Assessment of PD-L1, TROP2, and nectin-4 expression in penile squamous cell carcinoma.

OBJECTIVES: There is an unmet need for therapeutically relevant biomarkers for advanced penile squamous cell carcinoma (pSCC). Proposed immunohistochemistry (IHC)-based biomarkers include programmed death-ligand 1 (PD-L1), trophoblast cell-surface antigen 2 (TROP2), and nectin-4; however, there is a paucity of data pertaining to these biomarkers. Herein, we investigated the expression of PD-L1, TROP2, and nectin-4 in a well-annotated cohort of pSCCs. METHODS: A single-institution pathology archive was queried for patients who had a partial or total penectomy for pSCC between January 2000 and December 2022. Whole-slide sections were stained with antibodies against PD-L1 (22C3), TROP2, and nectin-4. Expression in tumor cells was quantified using H-scores (0-300). Associations between IHC expression, human papilloma virus (HPV) status, clinicopathologic findings, and outcome parameters were evaluated. RESULTS: This study included 121 patients. For PD-L1, the median combined positive and H-scores were 1 and 0, respectively; 32.7 % of the cases had an H-score>0. Compared to PD-L1-negative tumors, PD-L1-positive tumors had higher pT stage and grade. The median TROP2 and nectin-4 H-scores were 230 and 140, respectively, with high TROP2 and nectin-4, defined by an H-score>200, noted in 80.7 % and 10.9 % of cases, respectively. High-risk HPV-positive cases had higher TROP2 and nectin-4 scores compared to HPV-negative cases. Patients with high TROP2 expression had significantly more disease progression, and patients with high nectin-4 expression had significantly fewer deaths due to disease. CONCLUSIONS: High expression of TROP2 and nectin-4 in pSCC support evaluation of these markers as therapeutic targets pending validation of our findings.

Proteomic and Clinicopathologic Assessment of Penile Amyloidosis: A Single Institutional Review of 12 Cases.

OBJECTIVES: There is a paucity of data on penile amyloidosis. We aimed to assess the frequency of different amyloid types in surgical specimens from the penis involved by amyloidosis and correlate relevant clinicopathologic parameters with proteomic findings. METHODS: Since 2008, our reference laboratory has performed liquid chromatography/tandem mass spectrometry (LC-MS/MS) for amyloid typing. The institutional pathology archive and reference laboratory database were queried to retrospectively identify all penile surgical pathology specimens with LC-MS/MS results between January 1, 2008, and November 23, 2022. Archived H&E-stained and Congo red-stained sections were re-reviewed. RESULTS: Twelve cases of penile amyloidosis were identified, which represented 0.35% (n = 3,456) of penile surgical specimens. AL-type amyloid was most frequent (n = 7), followed by keratin-type amyloid (n = 3) and ATTR (transthyretin)-type amyloid (n = 2). AL-type amyloid cases often showed diffuse dermal/lamina propria deposition, whereas all keratin-type amyloid cases were localized to the superficial dermis. Two cases with keratin-type amyloid had concomitant cutaneous findings (penile intraepithelial neoplasia and condyloma). CONCLUSIONS: This series, the largest to date, demonstrates that penile amyloidosis has a heterogeneous proteomic landscape. To the best of our knowledge, this is the first study describing ATTR (transthyretin)-type penile amyloid.

Mass spectrometry-based assessment of prostate cancer-associated crystalloids reveals enrichment for growth and differentiation factor 15.

Intraluminal crystalloids are a common finding within malignant prostatic acini and are infrequently identified within benign glands. The proteomic composition of these crystalloids remains poorly understood and may provide insight regarding prostate cancer pathogenesis. Laser microdissection-assisted liquid chromatography-tandem mass spectrometry (LMD-LC-MS/MS) was performed to compare proteomic composition of corpora amylacea within benign acini (n = 9), prostatic adenocarcinoma-associated crystalloids (n = 8), benign (n = 8), and malignant prostatic acini (n = 6). The expression of candidate biomarkers was then measured in urine specimens from patients with (n = 8) and without prostate cancer (n = 10) using ELISA, and immunohistochemistry-based expression in adjacent prostate cancer and benign glands was assessed in 56 whole-slide sections from radical prostatectomy specimens. LMD-LC-MS/MS revealed enrichment for the C-terminal portion of growth and differentiation factor 15 (GDF15) in prostatic crystalloids. Although urinary GDF15 levels were higher in patients with prostatic adenocarcinoma compared to those without (median: 1561.2 versus 1101.3, arbitrary units), this did not meet statistical significance (P = 0.07). Immunohistochemistry for GDF15 revealed occasional positivity in benign glands (median H-score: 30, n = 56), and diffuse positivity in prostatic adenocarcinoma (median H-score: 200, n = 56, P < 0.0001). No significant difference was identified within different prognostic grade groups of prostatic adenocarcinoma, or within malignant glands with large cribriform morphology. Our results show that the C-terminal portion of GDF15 is enriched in prostate cancer-associated crystalloids, and higher GDF15 expression is seen in malignant rather than benign prostatic acini. Improved understanding of the proteomic composition of prostate cancer-associated crystalloids provides the rationale for evaluating GDF15 as a urine-based biomarker of prostate cancer.

