Products of tumor suppressor genes have a variety of functions within the cell. A large num ber of genes have now been isolated, as can be seen in the table. Multi-step carcinogenesis The development of cancer is a multi-step process characterized b y repeated cellular insults resulting in the accumulation of mutations. The steps involved in the development of colorectal cancer have been particularly well characterized (see \201gure). Single mutations (e.g. APC) can lead to benign cellular proliferation (familial polyposis coli) that predisposes to the development of malignancy. Mutations in DNA repair genes (e.g. MMR \200\224mis-match repair) speed up this process of mutation accumulation. p53 Other changes ? DCC DPC4 JV18 ? APC DNA hypomethylation K-ras Early adenoma Late adenoma Intermediate adenoma Normal epithelium Carcinoma Metastasis Dysplastic MMR deficiency Genetic alterations associated with colorectal carcinogenesis. Mutation frequencies in MMR- de \201cient cells are two to three times higher than in normal cells \200\224such MMR mut ations are found in > 70% of hereditary non-polyposis colorectal cancer (HNPCC) cases and > 65% of sporadic colorectal cancers exhibiting micro-satellite instability.These cases acco unt for 15 \200\22317% of total colorectal cancers. (Adapted from Kinzler and Vogelstein). OHO-03(11-36) 11/16/02 10:44 AM Page 32 BIOLOGY OF CANCER 32 Cytogenetics and cancer Cytogenetic analysis in cancer is used to con \201rm diagnosis, to aid differential diagno sis, and (possibly) to indicate prognosis. Nowell and Hungerford \201rst described a speci \201c chromosome abnor- mality associate d with chronic myeloid leukemia in 1960. Since then, numbers of chromosomal abnormalities a ssociated with cancer have grown exponentially. Abnormalities can be structural, numerical, or both; they may be restricted in distribution or found in many different tumor types. So me chromosomal alterations are useful in the diagnosis and prognosis of malignancies; the s igni \201cance of others is unknown. Nomenclature Last revised in 1995, cytogenetic abnormalities are described by the ISCN (Int ernational System for Human Cytogenetic Nomenclature). Summary of the nomenclature used: + or \200\223 Gain or loss of chromosome following the symbol. p q del Short arm of chromosome. Long arm of chromosome. Deletion. A deletion in the chromosome dis tal to breakpoint, or between two breakpoints if two are stated. Derivative. A chromosome d erived from a rearrangement. Rearrangement (if known) is described. Dicentric. Chromosome h as two centromeres derived from the two named chromosomes. der dic dup Duplication. A segment of chromosome de \201ned by two break- points is duplicated. Isochromosome. Chromosome is composed of two identical arms rather th an a p and q arm. Insertion. Segment from one chromosome is inserted into another. Inversio n. Segment of chromosome de \201ned by two break- points is inverted compared to its norma l orientation. Translocation. Swapping of segments distal to the stated breakpoints in name d chromosomes. i ins inv t Chromosomal abnormalities structural (cid:2) numerical Examples include: (cid:2) Chronic myeloid leukemia (cid:2) \200\224small chromosome 22 (Philadelphia chromosome) OHO-03(11-36) 11/16/02 10:44 AM Page 33 CYTOGENETICS AND CANCER 33 (cid:2) Acute myeloid leukemia \200\224many cytogenic abnormalities (80% of patients) (cid:2) Therapy-related AML (cid:2) Myelodysplastic syndromes \200\22450% have chromosomal abnormalities (cid:2) Myeloproliferative disorders (cid:2) Acute lymphocytic leukemia \200\22480% have chromosomal abnormalities (cid:2) Chronic lymphoproliferative disorders (cid:2) Lymphoma \200\224many structural and numerical chromosomal abnormalities (cid:2) Neuroblastoma \200\224deletion of 1p and ampli \201cation of N-myc (cid:2) Wilms \200\231 tumor (nephroblastoma) \200\224many chromosomal abnormalities reported Invasion methodology Tumor invasion and metastases is a complex, dynamic, multi-step proces s: Initial invasion of tumor through basement membrane. (cid:2) Movement into connective tissu e surrounding tumor cells. (cid:2) Invasion of tumor cells into blood vessels. (cid:2) (cid:2) Circulating tumor cells are arrested in blood vessels of a distant organ or tissue; tumor cells invade organ from blood vessels. (cid:2) Tumor cells then grow within tissue to form a metastatic tumor that may become clinically evident. (cid:2) Process of tumor invasion and metastases results from alterations in cell-to-cell a nd cell-to-matrix adhesion and increased matrix degradation. Extracellular matrix degradation Several stages during the process of tumor invasion and me tastases require increased degradation or breakdown of extracellular matrix or connective t issue surrounding tumor cells. The extracellular matrix is a complex mixture of proteins in cluding different types of