Validation of Long Mononucleotide Repeat Markers for Detection of Microsatellite Instability.

Mismatch repair deficiency (dMMR) predicts response to immune checkpoint inhibitor therapy in solid tumors. Long mononucleotide repeat (LMR) markers may improve the interpretation of microsatellite instability (MSI) assays. Our cohorts included mismatch repair (MMR) proficient and dMMR colorectal cancer (CRC) samples, MMR proficient and dMMR endometrial cancer (EC) samples, dMMR prostate cancer samples, MSI-high (MSI-H) samples of other cancer types, and MSI-low (MSI-L) samples of various cancer types. MMR status was determined by immunohistochemical staining and/or MSI Analysis System Version 1.2 (V1.2). The sensitivity and specificity of the LMR MSI panel for dMMR detection were both 100% in CRC. The sensitivity values of the MSI V1.2 and LMR MSI panels in EC were 88% and 98%, respectively, and the specificity values were both 100%. The sensitivity of the LMR panel was 75% in dMMR prostate cancer detected by immunohistochemistry. The 22 samples of other cancer types that were previously classified as MSI-H were also classified as MSI-H using the LMR MSI panel. For the 12 samples that were previously classified as MSI-L, 1 sample was classified as microsatellite stable using the LMR MSI panel, 8 as MSI-L, and 3 as MSI-H. The LMR MSI panel showed high concordance to the MSI V1.2 panel in CRC and greater sensitivity in EC. The LMR MSI panel improves dMMR detection in noncolorectal cancers.

Assessment of MYC/PTEN Status by Gene-Protein Assay in Grade Group 2 Prostate Biopsies.

This study leveraged a gene-protein assay to assess MYC and PTEN status at prostate cancer biopsy and examined the association with adverse outcomes after surgery. MYC gain and PTEN loss were simultaneously assessed by chromogenic in situ hybridization and immunohistochemistry, respectively, using 277 Grade Group 2 needle biopsies that were followed by prostatectomy. The maximal size of cribriform Gleason pattern 4 carcinoma (CRIB), the presence of intraductal carcinoma (IDC), and percentage of Gleason pattern 4 carcinoma at biopsy were also annotated. MYC gain or PTEN loss was present in 19% and 18% of biopsies, respectively, whereas both alterations were present in 9% of biopsies. Tumors with one or both alterations were significantly more likely to have non-organ-confined disease (NOCD) at radical prostatectomy. In logistic regression models, including clinical stage, tumor volume on biopsy, and presence of CRIB/IDC, cases with MYC gain and PTEN loss remained at higher risk for NOCD (odds ratio, 6.23; 95% CI, 1.74-24.55; P = 0.005). The area under the curve for a baseline model using CAPRA variables (age, prostate-specific antigen, percentage of core involvement, clinical stage) was increased from 0.68 to 0.69 with inclusion of CRIB/IDC status and to 0.75 with MYC/PTEN status. Dual MYC/PTEN status can be assessed in a single slide and is independently associated with increased risk of NOCD for Grade Group 2 biopsies.

Risk Stratification of Prostate Cancer Through Quantitative Assessment of PTEN Loss (qPTEN).

BACKGROUND: Phosphatase and tensin homolog (PTEN) loss has long been associated with adverse findings in early prostate cancer. Studies to date have yet to employ quantitative methods (qPTEN) for measuring of prognostically relevant amounts of PTEN loss in postsurgical settings and demonstrate its clinical application. METHODS: PTEN protein levels were measured by immunohistochemistry in radical prostatectomy samples from training (n = 410) and validation (n = 272) cohorts. PTEN loss was quantified per cancer cell and per tissue microarray core. Thresholds for identifying clinically relevant PTEN loss were determined using log-rank statistics in the training cohort. Univariate (Kaplan-Meier) and multivariate (Cox proportional hazards) analyses on various subpopulations were performed to assess biochemical recurrence-free survival (BRFS) and were independently validated. All statistical tests were two-sided. RESULTS: PTEN loss in more than 65% cancer cells was most clinically relevant and had statistically significant association with reduced BRFS in training (hazard ratio [HR] = 2.48, 95% confidence interval [CI] = 1.59 to 3.87; P < .001) and validation cohorts (HR = 4.22, 95% CI = 2.01 to 8.83; P < .001). The qPTEN scoring method identified patients who recurred within 5.4 years after surgery (P < .001). In men with favorable risk of biochemical recurrence (Cancer of the Prostate Risk Assessment - Postsurgical scores <5 and no adverse pathological features), qPTEN identified a subset of patients with shorter BRFS (HR = 5.52, 95% CI = 2.36 to 12.90; P < .001) who may be considered for intensified monitoring and/or adjuvant therapy. CONCLUSIONS: Compared with previous qualitative approaches, qPTEN improves risk stratification of postradical prostatectomy patients and may be considered as a complementary tool to guide disease management after surgery.