Renal Neoplasia in Polycystic Kidney Disease: An Assessment of Tuberous Sclerosis Complex-associated Renal Neoplasia and PKD1/TSC2 Contiguous Gene Deletion Syndrome.

To determine the incidence of renal neoplasia among patients undergoing nephrectomy for polycystic kidney disease (PKD), we queried our institutional nephrectomy registry (years 2000-2020). Approximately 4% (231 of 5757) of patients who underwent nephrectomy had PKD, and 26 of these 231 patients (11.3%) had renal neoplasia. Tumors from an additional two patients with PKD were also evaluated. Patients with PKD who had tuberous sclerosis complex (TSC)-associated renal neoplasia were screened for PKD1/TSC2 contiguous gene deletion syndrome (CGS) using single nucleotide polymorphism arrays. The median age of patients with PKD and renal neoplasia at nephrectomy was 54 yr. The median tumor size was 2.0 cm and the tumors were predominantly of low grade and stage. The tumors consisted of 23 renal cell carcinomas (RCCs), one epithelioid angiomyolipoma, and four angiomyolipomas. The median follow-up was 59.5 mo (n = 26) and only one patient with clear cell RCC developed metastases. Two patients with angiomyolipomas had PKD1/TSC2 CGS. Our results support screening of patients with PKD and TSC-associated renal neoplasia as well as TSC patients with cystic renal disease for CGS, as identification of patients with CGS can better define the manifestation and prognosis of CGS and guide counseling regarding patterns of inheritance. PATIENT SUMMARY: We identified patients with abnormal kidney cell growth (called renal neoplasia) among those undergoing removal of kidney tissue for polycystic kidney disease (PKD) and patients with a syndrome involving deletions in two genes, called PKD1/TSC2 contiguous gene deletion syndrome (CGS) at our institution. Of 231 PKD patients with removal of kidney tissue, 11.3% had renal neoplasia, and two patients with angiomyolipoma tumors had PKD1/TSC2 CGS. Detection of renal neoplasia associated with a condition called tuberous sclerosis complex in PKD may increase the identification of patients with PKD1/TSC2 CGS and guide patient counseling regarding outcomes and patterns of inheritance.

Assessment of Risk of Hereditary Predisposition in Patients With Melanoma and/or Mesothelioma and Renal Neoplasia.