collagen, elastin, \201bronectin, and laminin. Digestion of ex tracellular matrix is carried out by several groups of proteolytic enzymes. Major groups of enzymes implicated in tumor invasion and metastases are: Matrix metalloproteinases (MMPs) A family of zinc-containing enzymes involved in the degrad ation of the extracellular matrix; considerable evidence indicates that OHO-03(11-36) 11/16/02 10:44 AM Page 34 BIOLOGY OF CANCER 34 individual MMPs have an important role in tumor invasion and tumor spread while expression of individual MMPs in tumors may be associated with prognosis2. MMPs are broadly classi \201ed into collagenases, gelatinases, stromelysins, and the recently-identi \201ed membra ne-type MMPs. MMPs are secreted proteins, produced as pro-enzymes and activated by cleavage of a N-termin al propeptide. Gelatinases, particularly MMP-2, appear to be important in initial stages of tumor invasion as they degrade components of the basement membrane, while other MMPs contr ibute to later stages of tumor invasion. Membrane-type MMPs that are membrane-bound appear to be involved in activation of MMP-2, which is capable of activating other MMPs. Activity of MMPs is regulated by interaction with naturally occurring inhibitors, tissue in hibitors of metalloproteinases. Clinical interest in MMPs is considerable since the use of synthetic, low- molecular-weight, broad-spectrum inhibitors of MMPs can prevent tumor sprea d in human tumor xenografts. Several MMP inhibitors are being developed for clinical use. Plasmin system Urokinase-type plasminogen activator (uPA) is a serine protease that catalyz es the activation of plasminogen to plasmin \200\224a broad-spec- trum protease that in tur n can break down a variety of extracellular matrix components. Plasmin can also promote the activation of MMP-2, thus linking plasmin system and MMPs in tumor invasion. Cathepsins A group of lysosomal proteolytic enzymes that also degrade many com- ponents of the extracellular matrix. Widely expressed in tumor cells, stromal cells, and endothelial c ells. Cathepsins can be activated by uPA. Cell adhesion Tumor invasion and metastasis is also characterized by alterations in both ce ll-to-cell and cell-to-matrix adhesion. Cellular adhesion both to adjacent cells and surrou nding extracellular matrix is mediated by a variety of molecules including: Cadherins Transmembrane glycoproteins involved in cell adenomatous polypo- sis coli (APC) g ene product. The most important cadherin in relation to tumor invasion is E-cadherin whose expression is downregulated in various types of malignant tumor; loss of E-cadherin frequen tly appears to correlate with tumor invasion and metastasis. Integrins Transmembrane proteins involved in cell to matrix adhesion. Individual integrins are receptors for a variety of matrix proteins OHO-03(11-36) 11/16/02 10:44 AM Page 35 CYTOGENETICS AND CANCER 35 including speci \201c types of collagen, \201bronectin, and vitronectin. Cell signaling pathways and expression of MMPs is also partially regulated by integrins. Altered regulatio n (often downregulation) and expres- sion of integrins contributes to tumor cell invasion. CD44 A cell surface glycoprotein that functions as an adhesion molecule. CD44 variants can be expressed on the surface of a variety of tumor cells. Speci \201c splice variants of CD 44 are associated with increased tumor invasion. Angiogenesis New blood vessel formation (angiogenesis) is an important factor for continued growth and development of both malignant tumors and metastases. Development of new blood v essels in tumors is stimulat- ed by a wide variety of angiogenic factors produced by both t umor cells and stromal cells. In addition, several naturally occurring anti- angiogenic fac tors have been identi \201ed, most notably angiostatin and endostatin. New blood vessel fo rmation in tumors is a complex and dynamic process requiring: (cid:2) Proliferation of endo thelial cells from pre-existing capillaries or venules. (cid:2) Breakdown of extracellular matrix. (cid:2) Migration of endothelial cells. Growth a nd development of blood vessels within tumors requires the same factors (i.e. increased mat rix degradation and altered cell-to- cell and cell-to-matrix adhesion) that are crucial to tumor cell inva- sion. New blood vessel formation is important in allowing tumor cells to enter the circulation an d a high degree of tumor vascularity increas- es the likelihood of this. Newly formed blood vessels may be more per- meable to tumor cells. There is extensive interest in angiogenesis as a therapeutic target to prevent both tumor g rowth and metastases with both naturally occurring and synthetic anti-angiogenic compounds being intensively investigated for possible clinical use. Formation of metastases in speci \201c tissues Some tissues and organs are more susceptibl e to the formation of metastases (e.g. liver, lung, and bone), whereas metastases are relat ive- ly uncommon in other tissues (e.g. kidney and heart). Several factors have been propos ed to explain the formation of metastases in par- ticular tissues including the expression of speci \201c cell adhesion mole- cules in vascular endothelium of particular organs that are able to arrest circulating tumor cells. Another feature of metastases is the phenomeno n of dormancy or latency of metastatic tumors such that OHO-03(11-36) 11/16/02 10:44 AM Page 36 BIOLOGY OF CANCER 36 many years can elapse between the diagnosis and the apparent cura- tive treatment of the pr imary tumor and the clinical appearance of metastatic tumors. Dormancy appears to occur whe n growth of the metastatic tumor is balanced by an equivalent or even higher rate of tumor cell death by apoptosis. 1 Kinzler, K.W. and Vogelstein, B. (1996) The lessons from hereditary colorec- tal cancer. Cell 87, 159 \200\22370. 2 Murray, G.I., Duncan, M.E., O \200\231 Neil, P. et al. (1996) Matrix metallopro- teinase-1 is associated with poor prognosis in colorectal cancer. Nat Med 4, 461 \200\2232. Key website resources New England Journal of Medicine: http://www.nejm.org/collections/ molecularmedicine/TOC/1.htm National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/ OHO-04(37-48) 11/16/02 10:50 AM Page 37 Chapter 4 Pathology of cancer Tumor types 39 Histological identi \201cation 40 Tumor classi \201cation 43 Grading 44 Fr ozen section examination 45 Transmission election microscopy (TEM) 46 Immunocytochemistry ( ICC) 47 Flow cytometry 48 OHO-04(37-48) 11/16/02 10:50 AM Page 38 PATHOLOGY OF CANCER 38 Cancer is a genetic disease at cellular level. The \201nal result of synchro- nous or seq uential lesions usually involving more than one gene is the capability acquired by the tran sformed cell(s) to undergo clonal, autonomous and purposeless growth into measurable masses . These masses comprise tightly packed cells with a varying amount of extra- cellular matri x of variable density incorporating disorderly distrib- uted, newly formed blood and lympha tic vessels. Malignant cells \200\231 proliferation rate is in \202uenced by numerous fact ors, but is \201nally characterized by aggressiveness that consists of invasion of adjace nt normal tissues and distant metastatic spread by several routes. Each germ cell and each somatic cell \200\224some 200 cell types \200\224can be a tumor \200\231s starting point. Several hundred tumors exist due to the large but not unlimited c onstellation of genetic alterations responsible for neoplastic proliferation at various bod y sites, each with its own natural history. About 12 are responsible for over 80% of cancer deaths (carcinoma of breast, lung, colon and rectum, prostate, stom- ach, pancreas, urinar y tract, ovary, liver, kidney, melanoma, leukaemias and lymphomas). OHO-04(37-48) 11/16/02 10:50 AM Page 39 tumor TYPES 39 Tumor types Tumors can be related to four basic tissue types: (cid:2) Epithelial. (cid:2) Connective. ( cid:2) Musculoskeletal. (cid:2) Nervous, or precursors of germ cells. Benign tumors recapit ulate normal cell morphology, lack invasive- ness and metastatic potential, are slowly grow ing, but may cause severe damage and death because they are strategically placed (e.g. CNS, larynx). Benign tumors may transform into malignant (e.g. Schwannoma). Tumor cells proliferate autonomously, escaping to an unpre- dictable extent the control of hormones and growth factors. They show varying degrees of atypical morphology, in terms of degree of maturation, and differentiation. The malignant appearance (pheno- type) of cells include: (cid:2) Anaplasia (lack of differentiation). (cid:2) Cytoplasmic basophilia (incre ase of ribosomes). (cid:2) Nucleolar hypertrophy. and/or: Increase in number of the nucleoli. Increase of the mitotic index. (cid:2) (cid:2) (cid:2) Presence of bizarre mitotic \201gures. (cid:2) Variations in cell size and shape. Increase of nucleo-cytoplasmic ratio. (cid:2) Disorderly structure and deviations from the normal tissue architec- ture, such as loss of polarity, are re \202ected in atypical growth. OHO-04(37-48) 11/16/02 10:50 AM Page 40 PATHOLOGY OF CANCER 40 Histological identi \201cation Histological identi \201cation of the malignant nature is the critical issue; some lesions escape the criteria mentioned previously. Examples are: (cid:2) Small-sized renal cell car cinomas (erroneously called adenomas because of their size). (cid:2) Highly differentiated \200\230lipoma-like \200\231 liposarcomas. (cid:2) Controver sial pigmented skin lesions ( \200\230dysplastic \200\231 naevi). (cid:2) Questionable soft tissue tumors \200\224such as atypical \201bro- histiocytoma, \201bromatosis, haemangi opericytoma; myelodysplasia. While lesion classi \201cation uncertainty has decreased in r ecent years, \200\230borderline \200\231 ovarian tumors are