CDKN1B Deletions are Associated with Metastasis in African American Men with Clinically Localized, Surgically Treated Prostate Cancer.

PURPOSE: The potential biological determinants of aggressive prostate cancer in African American (AA) men are unknown. Here we characterize prostate cancer genomic alterations in the largest cohort to date of AA men with clinical follow-up for metastasis, with the aim to elucidate the key molecular drivers associated with poor prognosis in this population. EXPERIMENTAL DESIGN: Targeted sequencing was retrospectively performed on 205 prostate tumors from AA men treated with radical prostatectomy (RP) to examine somatic genomic alterations and percent of the genome with copy-number alterations (PGA). Cox proportional hazards analyses assessed the association of genomic alterations with risk of metastasis. RESULTS: At RP, 71% (145/205) of patients had grade group ≥3 disease, and 49% (99/202) were non-organ confined. The median PGA was 3.7% (IQR = 0.9%-9.4%) and differed by pathologic grade (P < 0.001) and stage (P = 0.02). Median follow-up was 5 years. AA men with the highest quartile of PGA had increased risks of metastasis (multivariable: HR = 13.45; 95% CI, 2.55-70.86; P = 0.002). The most common somatic mutations were SPOP (11.2%), FOXA1 (8.3%), and TP53 (3.9%). The most common loci altered at the copy number level were CDKN1B (6.3%), CHD1 (4.4%), and PTEN (3.4%). TP53 mutations and deep deletions in CDKN1B were associated with increased risks of metastasis on multivariable analyses (TP53: HR = 9.5; 95% CI, 2.2-40.6; P = 0.002; CDKN1B: HR = 6.7; 95% CI, 1.3-35.2; P = 0.026). CONCLUSIONS: Overall, PGA, somatic TP53 mutations, and a novel finding of deep deletions in CDKN1B were associated with poor prognosis in AA men. These findings require confirmation in additional AA cohorts.

PTEN Loss with ERG Negative Status is Associated with Lethal Disease after Radical Prostatectomy.

PURPOSE: Few groups have investigated the combined effects of PTEN loss and ERG expression on the outcomes of metastasis of or death from prostate cancer in surgically treated patients. We examined the association of PTEN/ERG status with lethal prostate cancer in patients treated with radical prostatectomy. MATERIALS AND METHODS: Included in analysis were 791 patients with clinically localized prostate cancer treated with radical prostatectomy at a single institution. Genetically validated immunohistochemistry assays for PTEN and ERG were performed on tissue microarrays. Multivariable Cox proportional hazard models were used to assess the association of PTEN/ERG status with lethal prostate cancer (defined as metastasis or prostate cancer specific death), adjusting for patient age, race, pathological grade and stage, and surgical margin status. RESULTS: Median followup in the cohort was 12.8 years. Of 791 cases 203 (25%) demonstrated PTEN loss and 330 of 776 (43%) were ERG positive. On multivariable analysis PTEN loss (HR 1.9, 95% CI 1.2-3.0, p=0.012) but not ERG expression (HR 0.6, 95% CI 0.4-1.1, p=0.11) was associated with an increased risk of lethal prostate cancer. The association of PTEN loss with lethal disease only remained among men with ERG negative tumors (HR 2.3, 95% CI 1.3-4.1, p=0.005) and not ERG positive tumors (HR 1.1, 95% CI 0.6-2.1, p=0.81). CONCLUSIONS: PTEN loss is associated with an increased risk of lethal prostate cancer after radical prostatectomy and this risk is most pronounced in the subgroup of patients with ERG negative tumors. This work corroborates the use of PTEN and ERG status for risk stratification in surgically treated patients.

Genomic Characterization of Prostatic Ductal Adenocarcinoma Identifies a High Prevalence of DNA Repair Gene Mutations.

