comprehensive global survey of thrombosis and cancer. The study was designed to collect data on the perceived risk and patterns of practice with regard to VTE in cancer patients undergoing surgical and medical management of their malignancy and to provide information on international and regional practice patterns, allowing for the design of research studies to answer the concerns of practicing clinicians. METHODS Literature reviews were performed to provide a current evidence base against which to compare the findings, and a survey was developed under the guidance of an advisory board. A paper-based reply-paid questionnaire was distributed globally between July and November 2001 to clinicians involved in cancer care and was made available on a dedicated website. FINDINGS A total of 3,891 completed responses were available for analysis. Brain and pancreatic tumors were considered to carry a high risk for VTE, and 80% of respondents considered the use of central venous lines to be associated with a high risk of VTE. Marked differences were seen in the use of thromboprophylaxis for surgical and medical cancer patients, with over 50% of surgeons reporting that they initiated thromboprophylaxis routinely, while most medical oncologists reported using thromboprophylaxis in less than 5% of medical patients. Low molecular weight heparin (LMWH) was the most popular method of thromboprophylaxis employed in both surgical and medical patients and was more favored by European than U.S. clinicians. Some 20% of respondents reported using aspirin for prophylaxis, despite there being no reliable evidence for this agent as effective in prevention in this population. For the treatment of VTE, LMWH was again the most common initial treatment, although, for the long-term, oral anticoagulation therapy was widely adopted. Many patients were treated for VTE on an outpatient basis, and secondary prevention of VTE was typically continued for 3 to 6 months after an episode of deep vein thrombosis or for longer in the case of pulmonary embolism. INTERPRETATION The results of the FRONTLINE survey demonstrate a need for guidelines to direct clinical practice in line with evidence-based data concerning cancer and VTE. Oncologists need to be educated regarding the true risks of VTE associated with certain cancers and on strategies for prevention and treatment to reduce the morbidity and mortality associated with VTE in all cancer patients. The study has also helped identify areas for future research.

Ovarian Surface Epithelium in Patients with Severe Ovarian Infertility: A Potential Source of Cells Expressing Markers of Pluripotent/Multipotent Stem Cells

The aim of this study was to confirm the presence of stem cells in the ovarian surface epithelium of patients with premature ovarian failure and no mature follicles and oocytes. In these patients, small round cells of unknown origin expressing SOX-2 marker of pluripotency were observed among the epithelial cells just after the ovarian surface epithelium scraping. These cells were an integral part of the ovarian surface epithelium. When the scraped cells were cultured in a medium with added follicular fluid to provide some ovarian niche, primitive oocyte-like cells and typical round-shaped cell clusters positively stained on alkaline phosphatase, and markers of pluripotency, such as SOX-2 and SSEA-4, were developed. These markers were expressed early and also later in the culture. Single oocyte-like cells expressed genes OCT4A, SOX-2, NANOG, NANOS, STELLA, CD9, LIN28, KLF4, GDF3, and MYC, characteristic for pluripotent stem cells. The results of this study confirmed the presence of putative stem cells in the ovarian surface epithelium of these patients and provided some basis to create a stem cell line in the future.

Geographical access to healthcare in Northern England and post-mortem diagnosis of cancer.

BACKGROUND There is some previous evidence that diagnosis of cancer at death, recorded as registry death certificate only records, is associated with problems of access to care. METHODS Records from the Northern and Yorkshire Cancer Registry for patients registered with breast, colorectal, lung, ovarian or prostate cancer between 1994 and 2002 were supplemented with measures of travel time to general practitioner and hospital services, and social deprivation. Logistic regression was used to identify predictors of records where diagnosis was at death. RESULTS There was no association between the odds diagnosis at death and access to primary care. For all sites except breast, the highest odds of being a cancer diagnosed at death fell among those living in the highest quartile of hospital travel time, although it was only statistically significant for colorectal and ovary tumours. Those in the most deprived and furthest travel time to hospital quartile were 2.6 times more likely to be a diagnosis at death case compared with those in the most affluent and proximal areas. CONCLUSIONS There is some evidence that poorer geographical access to tertiary care, in particular when coupled with social disadvantages, may be associated with increased odds of diagnosis at death.

Assessment of the cerebral circulation in adults with coarctation of the aorta.