still controversial; for some time they were classi \201ed as (cyst)adenocarcinomas of low malignant potential, but now as (cyst)adenomas with uncertain malignant potential or atypical proliferating tumors. Lesions preceding malignant tumors are as various as the natural histories of the subsequen t cancers. At some sites (e.g. uterine cervix and prostate) pre-malignant lesions may persi st for a long time, others are short-lived. Among the former, some lesions permit reproduci ble histological typing and even grading, whereas ductal carcinoma in situ of the breast ca n be sub-typed and graded according to cell differenti- ation. At other sites the current c riteria allow only a reproducible overall diagnosis of carcinoma in situ. Examples include: (cid:2) Bronchi. (cid:2) Prostate. (cid:2) Uterine cervix. (cid:2) Most organs lined by squ amous epithelium (skin, upper respir- atory and alimentary tract). (cid:2) Transitional epithelium (lower urinary tract). Melanoma in situ is recognized as a stage in melanoma tumor pro- gression. \200\230Dysplasia \200\231, though vague, is noneth eless well established and employed to de \201ne pre-malignant changes of, for example, la rynx, uterine cervix, and Barrett \200\231s esophagus. Malignant potential without well-de \201ned or reproducible histopathological features is attributable to hypercellular leiomyomas of the uterus, hyperplasia of plasma cells in the bone marrow accom- panied by monoclonal gammopathy, nodular hyperplasia of regener- ating c irrhotic liver, dysplastic naevi with random atypia, large congenital cutaneous naevi, and lymphomatoid granulomatosis. Although clonality is a basic feature of malignancy in early phases of proliferation before multiple subclones develop due to additional OHO-04(37-48) 11/16/02 10:50 AM Page 41 HISTOLOGICAL IDENTIFICATION 41 mutational events (genetic instability), it is not a synonym of malig- nancy as shown by th e curable, occasionally self-limited, local lesions of Langerhans \200\231 cell histiocytos is (eosinophilic granuloma) whose monoclonality was recently proven. Epithelial tumors comprise over 80% of neoplasms; adenoma being the most common epithelial benign growth and carcinoma the general term for the malignant counterpart. The latter may be addi- tionally classi \201ed according to: (cid:2) Origin (adenocarcinoma, Merkel-cell ca.). (cid:2) Structure (papillary ca., tubular ca.). (cid:2) Extracellular matrix (desmopl astic ca.). (cid:2) Cellular content (glycogen-rich ca., lipid-rich ca.). (cid:2) Cell prod ucts (mucinous ca., keratinizing ca.). (cid:2) Cell-size (small or large cell ca.). (cid:2) Cell shape (spindle cell ca.). The proliferating cell type is recalled in the names of tum ors of soft parts and of myoid origin, both benign and malignant (e.g. \201broma vs. \201brosarcoma, leiomyoma vs. leiomyosarcoma). Most of the latter are subject to grading, a nd several systems have been proposed. The central nervous system (CNS) displays an array of cell types from which several tumors may arise (astrocytes, ependymal cells, neuroblasts, etc.). A few CNS tumors are histologic ally benign (gangliocytoma, central neurocytoma); all others show a broad range of grades o f malignancy. Progression from low to high-grade malignancy is a common event. Mixed benign epithelial-stromal tumors are common in salivary glands (pleomorphic adenoma) and breast ( \201broadenoma). Despite phenotypic multi-cellularity, clonality was recently proven for these tumors, supporting a totipotential stem-cell divergence hypothesis. Devel opmental errors are probably responsible for teratomas, which mostly arise along the midlin e of the body and in the gonads \200\224these commonly display immature and malignant featu res in the testes and mature benign features in the ovaries. Tumor-like conditions are non-neoplastic lesions that mimic neo- plastic growth. Their impo rtance lies in the differential diagnostic work-up. Chronic reactive in \202ammatory respo nse may produce deceptively neoplastic-looking lesions such as nodular fasciitis of soft ti ssue. Other common lesions which simulate a neoplasm indicate: (cid:2) Aneurysmal bone cyst s. (cid:2) Traumatic neuromas. Intravascular papillary endothelial hyperplasia. (cid:2) (cid:2) Central giant-cell granuloma of bone. (cid:2) Reactive hyperplastic nodules of meso thelium. OHO-04(37-48) 11/16/02 10:50 AM Page 42 PATHOLOGY OF CANCER 42 Tumor-like conditions also include hamartomas (e.g. pulmonary and hepatic bile duct) \200 \224local circumscribed developmental errors \200\224 and choristomas which are aggregates of ectopic tissue (e.g. nasal glial heterotopia, accessory spleen). OHO-04(37-48) 11/16/02 10:50 AM Page 43 tumor CLASSIFICATION 43 Tumor classi \201cation Classi \201cation and typing of tumors still relies on histopathology and aspirated cell m aterial. Tissue and cellular samples are