PURPOSE: Ductal prostate cancer (dPC) is a rare variant of prostatic adenocarcinoma associated with poor outcomes. Although its histopathologic features are well characterized, the underlying molecular hallmarks of this aggressive subtype are not well described. We sought to provide a comprehensive overview of the spectrum of mutations associated with dPC. METHODS: Three case series across multiple institutions were assembled. All patients had a diagnosis of dPC, and histopathologic classification was confirmed by an expert genitourinary pathologist. Case series 1 included men who were prospectively enrolled in a tumor sequencing study at the University of Washington (n = 22). Case series 2 and 3 included archival samples from men treated at Johns Hopkins Hospital (n = 21) and University of Calgary (n = 8), respectively. Tumor tissue was sequenced on a targeted next-generation sequencing assay, UW-OncoPlex, according to previously published methods. The frequency of pathogenic/likely pathogenic mutations are reported. RESULTS: Overall, 25 patients (49%) had at least one DNA damage repair gene alteration, including seven (14%) with a mismatch repair gene mutation and 16 (31%) with a homologous repair mutation. Germline autosomal dominant mutations were confirmed or suspected in 10 patients (20%). Activating mutations in the PI3K pathway (n = 19; 37%), WNT pathway (n = 16; 31%), and MAPK pathway (n = 8; 16%) were common. CONCLUSION: This study strongly suggests that dPCs are enriched for actionable mutations, with approximately 50% of patients demonstrating DNA damage repair pathway alteration(s). Patients with dPC should be offered next-generation sequencing to guide standard-of-care treatment (eg, immune checkpoint inhibitors) or triaged toward an appropriate clinical trial (eg, poly [ADP-ribose] polymerase inhibitors).

TP53 missense mutation is associated with increased tumor-infiltrating T cells in primary prostate cancer.

The makeup of the tumor immune microenvironment may be associated with tumor somatic genomic alterations and plays a key role in tumor progression and response to immunotherapy. We examined the association of tumor-infiltrating T-cell density with TP53 status in surgically treated primary prostate cancer using 3 independent tissue microarray sets, including one set of tumors from grade-matched patients of European American or African American ancestry (n = 391), a retrospective case-cohort of intermediate- and high-risk patients enriched for adverse outcomes (n = 267), and a set of tumors with primary Gleason pattern 5 (n = 77). The presence of TP53 missense mutation, indicated by p53 nuclear accumulation using a genetically validated assay, was significantly associated with increased CD3+ T-cell density (median, 341 versus 231 CD3+ T cells/mm2; P = .004) in the matched European American and African American ancestry patient sets. The same association was present in patients of both ancestries when analyzed separately, despite the fact that p53 nuclear accumulation was less frequent among African American compared with European American tumors (7% versus 3%, P = .2). The validation cohorts of intermediate/high-risk and primary Gleason pattern 5 patients corroborated the association of increased CD3+ T-cell density with presence of p53 nuclear accumulation. In a pooled analysis of all sets, adjusting for clinicopathological variables, CD3+ and CD8+, but not FOXP3+, T-cell densities remained significantly higher in tumors with p53 nuclear accumulation compared with those without. TP53 mutation is associated with higher tumor-infiltrating T-cell density, which may be relevant in future clinical trials of immunotherapy in prostate cancer.

Effect of Preanalytic Variables on an Automated PTEN Immunohistochemistry Assay for Prostate Cancer.

CONTEXT.­: Phosphatase and tensin homolog (PTEN) is a promising prognostic and potentially predictive biomarker in prostate cancer. OBJECTIVE.­: To assess the effects of preanalytic variables on an analytically validated and fully automated PTEN immunohistochemistry assay. DESIGN.­: PTEN immunohistochemistry was performed on Ventana immunostaining systems. In benign prostate tissues, immunostaining intensity across variable conditions was assessed by digital image analysis. In prostate tumor tissues, immunostaining was scored visually. RESULTS.­: Delay of fixation for 4 hours or longer at room temperature or 48 hours or longer at 4°C and duration of formalin fixation did not significantly alter immunostaining intensity. Intensity of staining was highest in 10% formalin compared with other fixatives. Tumor tissues with PTEN loss processed using protocols from 11 academic institutions were all evaluable and scored identically. PTEN immunostaining of needle biopsies where tissue blocks had been stored for less than 10 years was more frequently scored as nonevaluable compared with blocks that had been stored for 10 years or longer. This effect was less evident for radical prostatectomy specimens, where low rates of nonevaluable staining were seen for 23 years or more of storage. Storage of unstained slides for 5 years at room temperature prior to immunostaining resulted in equivalent scoring compared with freshly cut slides. Machine-to-machine variability assessed across 3 Ventana platforms and 2 institutions was negligible in 12 tumors, and platform-to-platform variability was also minor comparing Ventana and Leica instruments across 77 tumors (κ = 0.926). CONCLUSIONS.­: Automated PTEN immunostaining is robust to most preanalytic variables in the prostate and may be performed on prostate tumor tissues subjected to a wide range of preanalytic conditions. These data may help guide assay development if PTEN becomes a key predictive biomarker.