AIMS There is a fivefold increase in the frequency of intracranial aneurysm (IA) in adults with coarctation of the aorta (CoA). Current guidelines for management of adults with CoA recommend computed tomography angiography (CTA) or magnetic resonance imaging of the intracranial vessels. However, this recommendation has not been universally accepted. The purpose of our study was to prospectively perform CTA of the intracranial vessels in adults with CoA to evaluate the prevalence and identify high-risk features of this complication. METHODS AND RESULTS From January 2008 to February 2011, adults ≥18 years of age with CoA were prospectively enrolled in a screening program with CTA of the intracranial vessels. Analyses of prognostic variables were performed with both Fisher's exact and two sample t-test. Forty-three patients (58% female, 33.55 ± 10.21 years) with CoA completed CTA of the intracranial vessels. Five patients (11%) were found to have IA. Patients with IA were older than those without (45.6 ± 8.17 vs. 30.89 ± 7.89, P = 0.0003). There were no statistically significant differences detected between measurements of fasting lipid profiles, C-reactive protein, brain natriuretic peptide, and homocysteine levels among CoA patients with and without IA (P = not significant). CONCLUSION Prospective screening of adults with CoA confirmed the increased prevalence of IA but also identified increased age as the sole risk factor. These data suggested that screening is justified particularly in the fourth and fifth decades of life. Further studies are required that focus on the development, natural history, and treatment of IA.

Hypoxia in cancer: significance and impact on clinical outcome

Hypoxia, a characteristic feature of locally advanced solid tumors, has emerged as a pivotal factor of the tumor (patho-)physiome since it can promote tumor progression and resistance to therapy. Hypoxia represents a “Janus face” in tumor biology because (a) it is associated with restrained proliferation, differentiation, necrosis or apoptosis, and (b) it can also lead to the development of an aggressive phenotype. Independent of standard prognostic factors, such as tumor stage and nodal status, hypoxia has been suggested as an adverse prognostic factor for patient outcome. Studies of tumor hypoxia involving the direct assessment of the oxygenation status have suggested worse disease-free survival for patients with hypoxic cervical cancers or soft tissue sarcomas. In head & neck cancers the studies suggest that hypoxia is prognostic for survival and local control. Technical limitations of the direct O2 sensing technique have prompted the use of surrogate markers for tumor hypoxia, such as hypoxia-related endogenous proteins (e.g., HIF-1α, GLUT-1, CA IX) or exogenous bioreductive drugs. In many—albeit not in all—studies endogenous markers showed prognostic significance for patient outcome. The prognostic relevance of exogenous markers, however, appears to be limited. Noninvasive assessment of hypoxia using imaging techniques can be achieved with PET or SPECT detection of radiolabeled tracers or with MRI techniques (e.g., BOLD). Clinical experience with these methods regarding patient prognosis is so far only limited. In the clinical studies performed up until now, the lack of standardized treatment protocols, inconsistencies of the endpoints characterizing the oxygenation status and methodological differences (e.g., different immunohistochemical staining procedures) may compromise the power of the prognostic parameter used.

Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses

BACKGROUND Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases. OBJECTIVES Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping. METHODS The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non-H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR. RESULTS Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non-H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95.4% and 64.6% for the HA2 and CS RRT PCRs respectively, and specificity 97.9% and 99.6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post-inoculation and LPAI H7N1 chicken infections sampled at 40-64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested. CONCLUSIONS The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.

Preparing the outbreak assistance laboratory network in the Netherlands for the detection of the influenza virus A(H1N1) variant.

BACKGROUND Late April 2009, human infection with variant influenza virus A(H1N1)v emerged in the Northern Americas posing a threat that this virus may become the next pandemic influenza virus. OBJECTIVES To prepare laboratories for surge capacity for molecular diagnosis of patients suspected for A(H1N1)v infection in the Netherlands. STUDY DESIGN A panel of 10 blinded specimens containing seasonal A(H1N1) or A(H3N2), or A/Netherlands/602/2009(H1N1)v influenza virus, or negative control was distributed to the outbreak assistance laboratories (OAL) together with influenza virus A (M-gene), swine influenza virus A (NP-gene) and influenza virus A(H1N1)v (H1v-gene) specific primers and probes and protocol (CDC Atlanta, USA). Laboratories were asked to implement and test this protocol. RESULTS All OAL were able to detect A(H1N1)v using the CDC M-gene reagents, the majority with similar sensitivity as the in-house M-gene based assays. RT-PCRs used in routine diagnostic setting in the OAL specifically designed to detect H1, H3, or NS1 from seasonal influenza A viruses, did not or at very low level cross-react with A(H1N1)v. The CDC swine NP-gene and H1v-gene RT-PCRs showed somewhat reduced sensitivity compared to the CDC and in-house M-gene RT-PCRs. In contrast, in-house developed A(H1N1)v specific H1v-gene and N1v-gene RT-PCRs showed equal sensitivity to CDC and in-house M-gene RT-PCRs. CONCLUSIONS The Dutch OAL are prepared for detection and specific identification of A(H1N1)v, although some level of cross-reactivity was observed with seasonal influenza viruses. Additionally, M-gene based generic influenza A virus detection is recommended to be able to detect emerging influenza A viruses in routine settings.