submitted fresh or \201xed (usually in formalin) with all relevant pieces of information (present and past clinical history, operative \201ndings, and radiological data). Description of gross features of the organ or surgical specimen containing the tumor is essential. Size, shape, color, consistency, appearance of the cut surface, and tumor-host interface (neoplastic pseudocapsule), presence/absence of u lceration, necrosis, and cystic spaces have to be recorded. Important histological features include:- (cid:2) Mitotic index. (cid:2) Lymphocytic in \201ltration (e.g. medullary carc inoma of breast, cuta- neous melanoma). (cid:2) Extent and the type of necrosis (e.g. breast cancer and soft tissue tumors). (cid:2) Presence/absence of peritumoral vascular invasion (e.g. germ cell tumors of testes). (cid:2) Type of lesions adjacent to the tumor (e.g. atrophic gastritis, carci- noma in situ). OHO-04(37-48) 11/16/02 10:50 AM Page 44 PATHOLOGY OF CANCER 44 Grading Grading of carcinomas is mandatory for adequate treatment (e.g. prostate, breast, endometri um, liver, kidneys) and several systems are available. Simplest consists of counting mitoti c \201gures in de \201ned microscopic areas. For some cancers micro-staging procedures a re routinely applied (melanoma, carcinomas of organs with a cavity such as urinary bladder, gut, endometrium, uterine cervix, vulva) and con- sist of depth of invasion, tumor thickne ss, and type of margins (pushing, in \201ltrating). For \201nal diagnosis pathological reports should employ standardized nomenclature and co ding using dedicated, computer-assisted report- ing systems. Where appropriate, checkpoints for technical quality assurance should be incorporated. Due to rapidly expanding immunolog ical, cytogenetic, and molecular techniques careful plan- ning of supplementary methods is required; a tissue bank of snap- frozen samples of tumors and the corresponding normal tiss ues of the same patient is recommended. OHO-04(37-48) 11/16/02 10:50 AM Page 45 FROZEN SECTION EXAMINATION 45 Frozen section examination Frozen section examination is still useful to establish/rule out malig- nancy and ascertain the status of surgical resection margins. On frozen section, tumor typing is often feasibl e. Enlarged non-metasta- tic lymph nodes and suspicious in situ or borderline lesions (brea st nodules, polyps, ovarian cysts) are often unsuitable for frozen section procedures and r equire a de \201nitive deferred diagnosis. Routine H and E staining of paraf \201n-embedd ed tissue section has to be supplement- ed by special stains, some of which are still used despite the advent of immunocytochemistry (ICC). Useful stains and methods are: (cid:2) Alc ian blue cationic dye at pH 2.5 for intracytoplasmic epithelial acid mucin (e.g. mucin-producing carcinomas). (cid:2) Periodic acid-Schiff reaction (PAS) which visualizes glycoproteins and glycogen (e. g. Ewing \200\231s sarcoma, alveolar sarcoma, some carci- nomas) and basement membranes. (cid:2) Romanowsky \200\223Giemsa panoptical method for hematological pro- liferation. (cid:2) Reticulin \201bre silver impregnation method. (cid:2) Stein \200\231s method for bile. (cid:2) Grimelius technique for argyrophilic substances. (cid:2) Several stains for m icro-organisms. Most enzymes are now identi \201ed by ICC, exploiting immuno- genic proper ties of enzymatic proteins; the majority are hydrolases, oxidases, and dehydrogenases. OHO-04(37-48) 11/16/02 10:50 AM Page 46 PATHOLOGY OF CANCER 46 Transmission electron microscopy (TEM) Diagnostically useful detectable structures include: (cid:2) Myo \201brillary elements. (c id:2) Neurosecretory dense-core granules. (cid:2) Birbeck \200\231s granules of Langerhans \200\231 cells. (cid:2) Melanosomes. (cid:2) Cell junctions. (cid:2) Weibel-Palade bodies. (cid:2) Some virions (HPV). (cid:2) Bacteria. Even more valuable are the results of a combi nation of immunological techniques with TEM. OHO-04(37-48) 11/16/02 10:50 AM Page 47 IMMUNOCYTOCHEMISTRY (ICC) 47 Immunocytochemistry (ICC) Use of ICC for detecting antigens in tissue sections for diagnostic pur- poses is now routi ne. Preservation of the antigen(s) and validation of antibodies are critical technical prer equisites, whereas interpretation of results supplemented by positive and negative controls are the histopathologist \200\231s responsibility. Many antibodies (most of them monoclona l) are now available, working on paraf \201n-embedded mate- rial with/without antigen retr ieval. Targets are: (cid:2) Structural antigens (e.g. intermediate \201laments). (cid:2) Matrix antigens. (cid:2) Functional antigens (secretory products). (cid:2) Lineage antigens (immunoglobulins). (cid:2) Membrane-bound antigens (receptors, glycoproteins). (cid:2) Mis cellaneous cell constituents. Rarely does a single antibody afford a speci \201c diagnosis ; mostly a small panel of antibodies yields