PTEN status assessment in the Johns Hopkins active surveillance cohort.

BACKGROUND: Up to half of men with Gleason score 6 (GS6) prostate cancers initially managed with active surveillance (AS) will eventually require definitive therapy, usually due to tumor grade reclassification during follow-up. We examined the association between PTEN status on biopsy and subsequent clinicopathologic outcomes in men with GS6 cancers who enrolled in AS. METHODS: We performed a case-control study of men enrolled in the Johns Hopkins AS cohort with diagnostic biopsy tissue available for immunohistochemical (IHC) staining. IHC was performed for PTEN using genetically validated protocols for all patients. Cases included men who underwent grade reclassification to GS ≥ 3 + 4 = 7 on biopsy within 2 years of follow-up (i.e., early reclassification) or reclassification to GS ≥ 4 + 3 = 7 on biopsy or radical prostatectomy during follow-up (i.e., extreme reclassification). Control patients were diagnosed with GS6 cancer and monitored on AS for at least 8 years without undergoing biopsy reclassification. RESULTS: Among 67 cases with adequate tissue, 31 men underwent early reclassification and 36 men underwent extreme reclassification. Cases were compared to 65 control patients with adequate tissue for assessment. On initial prostate biopsy, cases were older (median age 67 vs. 65, p = 0.024) and were less likely to meet very-low-risk criteria (64 vs 79%, p = 0.042) as compared to controls. Although not statistically significant, PTEN loss was observed in only 1 (1.5%) of 65 controls as compared to 6 (9%) of 67 cases (p = 0.062). CONCLUSIONS: PTEN loss was rare among men with GS6 prostate cancer enrolled in AS at Johns Hopkins. Despite this, PTEN loss was more frequent among men who underwent early or extreme reclassification to higher-grade cancer as compared to controls. Additional studies in larger low-risk cohorts may better elucidate a potential role for PTEN in selecting patients for AS.

Association of tumor-infiltrating T-cell density with molecular subtype, racial ancestry and clinical outcomes in prostate cancer.

The inflammatory microenvironment plays an important role in the pathogenesis and progression of tumors and may be associated with somatic genomic alterations. We examined the association of tumor-infiltrating T-cell density with clinical-pathologic variables, tumor molecular subtype, and oncologic outcomes in surgically treated primary prostate cancer occurring in patients of European-American or African-American ancestry. We evaluated 312 primary prostate tumors, enriched for patients with African-American ancestry and high grade disease. Tissue microarrays were immunostained for CD3, CD8, and FOXP3 and were previously immunostained for ERG and PTEN using genetically validated protocols. Image analysis for quantification of T-cell density in tissue microarray tumor spots was performed. Automated quantification of T-cell densities in tumor-containing regions of tissue microarray spots and standard histologic sections were correlated (r = 0.73, p < 0.00001) and there was good agreement between visual and automated T-cell density counts on tissue microarray spots (r = 0.93, p < 0.00001). There was a significant correlation between CD3+, CD8+, and FOXP3+ T-cell densities (p < 0.00001), but these were not associated with most clinical or pathologic variables. Increased T-cell density was significantly associated with ERG positivity (median 309 vs. 188 CD3+ T cells/mm2; p = 0.0004) and also with PTEN loss (median 317 vs. 192 CD3+ T cells/mm2; p = 0.001) in the combined cohort of matched European-American and African-American ancestry patients. The same association or a similar trend was present in patients of both ancestries when analyzed separately. When the African-American patients from the matched race set were combined with a separate high grade set of African-American cases, there was a weak association of increased FOXP3+ T-cell densities with increased risk of metastasis in multivariable analysis. Though high T-cell density is associated with specific molecular subclasses of prostate cancer, we did not find an association of T-cell density with racial ancestry.

p53 status in the primary tumor predicts efficacy of subsequent abiraterone and enzalutamide in castration-resistant prostate cancer.

