PTEN protein loss by immunostaining: Analytic validation and prognostic indicator for a high risk surgical cohort of prostate cancer patients

PURPOSE: Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome fluorescence in situ hybridization (FISH) spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemical (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease. EXPERIMENTAL DESIGN: PTEN IHC was validated by employing formalin fixed and paraffin embedded isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and 97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then assessed on 376 prostate tumor samples, and PTEN FISH or high resolution SNP microarray analysis was performed on a subset of these cases. RESULTS: PTEN protein loss, as assessed as a dichotomous IHC variable, was highly reproducible, correlated strongly with adverse pathologic features (e.g. Gleason score and pathological stage), detected between 75% and 86% of cases with PTEN genomic loss, and was found at times in the absence of apparent genomic loss. In a cohort of 217 high risk surgically treated patients, PTEN protein loss was associated with decreased time to metastasis. CONCLUSIONS: These studies validate a simple method to interrogate PTEN status in clinical specimens and support the utility of this test in future multi-center studies, clinical trials and ultimately perhaps for routine clinical care

Genomic and clinical characterization of stromal infiltration markers in prostate cancer

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154519/1/cncr32688_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154519/2/cncr32688.pd

MYC Overexpression Induces Prostatic Intraepithelial Neoplasia and Loss of Nkx3.1 in Mouse Luminal Epithelial Cells

Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate[1]. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina—features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These “Super-Lo-MYC” mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a “paradoxical” increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas

Tumor Frameshift Mutation Proportion Predicts Response to Immunotherapy in Mismatch Repair‐Deficient Prostate Cancer

Background: Genomic biomarkers that predict response to anti-PD1 therapy in prostate cancer are needed. Frameshift mutations are predicted to generate more neoantigens than missense mutations; therefore, we hypothesized that the number or proportion of tumor frameshift mutations would correlate with response to anti-PD1 therapy in prostate cancer. Methods: To enrich for response to anti-PD1 therapy, we assembled a multicenter cohort of 65 men with mismatch repair-deficient (dMMR) prostate cancer. Patient characteristics and outcomes were determined by retrospective chart review. Clinical somatic DNA sequencing was used to determine tumor mutational burden (TMB), frameshift mutation burden, and frameshift mutation proportion (FSP), which were correlated to outcomes on anti-PD1 treatment. We subsequently used data from a clinical trial of pembrolizumab in patients with nonprostatic dMMR cancers of various histologies as a biomarker validation cohort. Results: Nineteen of 65 patients with dMMR metastatic castration-resistant prostate cancer were treated with anti-PD1 therapy. The PSA50 response rate was 65%, and the median progression-free survival (PFS) was 24 (95% confidence interval 16-54) weeks. Tumor FSP, more than overall TMB, correlated most strongly with prolonged PFS and overall survival (OS) on anti-PD1 treatment and with density of CD8+ tumor-infiltrating lymphocytes. High FSP similarly identified patients with longer PFS as well as OS on anti-PD1 therapy in a validation cohort. Conclusion: Tumor FSP correlated with prolonged efficacy of anti-PD1 treatment among patients with dMMR cancers and may represent a new biomarker of immune checkpoint inhibitor sensitivity. Implications for practice: Given the modest efficacy of immune checkpoint inhibition (ICI) in unselected patients with advanced prostate cancer, biomarkers of ICI sensitivity are needed. To facilitate biomarker discovery, a cohort of patients with DNA mismatch repair-deficient (dMMR) prostate cancer was assembled, as these patients are enriched for responses to ICI. A high response rate to anti-PD1 therapy in these patients was observed; however, these responses were not durable in most patients. Notably, tumor frameshift mutation proportion (FSP) was identified as a novel biomarker that was associated with prolonged response to anti-PD1 therapy in this cohort. This finding was validated in a separate cohort of patients with nonprostatic dMMR cancers of various primary histologies. This works suggests that FSP predicts response to anti-PD1 therapy in dMMR cancers, which should be validated prospectively in larger independent cohorts

Gene expression signatures of neuroendocrine prostate cancer and primary small cell prostatic carcinoma

Abstract Background Neuroendocrine prostate cancer (NEPC) may be rising in prevalence as patients with advanced prostate cancer potentially develop resistance to contemporary anti-androgen treatment through a neuroendocrine phenotype. While prior studies comparing NEPC and prostatic adenocarcinoma have identified important candidates for targeted therapy, most have relied on few NEPC patients due to disease rarity, resulting in thousands of differentially expressed genes collectively and offering an opportunity for meta-analysis. Moreover, past studies have focused on prototypical NEPC samples with classic immunohistochemistry profiles, whereas there is increasing recognition of atypical phenotypes. In the primary setting, small cell prostatic carcinoma (SCPC) is frequently admixed with adenocarcinomas that may be clonally related, and a minority of SCPCs express markers typical of prostatic adenocarcinoma while rare cases do not express neuroendocrine markers. We derived a meta-signature of prototypical high-grade NEPC, then applied it to develop a classifier of primary SCPC incorporating disease heterogeneity. Methods Prototypical NEPC samples from 15 patients across 6 frozen tissue microarray datasets were assessed for genes with consistent outlier expression relative to adenocarcinomas. Resulting genes were used to determine subgroups of primary SCPCs (N=16) and high-grade adenocarcinomas (N=16) profiled by exon arrays using formalin-fixed paraffin-embedded (FFPE) material from our institutional archives. A subgroup classifier was developed using differential expression for feature selection, and applied to radical prostatectomy cohorts. Results Sixty nine and 375 genes demonstrated consistent outlier expression in at least 80% and 60% of NEPC patients, with close resemblance in expression between NEPC and small cell lung cancer. Clustering by these genes generated 3 subgroups among primary samples from our institution. Nearest centroid classification based on the predominant phenotype from each subgroup (9 prototypical SCPCs, 9 prototypical adenocarcinomas, and 4 atypical SCPCs) achieved a 4.5% error rate by leave-one-out cross-validation. The classifier identified SCPC-like expression in 40% (2/5) of mixed adenocarcinomas and 0.3-0.6% of adenocarcinomas from prospective (4/2293) and retrospective (2/355) radical prostatectomy cohorts, where both SCPC-like retrospective cases subsequently developed metastases. Conclusions Meta-analysis generates a robust signature of prototypical high-grade NEPC, and may facilitate development of a primary SCPC classifier based on FFPE material with potential prognostic implications