more reliable information. Anaplastic tumors wh ose differential diagnoses include carcinomas, large-cell lymphomas, sarcomas, and melanoma , and small round-cell tumors in childhood which include neuroblastoma, the Ewing \200\231s sarcoma fam- ily (including PNET), lymphomas, and some rhabdomyosarcomas, are particular t argets for immunocytochemical scrutiny. A second level of investigation is represented by the array of malig- nant lymphomas, both of Hodgkin \200\231s and non-Hodgkin type. Their categorization on the basis of the recent REAL classi \201cation is facili- tated by the antibodies that offer a cluster designation (CD). An additional use of ICC is tumor staging consisting of a search of malignant cells by mean s of appropriate antibodies in lymph nodes, bone marrow, in lumina of peritumoral vessels, and at surgical resec- tion margins. Markers of proliferation, diagnostic cell products, and expressions of (onco)-gene protein products are also targets of the immunocyto- chemical diagnostic approach. Antibodies again st chimeric fusion proteins are now available; the \201rst example was an anti-p80 (chime ric NPM-ALK protein) antibody to detect t(2;5) (p23;q35) in anaplastic large-cell lymphoma. OHO-04(37-48) 11/16/02 10:50 AM Page 48 PATHOLOGY OF CANCER 48 Flow cytometry Flow cytometry is used mainly to quantitate average cellular content of DNA (ploidy) and as sess the cycle \200\231s DNA-synthetic phase. Another useful application is quantitative an d qualitative evaluation of nuclear, cytoplasmic, or cell surface proteins stained with \202uorescent dyes. Classical cytogenetic analysis is useful in detecting consistent, non- random chromosomal l esions such as translocations and deletions. It is applied mainly to hematological and soft tissue tumors. OHO-05(49-62) 11/16/02 10:51 AM Page 49 Chapter 5 Etiology of cancer Viral oncogenesis 50 Chemical carcinogenesis 54 Radiation carcinogenesis and radiosensitivi ty syndromes 58 Hormones in the etiology of cancer 61 OHO-05(49-62) 11/16/02 10:51 AM Page 50 etiology OF CANCER 50 Viral oncogenesis Approximately 15% of tumors worldwide can be attributed to virus- es; the majority of these are represented by hepatocellular and cervical carcinoma and are the sequelae of infection by two DNA viruses, the hepatitis B virus and the human papillomavirus respectively. Other viruses directly linked to human tumors include: (cid:2) Epstein \200\223Barr Virus (EBV). (cid:2) Human herpes virus 8 (HHV-8). (cid:2) Hum an T-cell leukemia virus (HTLV-1). (cid:2) Human immunode \201ciency virus (HIV). (cid:2) Herpes simplex virus (HSV). (cid:2) Cytomegalovirus (CMV). Viruses indirectly linked include: (cid:2) HHV-3. (cid:2) HHV-6. Although viral infection plays a signi \201cant role in the initial step towards carcinogenesis, acquisition of malignant phenotype requires additional genetic alterations. Most tumor viruses are ubiquitous; prevalence of infection is much higher than the incidenc e of the respective form of tumor, and development of associated tumors requires many years of infection. This latent period may last decades. Other co-factors are necessary for deve lopment of virally-linked tumors, including genetic, immunological, hormonal, and environme ntal factors. DNA and RNA viruses Human papillomavirus (HPV) HPV are small, double-standard DNA viruses that speci \201cally infect squamous epithelial cells. HPV represents a heterogeneous group of viruses with ove r 70 different HPV genotypes identi \201ed. Only keratinocytes can be infected, and the vi rus has evolved a unique model of replication with absolute dependence upon the micro- envi ronment of differentiating squamous epithelium for infection, replication, viral capsid syn thesis, and particle assembly. HPV virus and tumors (cid:2) Skin warts \200\224types 1/2. (cid:2) Anogenital warts \200 \224types 6/11. (cid:2) Cervical cancer \200\224types 16, 18, 31, 33, 35. (cid:2) Anal tumo rs. OHO-05(49-62) 11/16/02 10:51 AM Page 51 VIRAL ONCOGENESIS 51 (cid:2) Vulval tumors. (cid:2) Penile tumors. Convincing epidemiological studies have impli cated an infectious agent in the etiology of cervical cancer. Longitudinal studies have est ablished high-grade cervical intra-epithelial neoplasia (CIN) as a pre-malignant precursor lesion to invasive cancer. Genital warts show an identical epidemiological pattern to cervi cal cancer and, in addi- tion, HPV induces cellular changes previously interpreted as cervi cal dysplasia in cervical smears and tissue. Molecular con \201rmation of this association using PCR has demonstrated \200\230high-risk \200\231 HPV types in high-grade CIN lesions and other squamous intra-epithelial lesions such as PIN: penile intraepithelial neoplasia, AIN: anal intraepithelial neoplasia, and VIN: vulva intraepithelial neoplasia. Studies of HPV-positive cervical cancer have shown integration of viral DNA; the