Implementing the ESHRE 'poor responder' criteria in research studies: methodological implications.

The Bologna criteria for defining poor ovarian response (POR) during IVF provide a useful template for new research in this field of assisted conception. However, designing studies around the European Society for Human Reproduction and Embryology POR criteria can be methodologically challenging, as the new definition includes various POR subpopulations with diverse baseline characteristics and unknown clinical prognosis. When designing RCTs, potential result bias may be introduced if women from each subpopulation are not evenly allocated between intervention groups. In the case of small or moderate-size RCTs, a single-sequence randomization method may not ensure balanced allocation between groups. Stratified randomization methods provide an alternative methodological approach. Depending on the chosen methodology, patient characteristics and outcomes within each intervention group may be better reported according to relevant subpopulations.

Reduced expression of oestrogen receptor beta in invasive breast cancer and its re-expression using DNA methyl transferase inhibitors in a cell line model.

To gain insights into the possible role of oestrogen receptor (ER) beta in breast carcinogenesis, immunohistochemical analysis of ER beta was performed on 512 breast specimens encompassing normal (n = 138), pure ductal carcinoma in situ (n = 16), invasive cancers (n = 319), lymph node metastases (n = 31), and recurrences (n = 8). Real-time polymerase chain reaction (PCR) was used to investigate the methylation status of the ER beta gene in the ER beta negative breast cancer cell lines SkBr3 and MDA-MB-435. A gradual reduction in, but not a complete loss of, ER beta expression was observed during the transition from normal and pre-invasive lesions to invasive cancers, where ER beta was lost in 21% of cases. This was more pronounced in invasive ductal than in lobular carcinomas, a significantly higher proportion of which were ER beta-positive (74% compared with 91%, respectively, p = 0.0004). Examination of paired primary cancers with their axillary lymph node metastases showed that if ER beta was present in the primary tumour, it persisted in the metastasis. Treatment of ER beta-negative cell lines with DNA methyl transferase inhibitors restored ER beta expression, providing experimental evidence that silencing of ER beta in breast carcinomas could be due to promoter hypermethylation. These results suggest that loss of ER beta expression is one of the hallmarks of breast carcinogenesis and that it may be a reversible process involving methylation.

Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer.

Cells or cell-free fluid of malignant pleural effusion could be important clinical specimen for epidermal growth factor receptor (EGFR) mutation screening in advanced non-small cell lung cancer (NSCLC) patients. However, their usefulness in mutation detection has not been well compared. In this study we recruited 26 East Asian NSCLC patients with malignant pleural effusion, determined the mutation status of EGFR in both cells and matched cell-free fluid with the use of sequencing and mutant-enriched PCR. After comparing the mutation spectrums, we found both the cells and cell-free pleural fluid may be feasible clinical specimen for EGFR mutation detection in unresectable NSCLC given sensitive genotyping assays employed. Direct sequencing could miss a significant portion of mutations in these heterogeneous specimens. More sensitive methods, such as mutant-enriched PCR and gene scan, could provide more reliable mutational information.

A novel set of DNA methylation markers in urine sediments for sensitive/specific detection of bladder cancer.

