Combined immunohistochemistry of PRAME and p16 in the differentiation of melanocytic neoplasms, with a detailed focus on acral lesions
Melanoma (MM) stands out as a highly aggressive skin neoplasm, with its incidence showing a relentless upward trend. Regrettably, the therapeutic outcomes achieved through conventional modalities like radiotherapy, chemotherapy, and immunotherapy in patients with advanced MM remain far from satisfactory . Within the realm of diagnostic pathology, the timely identification of MM continues to pose a formidable challenge, particularly with the limited utility of traditional immunohistochemical markers in distinguishing between benign lesions and MM. Despite notable advancements in molecular pathology techniques, such as fluorescence in situ hybridization and comparative genomic hybridization, for characterizing malignant melanocytic neoplasms, the widespread adoption of these approaches in clinical settings is impeded by their prohibitive cost implications . PRAME, initially identified as a CUL2 ubiquitin ligase subunit in reactive T cells from MM patients , exhibits a physiological expression pattern in tissues like the testis, ovaries, placenta, adrenal glands and endometrium . However, its expression becomes dysregulated in different cancer types, including lung, breast, kidney, ovarian, leukemia, synovial sarcoma, and mucinous liposarcoma, often correlating with aneuploidy and metastasis . Recent study has elucidated PRAME’s role in inducing genomic instability and augmenting reliance on the alternative base excision repair pathway, rendering cells susceptible to PRAP1/2 inhibition in uveal melanoma . These findings have catapulted PRAME into the limelight as a pivotal oncogenic driver and a promising target for immunotherapeutic interventions and diagnostic biomarkers . In the authentic milestone of PRAME evaluation in surgical pathology by Lezcano C et al. in 2018 , the IHC assessment of PRAME expression across primary MMs, metastatic MMs, and melanocytic nevi revealed diffuse positivity in 87% of metastatic and 83.2% of primary MM cases, while melanocytic nevi exhibited negative PRAME expression in 86.4% of cases. Some scholars have used PRAME IHC to distinguish MM that are more difficult to diagnose, such as mucosal MM of the head and neck region , and subungual and non-subungual acral melanocytic lesions . Nevertheless, PRAME positivity was also observed in some arcal nevi, dysplastic nevi, recurrent nevi, and Spitz nevi . Similarly, our prior investigations documented PRAME positivity rates of 89.9% and 93.1% in primary and metastatic MM, respectively , alongside positive expression in 6 out of 317 melanocytic nevi. Additionally, in 91 cases of in situ acral lentiginous melanoma (ALM) and 18 cases of in situ subungual MM, PRAME exhibited sensitivities of 75.8% and 77.8%, respectively, with 2 out of 40 cases of recurrent nevi of the ALM displaying positive staining . Collectively, these studies underscore PRAME as a relatively sensitive marker to differentiate melanocytic neoplasms, albeit with certain limitations particularly in situ acral melanoma (AM). The protein p16, encoded by CDKN2a situated on chromosome 9’s long arm, assumes significance in MM diagnosis . Studies showed a frequent absence of p16 expression in primary and metastatic MM, in stark contrast to its consistent expression in melanocytic nevi . In a clinicopathologic study of 50 acral Spitz nevus cases, acral Spitz nevi were characterized by strong and diffuse p16 expression, which differed from acral nevi and ALMs . Nevertheless, the utility of p16 immunostaining in discerning benign from malignant melanocytic lesions remains contentious. Since PRAME and p16 had their own strengths and weaknesses in the differentiation of melanocytic tumors, combined PRAME and p16 or other biomarkers (e.g., SOX10, HMB-45, Ki-67) IHC were utilized for better discrimination . Bahmad and co-workers had applied combinated PRAME and p16 IHC to distinguish melanocytic nevi(51 cases) from MMs(77 cases), and found that PRAME& p16 melanocytic lesion was unlikely to be a nevus, and most nevi exhibited PRAME &p16 pattern. In this study, we retrospectively analyzed the PRAME and p16 IHC results of confirmed 112 MMs and 53 melanocytic nevi, with the view to be able to improve the diagnostic rate of MM in the clinic. Since AM has poor survival and accounts for a considerable share of MM-associated morbidity and mortality worldwide which requires early diagnosis urgently, we specifically focused on acral samples(84 AMs and 38 acral nevi) and preinvasive ones.   