E6 and E7 genes are regularly overexpressed. Proteins encoded by E6 and E7 in high-risk HPV types are oncoproteins, p53 being targeted for degradation by E6 protein, whilst E7 protein inacti- vates retinoblastoma (Rb) protein. Conversely, the E6 and E7 proteins encoded by \200\230l ow-risk \200\231 HPV types 6/11, show dramatically lower activ- ity in these functions when compared to those encoded by \200\230high-risk \200\231 HPV types. The role of HPV types in other tumor types such as oesophageal, laryngeal, and oropharyngea l cancer needs to be further de \201ned. Epidemiological evidence suggests that HIV-infect ed patients have a higher incidence of HPV-associated anogenital tumors, namely cervical ca ncer in women and anal cancer in men. Hepatitis B (HBV) and C (HBC) viruses HBV is a small DNA virus of 3000 base pairs. Primary infection pro- duces either acute hepatitis B or a subclinical infection. The majority of i nfections resolve with clearance of virus and lifelong immunity; 5% fail to clear the virus and go on to develop a lifelong persistent hepatic infection, resulting in a spectrum of h epatocellular injury and the development of chronic persistent hepatitis or chronic active hepatitis. Chronic HBV infection is associated with a 100-fold increase in hepatocellular c arcinoma (HCC) risk. Hepatocellular injury is thought to trigger a proliferative response in the liver. This res ponse increases the chance for cellular mutations to occur over time and for cells to conse quently escape normal cellular growth control. However, cases of HHC do arise in non-cirrho tic livers, so raising the possibility of another mechanism, such as the existence of a spe ci \201c HBV oncogene. Unproven candidates include ORF X that encodes for a small regulato ry protein that may bind to and inactivate p53. Alternative molecular mechanisms may involv e viral genome inte- gration. However, no one common integrated viral encoding site has bee n demonstrated. OHO-05(49-62) 11/16/02 10:51 AM Page 52 etiology OF CANCER 52 Hepatitis C (HCV) is a single-stranded RNA virus and primary infection results in acute hep atitis C infection. HCV infection is linked to the development of HCC. Herpes viruses Epstein \200\223Barr virus is an endemic lymphotrophic herpes virus. Primary infection of the oropharyngeal epithelium produces pharyn- gitis and subsequent B lymphocy te in \201ltration. Infection results in lifelong latent infection within lymphoid tissue and B-lymphocytes. A number of EBV encoded proteins have oncogenic potential (EBNA 1 and LM P 1). T-cell surveillance restricts the ability of these proteins to deregulate cell growth /apoptosis with consequent emergence of a lymphoproliferative disorder. EBV is associated with a number of human tumors including: (cid:2) Endemic Burkitt \200\231s lymphoma (BL). (cid:2) Non-endemic/sporadic BL. (cid:2) U ndifferentiated nasopharyngeal carcinoma (NPC). (cid:2) Hodgkin \200\231s disease. (cid:2) Lymphoproliferative disease and immunoblastic lymphomas, in immunocompromised host. Endemic Burkitt \200\231s lymphoma (BL) The majority of tumors in endemic form of BL contai n EBV DNA sequences. In addition, malaria P. falciparum infection occurs co- incidentally. Malaria infection is thought to depress cytotoxic T-cell function, thus rendering the patie nt relatively cytotoxic T-cell de \201cient and allowing the EBV infection to escape T-cel l surveillance. The role of EBV in non-endemic and AIDS-related BL is complex and less de \201nitive. EBV DNA is detectable in only 10 \200\22315% of patients with non-endemic BL an d 50% of patients with AIDS-related BL. Immunoblastic lym- phomas associated with chronic i mmunosuppression in allograft recipi- ents or AIDS patients and undifferentiated NPC invari ably contain EBV DNA sequences. In Hodgkin \200\231s disease, 50% of tumors contain EBV-inf ected cells and, in addition, the EBV genome has been demon- strated in the Reed \200\223St ernberg cells of Hodgkin \200\231s disease. Kaposi \200\231s sarcoma (KS) Kaposi \200\231s sarcoma (KS) is commonly associated with AID S, but was originally described in elderly men of Mediterranean, Middle Eastern, or Eastern European ethnic origin \200\224so-called \200\230classic \200\231 KS. It has also been do cumented in chronically immunosuppressed organ transplant recipients. Within the HIV-infect ed population, variations in develop- ment of KS exists; the risk of KS is highest in HIV-i nfected homo- sexual/bisexual men, compared with only a minority of HIV-infected OHO-05(49-62) 11/16/02 10:51 AM Page 53 VIRAL ONCOGENESIS 53 hemophiliacs, thus supporting the role of a sexually transmitted agent in the development o f KS. Using PCR, herpes-like DNA sequences have been demonstrated in AIDS-associated KS lesions. These sequences have been shown to be non-human and closely related to gamma herpes viruses , EBV, and