PURPOSE This study aims to provide a better set of DNA methylation markers in urine sediments for sensitive and specific detection of bladder cancer. EXPERIMENTAL DESIGN Fifty-nine tumor-associated genes were profiled in three bladder cancer cell lines, a small cohort of cancer biopsies and urine sediments by methylation-specific PCR. Twenty-one candidate genes were then profiled in urine sediments from 132 bladder cancer patients (8 cases for stage 0a; 68 cases for stage I; 50 cases for stage II; 4 cases for stages III; and 2 cases for stage IV), 23 age-matched patients with noncancerous urinary lesions, 6 neurologic diseases, and 7 healthy volunteers. RESULTS Despite six incidences of four genes reported in 3 of 23 noncancerous urinary lesion patients analyzed, cancer-specific hypermethylation in urine sediments were reported for 15 genes (P < 0.05). Methylation assessment of an 11-gene set (SALL3, CFTR, ABCC6, HPR1, RASSF1A, MT1A, RUNX3, ITGA4, BCL2, ALX4, MYOD1, DRM, CDH13, BMP3B, CCNA1, RPRM, MINT1, and BRCA1) confirmed the existing diagnosis of 121 among 132 bladder cancer cases (sensitivity, 91.7%) with 87% accuracy. Significantly, more than 75% of stage 0a and 88% of stage I disease were detected, indicating its value in the early diagnosis of bladder cancer. Interestingly, the cluster of reported methylation markers used in the U.S. bladder cancers is distinctly different from that identified in this study, suggesting a possible epigenetic disparity between the American and Chinese cases. CONCLUSIONS Methylation profiling of an 11-gene set in urine sediments provides a sensitive and specific detection of bladder cancer.

Intrinsic genetic characteristics determine tumor-modifying capacity of fibroblasts: matrix metalloproteinase-3 5A/5A genotype enhances breast cancer cell invasion

Stromal fibroblasts can contribute to tumor invasion through the release of matrix metalloproteinases (MMPs). Population studies have suggested that single nucleotide polymorphisms (SNPs) in MMP genes influence levels of expression and may be associated with breast cancer risk and with disease progression. This study directly examined the impact of MMP SNP genotype on the ability of host fibroblasts to promote tumor cell invasion. Primary breast fibroblasts were isolated from patients with (n = 13) or without (n = 19) breast cancer, and their ability to promote breast cancer cell invasion was measured in in vitro invasion assays. Fibroblast invasion-promoting capacity (IPC) was analyzed in relation to donor type (tumor or non-tumor patient), MMP-1, MMP-3, and MMP-9 SNP genotype and MMP activity using independent samples t test and analysis of variance. All statistical tests were two-sided. Tumor-derived fibroblasts promoted higher levels of invasion than normal fibroblasts (p = 0.041). When IPC was related to genotype, higher levels of IPC were generated by tumor fibroblasts with the high-expressing MMP-3 5A/5A genotype compared with the 5A/6A and 6A/6A genotypes (p = 0.05 and 0.07, respectively), and this was associated with enhanced MMP-3 release. The functional importance of MMP-3 was demonstrated by enhanced invasion in the presence of recombinant MMP-3, whereas reduction occurred in the presence of a specific MMP-3 inhibitor. An inverse relationship was demonstrated between fibroblast IPC and the high-expressing MMP-1 genotype (p = 0.031), but no relationship was seen with MMP-9 SNP status. In contrast, normal fibroblasts showed no variation in IPC in relation to MMP genotype, with MMP-3 5A/5A fibroblasts exhibiting significantly lower levels of IPC than their tumor-derived counterparts (p = 0.04). This study has shown that tumor-derived fibroblasts exhibit higher levels of IPC than normal fibroblasts and that the MMP-3 5A/5A genotype contributes to this through enhanced MMP-3 release. Despite a high-expressing genotype, normal fibroblasts do not exhibit higher IPC or enhanced MMP release. This suggests that more complex changes occur in tumor-derived fibroblasts, enabling full expression of the MMP SNP genotype and these possibly are epigenetic in nature. The results do suggest that, in women with breast cancer, a high-expressing MMP-3 genotype may promote tumor progression more effectively.

Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks.

The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.