BACKGROUND: We tested whether tissue-based analysis of p53 and PTEN genomic status in primary tumors is predictive for subsequent sensitivity to abiraterone and enzalutamide in castration-resistant prostate cancer (CRPC). METHODS: We performed a retrospective analysis of 309 consecutive patients with CRPC treated with abiraterone or enzalutamide. Of these, 101 men (33%) had available primary tumor tissue for analysis. We screened for deleterious TP53 missense mutations and PTEN deletions using genetically validated immunohistochemical assays for nuclear accumulation of p53 protein and PTEN protein loss, with sequencing confirmation of TP53 mutations in a subset. Overall survival (OS) and progression-free survival (PFS) were compared between patients with and without p53 and/or PTEN alterations. RESULTS: Forty-eight percent of the evaluable cases had PTEN loss and 27% had p53 nuclear accumulation. OS and PFS did not differ according to PTEN status, but were significantly associated with p53 status. Median OS was 16.7 months (95% CI, 14-21.9 months) and 31.2 months (95% CI, 24.5-43.4) for men with and without p53 nuclear accumulation, respectively (HR 2.32; 95% CI 1.19-4.51; P = 0.0018). Similarly, median PFS was 5.5 months (95% CI, 3.2-9.9 months) and 10.9 months (95% CI, 8-15.2 months) in men with and without p53 nuclear accumulation, respectively (HR 2.14, 95%CI 1.20-3.81; P = 0.0008). In multivariable analyses, p53 status was independently associated with PFS (HR 2.15; 95% CI 1.03-4.49; P = 0.04) and a HR of 2.19 for OS (95% CI 0.89-5.40; P = 0.087). CONCLUSIONS: p53 inactivation in the primary tumor (but not PTEN loss) may be predictive of inferior outcomes to novel hormonal therapies in CRPC.

MSH2 Loss in Primary Prostate Cancer.

Purpose: Inactivation of mismatch repair (MMR) genes may predict sensitivity to immunotherapy in metastatic prostate cancers. We studied primary prostate tumors with MMR defects.Experimental Design: A total of 1,133 primary prostatic adenocarcinomas and 43 prostatic small cell carcinomas (NEPC) were screened by MSH2 immunohistochemistry with confirmation by next-generation sequencing (NGS). Microsatellite instability (MSI) was assessed by PCR and NGS (mSINGS).Results: Of primary adenocarcinomas and NEPC, 1.2% (14/1,176) had MSH2 loss. Overall, 8% (7/91) of adenocarcinomas with primary Gleason pattern 5 (Gleason score 9-10) had MSH2 loss compared with 0.4% (5/1,042) of tumors with any other scores (P < 0.05). Five percent (2/43) of NEPC had MSH2 loss. MSH2 was generally homogenously lost, suggesting it was an early/clonal event. NGS confirmed MSH2 loss-of-function alterations in all (12/12) samples, with biallelic inactivation in 83% (10/12) and hypermutation in 83% (10/12). Overall, 61% (8/13) and 58% (7/12) of patients had definite MSI by PCR and mSINGS, respectively. Three patients (25%) had germline mutations in MSH2 Tumors with MSH2 loss had a higher density of infiltrating CD8+ lymphocytes compared with grade-matched controls without MSH2 loss (390 vs. 76 cells/mm2; P = 0.008), and CD8+ density was correlated with mutation burden among cases with MSH2 loss (r = 0.72, P = 0.005). T-cell receptor sequencing on a subset revealed a trend toward higher clonality in cases versus controls.Conclusions: Loss of MSH2 protein is correlated with MSH2 inactivation, hypermutation, and higher tumor-infiltrating lymphocyte density, and appears most common among very high-grade primary tumors, for which routine screening may be warranted if validated in additional cohorts. Clin Cancer Res; 23(22); 6863-74. ©2017 AACR.

Analytic, Preanalytic, and Clinical Validation of p53 IHC for Detection of TP53 Missense Mutation in Prostate Cancer.