Study subjects 112 unequivocally diagnosed cases of MM were meticulously selected from the archives of our hospital, spanning from January 2022 and June 2023, including MM typically associated with CSD Pathway: 17 cases(11 superficial spreading MMs and 6 lentigo maligna MMs); MM not consistently associated with cumulative solar damage (no CSD) Pathway: 90 cases(84 AMs, 4 MMs arising in congenital nevus,1 MM arising in blue nevus and 1 mucosal MM); and Nodular MM: 5 cases. Concurrently, 53 cases of melanocytic nevus from the corresponding time-frame served as controls, including 38 acral nevi, 10 Spitz nevi, 2 dysplastic nevi, 2 recurrent nevi and 1 mucous nevus. Each case underwent meticulous, independent scrutiny by two seasoned dermato-pathologists (Qiuju Miao and Hao Chen), ensuring adherence to the diagnostic criteria delineated in the WHO-2018 Classification of Skin Tumors (4th edition) . Comprehensive clinical data of the patients were meticulously compiled and encapsulated within Table . IHC Immunohistochemical assessment targeting PRAME and p16 was conducted on all aforementioned specimens. Formalin-fixed paraffin-embedded tissue sections (4 μm thick) were performed according to our previously established method . The rabbit-derived monoclonal antibody PRAME (Clone number: EPR20330. Abcam, USA) was applied at a dilution of 1:400 and incubated for 30 min at ambient temperature. The chromogenic substrates 3,3’-diaminobenzidine or FAST RED were utilized for visualization, with hematoxylin employed for counterstaining. For the analysis of protein p16, an automated IHC platform (Auto-stainer link45 System, Dako, Denmark) was employed, utilizing a mouse-derived anti-human monoclonal antibody (Clone number: MX007, Fuzhou Maixin Biotechnology Development Co., LTD, China) as the first antibody. Result interpretation PRAME expression was interpreted as positive when its cumulative score was greater than four points, otherwise interpreted as negative. The cumulative score was made up of PRAME expression intensity score (weak: 1, medium: 2 and strong: 3) and positive percentage score (None: 0, 1-25%: 1, 26-50%: 2, 51-75%: 3 and > 75%: 4) in tumor samples according to our previous study  (Fig. ). The expression of p16 could be divided into reserved expression (uniform or checkerboard expression) or deficient expression (none or partial expression: <2%), which was interpreted as negative with deficient expression, otherwise interpreted as positive (Fig. ). The result interpretation was done double-blind by two dermatopathologists (Qiuju Miao and Hao Chen). In the event of disagreement, a discussion was held to reach a consensus on the IHC expression intensity and positive percentage score. Statistical analysis Statistical analysis was performed using SPSS (26.0 version; IBM Corp., SPSS Statistics for Windows Version 26.0, Armonk, NY, USA). The χ2 test was used to assess whether PRAME, p16, and their combined patterns have a significant association with MM or melanocytic nevus. Their positivity and negativity, sensitivity and specificity, and ROC and AUC in MM are obtained simultaneously. P < 0.05 was considered to be statistically significant.   112 unequivocally diagnosed cases of MM were meticulously selected from the archives of our hospital, spanning from January 2022 and June 2023, including MM typically associated with CSD Pathway: 17 cases(11 superficial spreading MMs and 6 lentigo maligna MMs); MM not consistently associated with cumulative solar damage (no CSD) Pathway: 90 cases(84 AMs, 4 MMs arising in congenital nevus,1 MM arising in blue nevus and 1 mucosal MM); and Nodular MM: 5 cases. Concurrently, 53 cases of melanocytic nevus from the corresponding time-frame served as controls, including 38 acral nevi, 10 Spitz nevi, 2 dysplastic nevi, 2 recurrent nevi and 1 mucous nevus. Each case underwent meticulous, independent scrutiny by two seasoned dermato-pathologists (Qiuju Miao and Hao Chen), ensuring adherence to the diagnostic criteria delineated in the WHO-2018 Classification of Skin Tumors (4th edition) . Comprehensive clinical data of the patients were meticulously compiled and encapsulated within Table .   