Gastric juice miR-129 as a potential biomarker for screening gastric cancer

MicroRNAs (miRNAs) play crucial roles during the occurrence and development of gastric cancer. Conventional serological tests for screening gastric cancer have limits on sensitivity and specificity. Several miRNAs in peripheral blood have been used as biomarkers of gastric cancer. However, most of these miRNAs are shared by several types of cancer. Thanks to the tissue specificity of gastric juice, here we examined the feasibility of using gastric juice miR-129-1/2, which are aberrantly expressed in gastric cancer, to screen gastric cancer. Total of 141 gastric juices samples from gastric cancer, gastric ulcer, atrophic gastritis, and minimal gastritis patients or subjects with normal mucosa were collected by gastroscopy. The gastric juice miR-129-1/2 levels were detected by quantitative reverse transcription-polymerase chain reaction. A receiver operating characteristic (ROC) curve was constructed for differentiating patients with gastric cancer from patients with benign gastric diseases. We showed that, compared with patients with benign gastric diseases, patients with gastric cancer had significantly lower levels of gastric juice miR-129-1-3p and miR-129-2-3p. The areas under ROC curve (AUC) were 0.639 and 0.651 for miR-129-1-3p and miR-129-2-3p, respectively. Using the parallel combination test, the AUC was up to 0.656. In summary, our results suggest that gastric juice miR-129-1-3p and miR-129-2-3p are potential biomarkers for the screening gastric cancer, and the detection of gastric juice miRNAs is a convenient non-invasion method for the diagnosis of gastric cancer.

Polymorphisms in EGFR and IL28B are associated with spontaneous clearance in an HCV-infected iranian population

Although most hepatitis C virus (HCV)-infected individuals develop chronic infection, about 25% of them are able to clear the virus spontaneously without any therapeutic intervention. The aim of the present study was to identify genes associated with spontaneous HCV clearance in a population of Iranian patients. We genotyped 110 single-nucleotide polymorphisms (SNPs) in 59 selected—candidate—genes in a cohort of 107 HCV-infected participants who spontaneously cleared the infection and 176 participants whose infection persisted. Three out of the 110 SNPs were found to be associated with HCV outcome (P-values<0.03). rs11506105 in EGFR (epidermal growth factor receptor gene), and rs11881222 and rs12979860 in IL28B (interferon-λ3 gene). Multivariate logistic regression of the three markers showed that the A/A genotypes in both rs11506105 (EFGR) and rs11881222 (IL28B), and the C/C genotype in rs12979860 (IL28B) are associated with HCV clearance (recessive model: odds ratio (OR)=2.06, 95% confidence interval (95% CI)=1.09–3.88, P=0.025; OR=2.09, 95% CI=1.23–3.60, P=0.007; and OR=1.95, 95% CI=1.15–3.35, P=0.014 for rs11506105, rs12979860 and rs11881222, respectively). In conclusion, EGFR and IL28B SNPs are strong independent predictive markers of spontaneous viral clearance.

A Teratocarcinoma-Like Human Embryonic Stem Cell (hESC) Line and Four hESC Lines Reveal Potentially Oncogenic Genomic Changes

The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis.

Microtubule-associated histone deacetylase 6 supports the calcium store sensor STIM1 in mediating malignant cell behaviors.

Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) storage sensor that promotes cell growth, migration, and angiogenesis in breast and cervical cancers. Here, we report that the microtubule-associated histone deacetylase 6 (HDAC6) differentially regulates activation of STIM1-mediated store-operated Ca(2+) entry (SOCE) between cervical cancer cells and normal cervical epithelial cells. Confocal microscopy of living cells indicated that microtubule integrity was necessary for STIM1 trafficking to the plasma membrane and interaction with Orai1, an essential pore subunit of SOCE. Cancer cells overexpressed both STIM1 and Orai1 compared with normal cervical epithelial cells. HDAC6 upregulation in cancer cells was accompanied by hypoacetylated α-tubulin. Tubastatin-A, a specific HDAC6 inhibitor, inhibited STIM1 translocation to plasma membrane and blocked SOCE activation in cancer cells but not normal epithelial cells. Genetic or pharmacologic inhibition of HDAC6 blocked STIM1 membrane trafficking and downstream Ca(2+) influx, as evidenced by total internal reflection fluorescent images and intracellular Ca(2+) determination. In contrast, HDAC6 inhibition did not affect interactions between STIM1 and the microtubule plus end-binding protein EB1. Analysis of surgical specimens confirmed that most cervical cancer tissues overexpressed STIM1 and Orai1, accompanied by hypoacetylated α-tubulin. Together, our results identify HDAC6 as a candidate target to disrupt STIM1-mediated SOCE as a general strategy to block malignant cell behavior.

Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal disease with a 5-year survival rate of 4%. A key hallmark of PDAC is extensive stromal involvement, which makes capturing precise tumor-specific molecular information difficult. Here we have overcome this problem by applying blind source separation to a diverse collection of PDAC gene expression microarray data, including data from primary tumor, metastatic and normal samples. By digitally separating tumor, stromal and normal gene expression, we have identified and validated two tumor subtypes, including a 'basal-like' subtype that has worse outcome and is molecularly similar to basal tumors in bladder and breast cancers. Furthermore, we define 'normal' and 'activated' stromal subtypes, which are independently prognostic. Our results provide new insights into the molecular composition of PDAC, which may be used to tailor therapies or provide decision support in a clinical setting where the choice and timing of therapies are critical.