Purpose:TP53 missense mutations may help to identify prostate cancer with lethal potential. Here, we preanalytically, analytically, and clinically validated a robust IHC assay to detect subclonal and focal TP53 missense mutations in prostate cancer.Experimental Design: The p53 IHC assay was performed in a CLIA-accredited laboratory on the Ventana Benchmark immunostaining system. p53 protein nuclear accumulation was defined as any p53 nuclear labeling in >10% of tumor cells. Fifty-four formalin-fixed paraffin embedded (FFPE) cell lines from the NCI-60 panel and 103 FFPE prostate cancer tissues (88 primary adenocarcinomas, 15 metastases) with known TP53 mutation status were studied. DU145 and VCaP xenografts were subjected to varying fixation conditions to investigate the effects of preanalytic variables. Clinical validation was performed in two partially overlapping radical prostatectomy cohorts.Results: p53 nuclear accumulation by IHC was 100% sensitive for detection of TP53 missense mutations in the NCI-60 panel (25/25 missense mutations correctly identified). Lack of p53 nuclear accumulation was 86% (25/29) specific for absence of TP53 missense mutation. In FFPE prostate tumors, the positive predictive value of p53 nuclear accumulation for underlying missense mutation was 84% (38/45), whereas the negative predictive value was 97% (56/58). In a cohort of men who experienced biochemical recurrence after RP, the multivariable HR for metastasis among cases with p53 nuclear accumulation compared with those without was 2.55 (95% confidence interval, 1.1-5.91).Conclusions: IHC is widely available method to assess for the presence of deleterious and heterogeneous TP53 missense mutations in clinical prostate cancer specimens. Clin Cancer Res; 23(16); 4693-703. ©2017 AACR.

Somatic molecular subtyping of prostate tumors from HOXB13 G84E carriers.

A recurrent germline mutation (G84E) in the HOXB13 gene is associated with early onset and family history-positive prostate cancer in patients of European descent, occurring in up to 5% of prostate cancer families. To date, the molecular features of prostate tumors occurring in HOXB13 G84E carriers have not been studied in a large cohort of patients. We identified 101 heterozygous carriers of G84E who underwent radical prostatectomy for prostate cancer between 1985 and 2011 and matched these men by race, age and tumor grade to 99 HOXB13 wild-type controls. Immunostaining for HOXB13, PTEN, ERG, p53 and SPINK1 as well as RNA in situ hybridization for ETV1/4/5 were performed using genetically validated assays. Tumors from G84E carriers generally expressed HOXB13 protein at a level comparable to benign and wild-type glands. ETS gene expression (either ERG or ETV1/4/5) was seen in 36% (36/101) of tumors from G84E carriers compared to 68% (65/96) of the controls (p < 0.0001). PTEN was lost in 11% (11/101) of G84E carriers compared to 25% (25/99) of the controls (p = 0.014). PTEN loss was enriched among ERG-positive compared to ERG-negative tumors in both groups of patients. Nuclear accumulation of the p53 protein, indicative of underlying TP53 missense mutations, was uncommon in both groups, occurring in 1% (1/101) of the G84E carriers versus 2% (2/92) of the controls (p = NS). Taken together, these data suggest that genes other than ERG and PTEN may drive carcinogenesis/progression in the majority of men with germline HOXB13 mutations.

PTEN Loss in Gleason Score 3 + 4 = 7 Prostate Biopsies is Associated with Nonorgan Confined Disease at Radical Prostatectomy.

PURPOSE: Men with intermediate risk prostate cancer have widely variable outcomes. Some suggest that active surveillance or less invasive therapies (brachytherapy or focal therapy) may be appropriate for some men with Gleason score 3 + 4 = 7 disease. Molecular markers may help further distinguish prostate cancers with aggressive behavior. We tested whether loss of the PTEN (phosphatase and tensin homolog) tumor suppressor in 3 + 4 = 7 tumor biopsies is associated with adverse pathology at prostatectomy. MATERIALS AND METHODS: We queried prostate needle biopsies from 2000 to 2014 with a maximum Gleason score of 3 + 4 = 7 followed by prostatectomy. A total of 260 cases had PTEN status evaluable by clinical grade immunohistochemistry. Biopsy PTEN status was correlated with preoperative and postoperative clinicopathological parameters. RESULTS: PTEN loss was detected in 27% of 3 + 4 = 7 biopsies. Loss of PTEN was less common in tumors of African American men compared to European American men (9% vs 31%, p = 0.002). At prostatectomy, tumors with PTEN loss were more likely to show nonorgan confined disease compared to those with PTEN intact (52% vs 27%, p <0.001). In logistic regression models including age, race, prostate specific antigen, clinical stage and biopsy tumor involvement, PTEN loss at biopsy remained significantly associated with an increased risk of nonorgan confined disease (HR 2.46, 95% CI 1.34-4.49, p = 0.004). On ROC analysis, the AUC for models including prostate specific antigen and clinical stage was increased from 0.61 to 0.67 upon inclusion of PTEN status. CONCLUSIONS: PTEN loss in a Gleason score 3 + 4 = 7 biopsy is independently associated with an increased risk of nonorgan confined disease at prostatectomy. It adds to the preoperative parameters commonly used to predict pathological stage.