Immunohistochemical assessment targeting PRAME and p16 was conducted on all aforementioned specimens. Formalin-fixed paraffin-embedded tissue sections (4 μm thick) were performed according to our previously established method . The rabbit-derived monoclonal antibody PRAME (Clone number: EPR20330. Abcam, USA) was applied at a dilution of 1:400 and incubated for 30 min at ambient temperature. The chromogenic substrates 3,3’-diaminobenzidine or FAST RED were utilized for visualization, with hematoxylin employed for counterstaining. For the analysis of protein p16, an automated IHC platform (Auto-stainer link45 System, Dako, Denmark) was employed, utilizing a mouse-derived anti-human monoclonal antibody (Clone number: MX007, Fuzhou Maixin Biotechnology Development Co., LTD, China) as the first antibody.   PRAME expression was interpreted as positive when its cumulative score was greater than four points, otherwise interpreted as negative. The cumulative score was made up of PRAME expression intensity score (weak: 1, medium: 2 and strong: 3) and positive percentage score (None: 0, 1-25%: 1, 26-50%: 2, 51-75%: 3 and > 75%: 4) in tumor samples according to our previous study  (Fig. ). The expression of p16 could be divided into reserved expression (uniform or checkerboard expression) or deficient expression (none or partial expression: <2%), which was interpreted as negative with deficient expression, otherwise interpreted as positive (Fig. ). The result interpretation was done double-blind by two dermatopathologists (Qiuju Miao and Hao Chen). In the event of disagreement, a discussion was held to reach a consensus on the IHC expression intensity and positive percentage score.   Statistical analysis was performed using SPSS (26.0 version; IBM Corp., SPSS Statistics for Windows Version 26.0, Armonk, NY, USA). The χ2 test was used to assess whether PRAME, p16, and their combined patterns have a significant association with MM or melanocytic nevus. Their positivity and negativity, sensitivity and specificity, and ROC and AUC in MM are obtained simultaneously. P < 0.05 was considered to be statistically significant.   For total samples In patients diagnosed with MM, the prevalence of PRAME expression was 82.1% (92/112), with 17.9% (20/112) exhibiting negative expression. Additionally, the positivity rate for p16 was 68.75% (77/112), while 31.25% (35/112) showed negative expression. Conversely, melanocytic nevi demonstrated a PRAME positivity rate of 5.7% (3/53) and a negativity rate of 94.3% (50/53). The positivity rate for p16 in melanocytic nevi was 94.3% (50/53), with a negativity rate of 5.7% (3/53). The sensitivity of PRAME for diagnosing MM was 82.1%, with a specificity of 94.3% (AUC = 0.882; 95% CI: 0.827–0.938, P < 0.001). Conversely, the sensitivity of p16 for MM diagnosis was 31.25%, with a specificity of 94.3% (AUC = 0.628; 95% CI: 0.542–0.714, P = 0.008). Statistical analysis via the χ2-test underscored the significance of PRAME ( P < 0.001) and p16 ( P < 0.001) in distinguishing MM from melanocytic nevus (Fig. ). Of the 112 MM cases, 96 exhibited the immunophenotype PRAME/p16, while 31 cases displayed PRAME &p16. In contrast, among the 53 melanocytic nevi cases, PRAME/p16 was expressed in only 6 cases (3 spitz nevi, 2 acral nevi and 1 recurrent nevus), while PRAME &p16 in 0 cases. The sensitivity and specificity of PRAME/p16 for MM were 85.7% and 88.7% (AUC = 0.872; 95%CI: 0.810–0.934, P < 0.001), while for PRAME  &p16, the values were 27.7% and 100% (AUC = 0.638; 95%CI: 0.555–0.722, P = 0.004). For acral samples and non-acral samples In 122 acral samples, PRAME exhibited a specificity of 97.4% and a sensitivity of 82.1% for MM ( AUC = 0.898; 95%CI: 0.839–0.956, P <0.001), with sensitivities of 90.9% and 72.5% for invasive and preinvasive MM, respectively. Besides, the sensitivity and specificity of p16 for MM were 27.4% and 97.4% (AUC = 0.624; 95%CI: 0.524–0.724, P = 0.029). PRAME/p16 demonstrated a specificity of 94.7% and a sensitivity of 86.9% for MM( AUC = 0.908; 95%CI: 0.849–0.968, P <0.001), with sensitivities of 90.9% and 82.5% for invasive and preinvasive MM, respectively. PRAME/p16 exhibited a sensitivity of 22.6% and a specificity of 100% for MM (AUC = 0.613; 95%CI: 0.513–0.714, P = 0.046) (Fig. ). In non-acral samples, the sensitivity and specificity of PRAME/p16 for MM were 82.1% and 73.3% (AUC = 0.777; 95%CI: 0.622–0.933; P = 0.003), while for PRAME &p16, the values were 42.9% and 100% (AUC = 0.714; 95%CI: 0.564–0.864; P = 0.022) (Fig. ). Tables and compared additional diagnostic indicators in acral and non-acral samples and in invasive and preinvasive cases, respectively.   