Importance: In BAP1 tumor predisposition syndrome, clear cell renal cell carcinoma (RCC) is frequently associated with melanoma and/or mesothelioma, while germline MITF p.E318K alterations are being increasingly reported in melanoma/RCC. Limited data exist on the co-occurrence of melanoma and/or mesothelioma with renal neoplasia and the prevalence of associated germline alterations. Objective: To assess the frequency of melanoma and/or mesothelioma co-occurring with renal neoplasia using our institutional nephrectomy registry and to determine the prevalence of BAP1 and MITF alterations within this cohort. Design, Setting, and Participants: In this genetic association study, medical records from 8295 patients from 1970 to 2018, renal neoplasia co-occurring with melanoma and/or mesothelioma within a single institutional nephrectomy registry was reevaluated based on contemporary histopathologic criteria and the medical records were reviewed. Data were analyzed from September 2019 to May 2021. Main Outcomes and Measures: Identified cases were screened for BAP1 loss using immunohistochemistry; while patients with melanoma and clear cell RCC were screened for MITF p.E318K alterations. Tumors from patients with potential germline alterations were analyzed with comprehensive molecular profiling using a 514-gene next generation sequencing panel. Results: Of a total of 8295 patients, 93 (1.1%; 95% CI, 0.9%-1.4%) had melanoma and/or mesothelioma co-occurring with renal neoplasia (cutaneous melanoma, n = 76; uveal melanoma, n = 11; mesothelioma, n = 6). A total of 69 (74.2%) were male; 24 (25.8%) were female; median age at diagnosis of renal neoplasia was 63 years (IQR, 58-70 years) and the median duration of follow-up was 8.5 years (IQR, 5.0-14.6 years). Two patients with clear cell RCC had germline BAP1 alterations in the setting of cutaneous melanoma and mesothelioma. Two patients with hybrid oncocytic tumors had biallelic inactivation of FLCN in a setting of Birt-Hogg-Dubé (BHD) syndrome associated with uveal melanoma and mesothelioma. Tumor-only screening of clear cell RCC associated with cutaneous (n = 53) and uveal melanoma (n = 6) led to the identification of 1 patient with a likely germline MITF p.E318K alteration. After excluding benign renal neoplasia (such as oncocytoma and angiomyolipoma), alterations of BAP1, FLCN, and MITF were identified in 5 of 81 patients (6.2%) with melanoma and/or mesothelioma and renal neoplasia. In contrast to hybrid oncocytic tumors in BHD, no unique genotype-phenotype correlations were seen for clear cell RCC with pathogenic BAP1/ MITF alterations and VHL loss of function variants. Four of 5 cases (80%) met current National Comprehensive Cancer Network criteria for germline testing based on a combination of age, multifocality, histologic findings, and family history. Conclusions and Relevance: In this genetic association study, findings support the continued use of these National Comprehensive Cancer Network criteria and suggest more stringent screening may be warranted in this patient population.

Assessment of isochromosome 12p and 12p abnormalities in germ cell tumors using fluorescence in situ hybridization, single-nucleotide polymorphism arrays, and next-generation sequencing/mate-pair sequencing.

The identification of isochromosome 12p [i(12p)] and 12p gains have significant clinical utility in the diagnosis of germ cell tumors (GCTs). We have summarized the results of fluorescence in situ hybridization (FISH) assays to identify i(12p), performed in a Clinical Laboratory Improvement Amendments (CLIA)-validated setting for 536 specimens. In addition, the American Association for Cancer Research (AACR) Project GENIE registry and The Cancer Genome Atlas (TCGA) data sets were evaluated for chromosome 12p gains, and a limited number of cases were concurrently evaluated using FISH, single-nucleotide polymorphism (SNP) arrays and next-generation sequencing (NGS; including mate-pair sequencing). Specimens submitted for FISH testing were frequently from potential sites of metastases (male: 70.9% and female: 69.3%), and polysomy of chromosome 12 with or without concurrent i(12p) was a frequent finding, seen in 3% (16/536) and 35% (186/536) of cases, respectively. Our analysis suggests that 12p gains are likely to be present in approximately 73% of male GCT and in 32% of female GCT (AACR GENIE, n = 555). When comparing TCGA cases of testicular GCT (n = 149) to combined cases of sarcoma, colorectal, prostate, and urothelial carcinoma (n = 1754), 12p gains had a sensitivity of 77.2% and specificity of 97.3% for GCT. Some advantages of FISH over SNP arrays/NGS include relatively lower cost, rapid turnaround time, the ability to analyze biopsy material with a limited number of tumor cells (50 cells), and the ability to distinguish i(12p) from polysomy. The ability to spatially restrict the analysis to cells of interest is critical, as specimens submitted for testing often have low tumor purity. Disadvantages include false negative results due to an inability to detect segmental gains due to FISH probe design. With the availability of numerous testing modalities, including FISH, SNP arrays, and NGS-based assays, a nuanced understanding of the advantages and disadvantages of each methodology, as has been presented in this study, may inform appropriate testing strategies.

Primary Female Urethral Carcinoma: Proposed Staging Modifications Based on Assessment of Female Urethral Histology and Analysis of a Large Series of Female Urethral Carcinomas.

