MSH2 Loss in Primary Prostate Cancer

Abstract

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.

FIGURE 1. Representative MSH2 immunostaining in formalin fixed and paraffin embedded primary prostate tumors with biallelic MSH2 inactivation

Top row: Gleason score 5+4=9 prostate tumors with intact nuclear immunostaining and wild type MSH2 gene. Second and third rows: tumors with loss of MSH2 expression and somatic two copy MSH2 genomic inactivation. Although in some sections a weakly positive cytoplasmic stain of unknown significance can be observed, the nuclei remain negative in all tumor cells, with intact staining in stromal cells, lymphocytes and benign epithelium in all cases as an internal positive control. Bottom row: Representative MSH2 immunostaining in formalin fixed and paraffin embedded primary prostate tumors with germline and somatic MSH2 gene inactivation. Both tumors lack nuclear staining for MSH2. Adjacent benign prostatic glands and stromal cells maintain nuclear expression of MSH2 as an internal control. All photomicrographs are reduced from 200×.

FIGURE 2. Representative MSH2 immunostaining in formalin fixed and paraffin embedded small cell neuroendocrine carcinoma (NEPC) of the prostate

Standard histologic tissue sections of a small cell carcinoma (35595) shows robust MSH2 nuclear staining while two other small cell carcinoma tumors (35592 and 35566) lacking nuclear staining with intact stromal and lymphocyte staining. All photomicrographs are reduced from 200×.

FIGURE 3. Representative electropherograms of colorectal carcinoma and prostatic adenocarcinoma cases that are MSI-H

MSI-PCR testing (Promega panel) for representative colorectal carcinoma and primary prostate carcinoma samples. Colorectal tumor sample shows a clear bi-modal pattern with distinct peak shifts in NR-21, BAT-25, MONO-27 mononucleotide markers (new peaks present in tumor sample but absent in normal sample are indicated by vertical arrows). In contrast, the MSI prostate tumor sample shows a bi-modal shift in NR-21 of only six bases (indicated by vertical arrow) and a subtle shift of MONO-27 (“shoulder” morphology, indicated by horizontal arrow).

FIGURE 4. CD8+ tumor infiltrating lymphocyte density in primary prostate tumors with MSH2 loss

(A) Immunostaining for CD3 identifies a high number of tumor infiltrating lymphocytes in a prostate tumor with MSH2 loss, case 71503 (upper panel). Aperio image analysis software is useful to identify CD3+ cells (red) in selected tumor regions and surrounding tumor and stromal nuclei (blue) (lower panel). (B) Immunostaining for CD8 identifies a high number of tumor infiltrating lymphocytes in a prostate tumor with MSH2 loss, case 71503 (upper panel). Aperio image analysis software is useful to identify CD8+ cells (red) in selected tumor regions and surrounding nuclei (blue) (lower panel). CD8 and CD3 cell counts were highly correlated in all cases with MSH2 loss and controls without MSH2 loss. (C) Mean density of CD8+ infiltrating lymphocytes are significantly higher in cases with MSH2 loss compared to matched control tumors with MSH2 intact and primary Gleason pattern 5. (D) Density of CD8+ infiltrating lymphocytes is significantly correlated with mutation burden among tumors with MSH2 loss.