Premature chromosome condensation: evidence for in vivo cell fusion in human malignant tumours.

Premature chromosome condensation (PCC) was studied in direct preparations of tissues from patients with haematological diseases and carcinomas of various histological types. PCC was found in 6 out of 166 malignancies (128 haematological cases, 35 carcinomas and 3 malignant effusions) analysed with the GTG-technique. Chromosome analysis revealed S-phase and G1-phase PCC in each case; the frequency of PCC varied between 1, 4 and 8.6% of the metaphases analysed. It is suggested that PCC chromosomes, which represent cell fusion in vivo, are not very rare in naturally-occurring human malignancies, and that cell fusion may affect the malignant phenotype. In conjunction with other factors they may also explain the heterogeneity of tumour cell populations.

Independent Association of Lobular Involution and Mammographic Breast Density With Breast Cancer Risk

BACKGROUND Lobular involution, or age-related atrophy of breast lobules, is inversely associated with breast cancer risk, and mammographic breast density (MBD) is positively associated with breast cancer risk. METHODS To evaluate whether lobular involution and MBD are independently associated with breast cancer risk in women with benign breast disease, we performed a nested cohort study among women (n = 2666) with benign breast disease diagnosed at Mayo Clinic between January 1, 1985, and December 31, 1991 and a mammogram available within 6 months of the diagnosis. Women were followed up for an average of 13.3 years to document any breast cancer incidence. Lobular involution was categorized as none, partial, or complete; parenchymal pattern was classified using the Wolfe classification as N1 (nondense), P1, P2 (ductal prominence occupying <25%, or >25% of the breast, respectively), or DY (extremely dense). Hazard ratios (HRs) and 95% confidence intervals (CIs) to assess associations of lobular involution and MBD with breast cancer risk were estimated using adjusted Cox proportional hazards model. All tests of statistical significance were two-sided. RESULTS After adjustment for MBD, having no or partial lobular involution was associated with a higher risk of breast cancer than having complete involution (none: HR of breast cancer incidence = 2.62, 95% CI = 1.39 to 4.94; partial: HR of breast cancer incidence = 1.61, 95% CI = 1.03 to 2.53; P(trend) = .002). Similarly, after adjustment for involution, having dense breasts was associated with higher risk of breast cancer than having nondense breasts (for DY: HR of breast cancer incidence = 1.67, 95% CI = 1.03 to 2.73; for P2: HR of breast cancer incidence = 1.96, 95% CI = 1.20 to 3.21; for P1: HR of breast cancer incidence = 1.23, 95% CI = 0.67 to 2.26; P(trend) = .02). Having a combination of no involution and dense breasts was associated with higher risk of breast cancer than having complete involution and nondense breasts (HR of breast cancer incidence = 4.08, 95% CI = 1.72 to 9.68; P = .006). CONCLUSION Lobular involution and MBD are independently associated with breast cancer incidence; combined, they are associated with an even greater risk for breast cancer.

Epithelium-intrinsic NAIP/NLRC4 inflammasome drives infected enterocyte expulsion to restrict Salmonella replication in the intestinal mucosa.

The gut mucosal epithelium separates the host from the microbiota, but enteropathogens such as Salmonella Typhimurium (S.Tm) can invade and breach this barrier. Defenses against such acute insults remain incompletely understood. Using a murine model of Salmonella enterocolitis, we analyzed mechanisms limiting pathogen loads in the epithelium during early infection. Although the epithelium-invading S.Tm replicate initially, this intraepithelial replicative niche is restricted by expulsion of infected enterocytes into the lumen. This mechanism is compromised if inflammasome components (NAIP1-6, NLRC4, caspase-1/-11) are deleted, or ablated specifically in the epithelium, resulting in ∼100-fold higher intraepithelial loads and accelerated lymph node colonization. Interestingly, the cytokines downstream of inflammasome activation, interleukin (IL)-1α/β and IL-18, appear dispensable for epithelial restriction of early infection. These data establish the role of an epithelium-intrinsic inflammasome, which drives expulsion of infected cells to restrict the pathogen's intraepithelial proliferation. This may represent a general defense mechanism against mucosal infections.