Primary female urethral carcinoma is rare. Limited clinicopathologic information has hindered development of staging criteria in this disease. We analyzed 29 primary female urethral carcinoma resections from 3 academic medical centers to characterize histopathologic features, clinical outcomes, and applicability of current and a novel modified staging criteria. We complemented this analysis with review of fully embedded female autopsy urethras to detail anterior and posterior urethral wall histology. Primary female urethral carcinoma subtypes included urothelial carcinoma in situ (3/29, 10%), adenocarcinoma in situ (1/29; 3%), invasive urothelial carcinoma (13/29, 45%), clear cell carcinoma (5/29, 17%), adenocarcinoma not otherwise specified (4/29, 14%) and squamous cell carcinoma (3/29, 10%). Only 6/29 cases (21%) were originally assigned a stage at diagnosis. Using histologic landmarks specific to the female urethra, we modified existing eighth edition American Joint Committee on Cancer urethral staging to a histology-based female urethral carcinoma staging (UCS) system. UCS stages were defined as pTa/pTisUCS (noninvasive carcinoma), pT1UCS (subepithelial tissue invasion), pT2UCS (periurethral muscle invasion), pT3UCS (vaginal adventitia or surrounding fibrovascular tissue), and pT4UCS (anterior wall fibroadipose tissue or posterior vaginal wall). UCS staging was applicable to all cases and showed stepwise changes in disease recurrence with increasing stage and was statistically significant for disease-specific and overall survival in contrast to the American Joint Committee on Cancer staging system. This study of one of the largest cohort of primary female urethral carcinomas provides a modified histology-based staging system specific to female urethral anatomy that provides outcomes-related information, which may be further validated by larger multi-institutional studies.

Evaluation of the National Comprehensive Cancer Network and American Urological Association renal cell carcinoma surveillance guidelines.

PURPOSE: The National Comprehensive Cancer Network (NCCN) and American Urological Association (AUA) provide guidelines for surveillance after surgery for renal cell carcinoma (RCC). Herein, we assess the ability of the guidelines to capture RCC recurrences and determine the duration of surveillance required to capture 90%, 95%, and 100% of recurrences. PATIENTS AND METHODS: We evaluated 3,651 patients who underwent surgery for M0 RCC between 1970 and 2008. Patients were stratified as AUA low risk (pT1Nx-0) after partial (LR-partial) or radical nephrectomy (LR-radical) or as moderate/high risk (M/HR; pT2-4Nx-0/pTanyN1). Guidelines were assessed by calculating the percentage of recurrences detected when following the 2013 and 2014 NCCN and AUA recommendations, and associated Medicare costs were compared. RESULTS: At a median follow-up of 9.0 years (interquartile range, 5.7 to 14.4 years), a total of 1,088 patients (29.8%) experienced a recurrence. Of these, 390 recurrences (35.9%) were detected using 2013 NCCN recommendations, 742 recurrences (68.2%) were detected using 2014 NCCN recommendations, and 728 recurrences (66.9%) were detected using AUA recommendations. All protocols missed the greatest amount of recurrences in the abdomen and among pT1Nx-0 patients. To capture 95% of recurrences, surveillance was required for 15 years for LR-partial, 21 years for LR-radical, and 14 years for M/HR patients. Medicare surveillance costs for one LR-partial patient were $1,228.79 using 2013 NCCN, $2,131.52 using 2014 NCCN, and $1,738.31 using AUA guidelines. However, if 95% of LR-partial recurrences were captured, costs would total $9,856.82. CONCLUSION: If strictly followed, the 2014 NCCN and AUA guidelines will miss approximately one third of RCC recurrences. Improved surveillance algorithms, which balance patient benefits and health care costs, are needed.

Maintaining clinical tissue archives and supporting human research: challenges and solutions.

CONTEXT: The increasing number of requests for use of clinically archived tissue in translational research poses unique challenges. Conflicts may arise between pathologists who are responsible for overseeing and preserving the tissues and investigators who need these materials for research purposes. OBJECTIVES: To evaluate the status of our institution's Tissue Registry Archive and to develop updated written policies and procedures to support a new modern and robust tracking system with features of a library loan system. DESIGN: An observational study was performed. RESULTS: We found the existing process for managing loans of tissue (slides and paraffin blocks) to be insufficient for the complexity and volume of this task. After extensive customization, a new tracking system was implemented in January 2008. Analysis of the first year of the system's use (2008) showed that of the 206,330 slides and 51,416 blocks loaned out in 2008, 92% and 94%, respectively, were returned by the due date. These rates were markedly improved from those before the new system: 61% and 47%, respectively, in 2005. Material permanently "lost" in 2008 represented only 0.02% of slides and 0.05% of blocks, none of which was the only diagnostic material for the case. CONCLUSIONS: With expanding needs for archived tissues for clinical care and growing demands for translational research, it is essential that pathology departments at institutions with large tissue-based research endeavors have a tracking and management system in place to meet clinical, educational, and research needs, as well as legal requirements.