In patients diagnosed with MM, the prevalence of PRAME expression was 82.1% (92/112), with 17.9% (20/112) exhibiting negative expression. Additionally, the positivity rate for p16 was 68.75% (77/112), while 31.25% (35/112) showed negative expression. Conversely, melanocytic nevi demonstrated a PRAME positivity rate of 5.7% (3/53) and a negativity rate of 94.3% (50/53). The positivity rate for p16 in melanocytic nevi was 94.3% (50/53), with a negativity rate of 5.7% (3/53). The sensitivity of PRAME for diagnosing MM was 82.1%, with a specificity of 94.3% (AUC = 0.882; 95% CI: 0.827–0.938, P < 0.001). Conversely, the sensitivity of p16 for MM diagnosis was 31.25%, with a specificity of 94.3% (AUC = 0.628; 95% CI: 0.542–0.714, P = 0.008). Statistical analysis via the χ2-test underscored the significance of PRAME ( P < 0.001) and p16 ( P < 0.001) in distinguishing MM from melanocytic nevus (Fig. ). Of the 112 MM cases, 96 exhibited the immunophenotype PRAME/p16, while 31 cases displayed PRAME &p16. In contrast, among the 53 melanocytic nevi cases, PRAME/p16 was expressed in only 6 cases (3 spitz nevi, 2 acral nevi and 1 recurrent nevus), while PRAME &p16 in 0 cases. The sensitivity and specificity of PRAME/p16 for MM were 85.7% and 88.7% (AUC = 0.872; 95%CI: 0.810–0.934, P < 0.001), while for PRAME  &p16, the values were 27.7% and 100% (AUC = 0.638; 95%CI: 0.555–0.722, P = 0.004).   In 122 acral samples, PRAME exhibited a specificity of 97.4% and a sensitivity of 82.1% for MM ( AUC = 0.898; 95%CI: 0.839–0.956, P <0.001), with sensitivities of 90.9% and 72.5% for invasive and preinvasive MM, respectively. Besides, the sensitivity and specificity of p16 for MM were 27.4% and 97.4% (AUC = 0.624; 95%CI: 0.524–0.724, P = 0.029). PRAME/p16 demonstrated a specificity of 94.7% and a sensitivity of 86.9% for MM( AUC = 0.908; 95%CI: 0.849–0.968, P <0.001), with sensitivities of 90.9% and 82.5% for invasive and preinvasive MM, respectively. PRAME/p16 exhibited a sensitivity of 22.6% and a specificity of 100% for MM (AUC = 0.613; 95%CI: 0.513–0.714, P = 0.046) (Fig. ). In non-acral samples, the sensitivity and specificity of PRAME/p16 for MM were 82.1% and 73.3% (AUC = 0.777; 95%CI: 0.622–0.933; P = 0.003), while for PRAME &p16, the values were 42.9% and 100% (AUC = 0.714; 95%CI: 0.564–0.864; P = 0.022) (Fig. ). Tables and compared additional diagnostic indicators in acral and non-acral samples and in invasive and preinvasive cases, respectively.   The immunohistochemical diagnosis of MM presents a formidable challenge, as it necessitates discrimination from common melanocytic nevi, Spitz tumors, and atypical nevi while exhibiting poor diagnostic consistency . Notably, a study involving 187 pathologists (113 general pathologists and 74 dermatopathologists) analyzing 24 melanocytic nevi inclusive mild dysplaia(class I from MPATH-Dx classifictaion) found a consistency less than 70% even in comparatively easy diagnoses . This lack of uniformity underscores the considerable risk of misdiagnosis and subsequent adverse outcomes, a concern shared among clinicians and pathologists alike. IHC stands as a primary ancillary method for distinguishing between benign and malignant melanocytic lesions. However, conventional markers like HMB45, Melan-A, S100, and Sox10 primarily affirm melanocytic lineage rather than differentiate benign and malignant tumors. Recent attention has turned to PRAME as a promising marker for MM and melanocytic nevus identification due to its relatively robust sensitivity and specificity . Nevertheless, PRAME’s reduced expression in preinvasive MM and partial expression in melanocytic nevi still contribute to diagnostic challenges . PRAME appears as nuclear staining in MM. There are two main criteria used for PRAME  lesions, (1) significant diffuse positivity, (2) cumulative scoring methods integrating expression intensity and positive percentage . The criteria have helped diagnosis, for instance, an arbitrary PRAME IHC score of < 5 versus ≥ 5 adopted by Santandrea, et al. could accurately lead to a differentiation of 82.5% of benign and 87.1% of malignant lesions , while Ricci and co-colleagues showed that the first-rank cut-off (< 60% versus ≥ 60%) of PRAME cells to make a distinction between benign nevus and mucosal MM of the head and neck region . Thus, the consensus cut-off value of PRAME for MM and its subtypes requires further investigation. Similarly, p16 expression fluctuates widely in melanocytic lesions due to gene deletions