ERG and PTEN status of isolated high-grade PIN occurring in cystoprostatectomy specimens without invasive prostatic adenocarcinoma.

High-grade prostatic intraepithelial neoplasia (HGPIN) is widely believed to represent a precursor to invasive prostatic adenocarcinoma. However, recent molecular studies have suggested that retrograde spread of invasive adenocarcinoma into pre-existing prostatic ducts can morphologically mimic HGPIN. Thus, previous molecular studies characterizing morphologically identified HGPIN occurring in radical prostatectomies or needle biopsies with concurrent invasive carcinoma may be partially confounded by intraductal spread of invasive tumor. To assess ERG and PTEN status in HGPIN foci likely to represent true precursor lesions in the prostate, we studied isolated HGPIN occurring without associated invasive adenocarcinoma in cystoprostatectomies performed at Johns Hopkins between 2009 and 2014. Of 344 cystoprostatectomies, 33% (115/344) contained invasive prostatic adenocarcinoma in the partially submitted prostate (10 blocks/case on average) and were excluded from the study. Of the remaining cases without sampled cancer, 32% (73/229) showed 133 separate foci of HGPIN and were immunostained for ERG and PTEN using genetically validated protocols. Of foci of HGPIN with evaluable staining, 7% (8/107) were positive for ERG. PTEN loss was not seen in any HGPIN lesion (0/88). Because these isolated HGPIN foci at cystoprostatectomy are unlikely to represent retrograde spread of invasive tumor, our study suggests that ERG rearrangement, but not PTEN loss, is present in a minority of potential neoplastic precursor lesions in the prostate.

Analytic validation of a clinical-grade PTEN immunohistochemistry assay in prostate cancer by comparison with PTEN FISH.

PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated, and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to four-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for the absence of PTEN gene deletion (549/602 tumors with two copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for the presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for the presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed two intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had two copies of the PTEN gene detected by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions, or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number.

Analytic Validation of RNA In Situ Hybridization (RISH) for AR and AR-V7 Expression in Human Prostate Cancer.

PURPOSE: RNA expression of androgen receptor splice variants may be a biomarker of resistance to novel androgen deprivation therapies in castrate-resistant prostate cancer (CRPC). We analytically validated an RNA in situ hybridization (RISH) assay for total AR and AR-V7 for use in formalin-fixed paraffin-embedded (FFPE) prostate tumors. EXPERIMENTAL DESIGN: We used prostate cell lines and xenografts to validate chromogenic RISH to detect RNA containing AR exon 1 (AR-E1, surrogate for total AR RNA species) and cryptic exon 3 (AR-CE3, surrogate for AR-V7 expression). RISH signals were quantified in FFPE primary tumors and CRPC specimens, comparing to known AR and AR-V7 status by IHC and RT-PCR. RESULTS: The quantified RISH results correlated significantly with total AR and AR-V7 levels by RT-PCR in cell lines, xenografts, and autopsy metastases. Both AR-E1 and AR-CE3 RISH signals were localized in nuclear punctae in addition to the expected cytoplasmic speckles. Compared with admixed benign glands, AR-E1 expression was significantly higher in primary tumor cells with a median fold increase of 3.0 and 1.4 in two independent cohorts (P < 0.0001 and P = 0.04, respectively). While AR-CE3 expression was detectable in primary prostatic tumors, levels were substantially higher in a subset of CRPC metastases and cell lines, and were correlated with AR-E1 expression. CONCLUSIONS: RISH for AR-E1 and AR-CE3 is an analytically valid method to examine total AR and AR-V7 RNA levels in FFPE tissues. Future clinical validation studies are required to determine whether AR RISH is a prognostic or predictive biomarker in specific clinical contexts. Clin Cancer Res; 22(18); 4651-63. ©2016 AACR.