Comparison of digital image analysis versus visual assessment to assess survivin expression as an independent predictor of survival for patients with clear cell renal cell carcinoma.

We previously used quantitative digital image analysis to report that high immunohistochemical tumor expression levels of survivin independently predict poor outcome among patients with clear cell renal cell carcinoma. However, given the cumbersome and costly nature of digital image analysis, we evaluated simple visual assessment as an alternative to digital image analysis for assessing survivin as a predictor of clear cell renal cell carcinoma patient outcomes. We identified 310 patients treated surgically for unilateral, sporadic, clear cell renal cell carcinoma at our institution between 1990 and 1994. Survivin expression was quantified independently by digital image analysis and visual assessment in paraffin slides using a commercially available antibody. We examined the agreement between the 2 methods using the kappa statistic and then used Cox regression to compare the ability of the 2 methods to predict renal cell carcinoma death. The kappa statistic comparing high survivin expression determined by digital image analysis versus visual assessment was .68, indicating substantial agreement between the 2 methods. Moreover, even after multivariate adjustment, the association of high survivin expression with risk of renal cell carcinoma death was similar for both visual assessment (risk ratio = 2.01; 95% confidence interval, 1.26-3.22) and digital image analysis (risk ratio = 1.75; 95% confidence interval, 1.10-2.80). Finally, among patients with "moderate risk" (Stage, Size, Grade, and Necrosis scores 3-6) and "high risk" (Stage, Size, Grade, and Necrosis scores 7 or greater) clear cell renal cell carcinoma, high survivin expression determined by visual assessment was significantly associated with poorer survival (P = .006 and P = .017, respectively). Herein, we demonstrate substantial agreement between survivin quantification by digital image analysis and visual assessment. We further confirm that high survivin expression assessed by visual assessment remains an independent predictor of aggressive clear cell renal cell carcinoma behavior. Thus, visual assessment represents an economical, widely available, and reliable method to assess survivin as a predictor of clear cell renal cell carcinoma patient outcomes.

Conventional assessment of needle biopsy specimens is more useful than digital image analysis of proliferation and DNA ploidy in prediction of positive surgical margins at radical prostatectomy.

OBJECTIVES: The preoperative prediction of the likelihood of positive surgical margins (+SMs) at radical retropubic prostatectomy (RRP) may be useful for counseling and determining the surgical approach. The aim of this study was to assess the additional value of digital image analysis (DIA) of ploidy and proliferation on needle biopsies, in addition to the known preoperative predictors of +SMs at RRP. METHODS: We identified 454 patients treated by RRP at our institution from 1995 to 1998 for prostate cancer verified by transrectal ultrasound-guided biopsy, with a specimen adequate for DIA. Patients receiving preoperative hormonal therapy were excluded. The clinical features, transrectal ultrasound-guided biopsy findings, and DIA evaluation of MIB-I immunostaining and DNA ploidy were assessed in a multivariate logistic regression model to predict for +SMs at RRP. RESULTS: The mean +/- SD age at treatment was 64.5 +/- 6.5 years, the percentage of positive cores was 40.4% +/- 24.3%, the median prostate-specific antigen level was 6.3 ng/mL (range 0.6 to 112.0), median biopsy Gleason score was 6 (range 4 to 9), and median percentage of diploid nuclei was 67% (range 0% to 100%). Of the 454 patients, 185 (40.7%) had +SMs; this finding was time dependent (1995 to 1996, 45% and 1997 to 1998, 31%; P = 0.004). Univariately, preoperative prostate-specific antigen, biopsy Gleason score, extent of cancer on biopsy, MIB-1 expression, percentage of diploid or nondiploid nuclei, and year of surgery were predictive for +SMs. On multivariate analysis, the preoperative prostate-specific antigen level, biopsy Gleason score, percentage of positive cores, and year of surgery remained significant. CONCLUSIONS: The results of our study have shown that the likelihood of +SMs at RRP is best predicted on the basis of conventional prognostic factors. The DIA features of needle biopsies did not provide additional predictive power.