or copy number alterations (CNA) . This variability makes p16 unreliable as a standalone identifier. For instance, p16 positivity ranged from 12 to 93% in primary cutaneous invasive MMs, 0–71% in metastatic MMs, and 61–100% in melanocytic nevi . In our study encompassing 165 melanocytic neoplasms, PRAME exhibited a sensitivity of 82.1% and specificity of 94.3% for MM identification (AUC = 0.882). However, a notable portion of MM cases remained challenging to diagnose. PRAME’s positivity in MM in situ was only 70.8% (34/48), in AM in situ was only 72.5%(29/40). In a study on diagnostic utility of PRAME IHC for subungual and non-subungual acral melanocytic lesions, any PRAME expression (1 + to 4+) was identified in 73% (16/22) subungual MMs and 95%(19/20) AMs, respectively. One of 14 (7%) acral nevi expressed PRAME . Therefore, PRAME is not a specicial biomarker for MM, emphasizing its limitations in differentiating benign and malignant tumors. Conversely, p16 showed high positivity in melanocytic nevi (94.3%) but unexpectedly appeared in 68.75% of MM cases, far exceeding anticipated levels. This may be hemozygous or heterozygous deletion and alterations via DNA methylation and CNV of CDKN2A , and indicates that p16 IHC alone has a limited capacity in differentiating melanocytic nevi from MM. Therefore, there is a requirement to explore the diagnostic and differential ability of combined IHC marker patterns. It is currently believed that PRAME is a proto-oncogene and p16 is a tumor suppressor. In addition, PRAME was significantly related to MM, and p16 was closely connected with melanocytic nevi . Combining PRAME and p16 IHC could enhance diagnostic accuracy. In a single-center retrospective cohort study-reported by Bahmad, et al., PRAME and PRAME&p16  had a sensitivity and specificity of 89.6% and 96.1%(AUC = 0.928; 95% CI: 0.878–0.979; P = 0.009), 27.3% and 100%(AUC = 0.636; 95% CI: 0.542–0.731; P = 0.009), respectively, for MM versus melanocytic nevus; most melanocytic nevi (48/51, 94.1%) were PRAME&p16 with a minority of MM . In this study, our findings showed that PRAME &p16 pattern achieved 100% specificity for MM but with lower sensitivity (27.7%), almost consistent with the result of PRAME &p16 in Bahmad et al.’ study. As for PRAME and p16, our result also had a similar positivity of MM and melanocytic nevus compared with that of Bahmad, et al. ’ study . Unsimilarly, PRAME/p16 pattern offered higher sensitivity (85.7%) and specificity (88.7%) in total samples (AUC = 0.872; 95%CI:0.810–0.934), with exceptional performance in AM(sensitivity 86.9%, specificity 94.7%, AUC = 0.908; 95%CI: 0.849–0.968) prevalent in Asia, and increase the sensitivity for preinvasive AM by 10% compared with PRAME alone. Thus, either PRAME-positive or p16-negative (PRAME/p16 balanced the requirements of diagnostic sensitivity and specificity, and also demonstrated good predictive ability in MM, especially in AM in situ. Despite these promising findings, our study had several limitations. Firstly, there were some biases in the statistical analysis of this study due to the uneven sample sizes of MM and melanocytic nevus, with the implication that additional PRAME combinated p16 IHC investigations are still required. Secondly, our selection of these clearly diagnosed specimens was a “priori ” and also a bias as it eliminated “difficult and suspicious” cases. Thirdly, the sample size was relatively small, particularly for some MM subtypes (e.g. mucosal MM, MM arising in blue nevus) and staging, which may have limited the precision of our results. Fourthly, establishing a cut-off value for new patterns may require a larger number of samples and quantitative analysis. Lastly, our study was not multi-center studies which are warranted to further validate our findings and assess their generalizability.   Our study demonstrates that PRAME exhibits a sensitivity of 82.1% and a specificity of 94.3% in diagnosing MM, while p16 lacks specificity in discerning benign lesions from MM. Combining PRAME and p16, PRAME & p16 showed 27.7% sensitivity and 100% specificity for MM diagnosis, significantly reducing the misdiagnosis rate. The combination of PRAME or p16 improved the overall sensitivity and specificity for MM, particularly in preinvasive AM. Furthermore, the combined IHC of PRAME and p16 outperformed its application in acral lesions compared to non-acral lesions, thus contributing significantly to the differentiation of melanocytic neoplasms.   