the simian herpes virus (HVS). These herpes-like DNA sequences have been identi \201ed in all KS tissue and are thought to represent a new herpes virus, HHV 8 or KS-assoc iated herpes virus (KSHV). This supports earlier epidemiological data suggesting an infecti ve agent in the development of this tumor. Like EBV, HHV8 is lymphotrophic and has also bee n identi \201ed in peripheral blood mononuclear cells and two lymphoproliferative conditio ns, the rare body cavity lymphoma and in multicentric Castleman \200\231s disease. Retroviruses Sero-epidemiological and molecular data have established a causal role for HTL V-1 in adult T-cell leukemia (ATL). ATL presents with either a leukaemic or non-Hodgkin \200\231s T-cell lymphoma type picture, characterized by malignant organ in \201ltration, immunode \201ciency, lymphadenopathy, and hypercalcaemia. HTLV-1 is also associated with a chronic non-malignant disorder, tropical spastic paraparesis. HTLV-1 is trophic for CD4+ h uman T-cells, resulting in transforma- tion of infected cells. Human immunode \201ciency virus A number of speci \201c tumors are associated with HIV in fection and are AIDS-de \201ning. However, this association is not due to a direct oncogen ic effect of the HIV virus, but is the result of chronic immunosuppression. A long natural history of virus-associated tumors is accelerated in HIV-infected patients, again the resul t of failure of immune surveillance. OHO-05(49-62) 11/16/02 10:51 AM Page 54 etiology OF CANCER 54 Chemical carcinogenesis Chemical carcinogens are a principal cause of cancer and can be induced by certain lifestyl es e.g. smoking tobacco, exposure to chemi- cals in the community or workplace, some drug t aking. Chemical car- cinogenesis is a multi-stage process. Initiation and promotion When cells are initially exposed to a carcinogenic agent, genetic car- cinogens interact with DNA to form an adduct or to induce chemical alterations of DNA. DNA adducts may result in damage by inducing point mutations, deletions, or chromosomal tr anslocations. When changes are induced in genes involved in cell growth regulation or diffe rentiation (e.g. oncogenes and tumor suppressor genes), cell transformation can result. The majority of chemical carcinogens are metabolically activated, leading to the formation of reactive species \200\224the ultimate carcinogen. Metabolism is frequently mediated by c ytochrome P450 but in tissues and organs with low levels of cytochrome P450, the peroxidase component of prostaglandin synthetase or lipozygenase can activate. Metabolism involves en zymes mainly in the body \200\231s entry routes \200\224 respiratory tract, gastrointestina l tract, and liver \200\224though metabolism may occur elsewhere, leading to organ-speci \201c tumors. Cytochrome P450 exists as a number of isoforms, each having a dif- ferent substrate selecti vity. Activity and substrate speci \201city of both activating and detoxifying enzymes var ies from organ to organ, indi- vidual to individual, and species to species, resulting in o rgan- and species-speci \201c carcinogencity. If the affected cell neither dies nor revert s to a normal cell through error-free DNA repair, initiation becomes irreversible after the cell undergoes replication. The initiated tumor cell, with altered genotype and phenotype, may remain dormant for a lon g time before becoming a tumor, in the presence of a promoter, which may be one of a wide v ariety of chemicals. Promotion is reversible, so for an initiated cell to continue to repli cate, it must be exposed to a promoter more or less continu- ously. Carbon tetrachloride is a well-known example of a promoter resulting in liver cancer in animal studies. Hormones c an also act as promoters e.g. thyroid hormone is a promoter of thyroid cancer. Like initiat ion, progression is an irreversible process. Epigenetic carcinogens Epigenetic carcinogens do not damage DNA, but act at a later stage t hrough non-genotoxic mechanisms. They are frequently promoters. OHO-05(49-62) 11/16/02 10:51 AM Page 55 CHEMICAL CARCINOGENESIS 55 Excretion, with or without EXCRETED CHEMICAL CHEMICAL metabolism Intermediary metabolites (Proximate carcinogen) Bioactivation Covalent binding with REACTIVE METABOLITE (Ultimate carcinogen) CONJUGATED CHEMICAL nucleophiles e.g. reduced glutathione Covalent binding to macromolecules ALTERED GENETIC MATERIAL REPAIRED GENETIC MATERIAL (NORMAL CELL) Replication INITIATED tumor CELLS Promotion DIFFERENTIATED NEOPLASM Conversion and progression CANCER Genotoxic carcinogens and induction of cancer. Their mode of action is based on induction of cell division. They may be mitogenic agents, cytotoxic agents, or immunosuppressive agents. Peroxisome proliferators are an interesting class of epigenetic carcinogens, a chemically h eterogeneous group of compounds, OHO-05(49-62) 11/16/02 10:51 AM Page 56 etiology OF CANCER 56