Abstract
Patients with neuroendocrine prostate cancer (NEPC) are often managed with immunotherapy regimens extrapolated from small-cell lung cancer (SCLC). We sought to evaluate the tumor immune landscape of NEPC compared with other prostate cancer types and SCLC.
In this retrospective study, a cohort of 170 patients with 230 RNA-sequencing and 104 matched whole-exome sequencing data were analyzed. Differences in immune and stromal constituents, frequency of genomic alterations, and associations with outcomes were evaluated.
In our cohort, 36% of the prostate tumors were identified as CD8+ T-cell inflamed, whereas the remaining 64% were T-cell depleted. T-cell–inflamed tumors were enriched in anti-inflammatory M2 macrophages and exhausted T cells and associated with shorter overall survival relative to T-cell–depleted tumors (HR, 2.62; P < 0.05). Among all prostate cancer types in the cohort, NEPC was identified to be the most immune depleted, wherein only 9 out of the 36 total NEPC tumors were classified as T-cell inflamed. These inflamed NEPC cases were enriched in IFN gamma signaling and PD-1 signaling compared with other NEPC tumors. Comparison of NEPC with SCLC revealed that NEPC had poor immune content and less mutations compared with SCLC, but expression of checkpoint genes PD-L1 and CTLA-4 was comparable between NEPC and SCLC.
NEPC is characterized by a relatively immune-depleted tumor immune microenvironment compared with other primary and metastatic prostate adenocarcinoma except in a minority of cases. These findings may inform development of immunotherapy strategies for patients with advanced prostate cancer.
Histologic transformation from prostate adenocarcinoma to small-cell neuroendocrine prostate cancer (NEPC) is an emerging resistance mechanism seen in up to 15% of advanced prostate cancers. There are no approved therapies for NEPC, and treatment, including immunotherapy, is often extrapolated from small-cell lung cancer, a tumor type with similar histological features. We characterized the immunogenomic landscape of the tumor microenvironment (TME) in NEPC compared with prostate adenocarcinoma and small-cell lung carcinoma. We found that NEPC is relatively the most T-cell immune depleted, except for a subset of inflamed tumors. TME profiling, as reported in this study, may inform patient selection and immunotherapy strategies for patients with advanced prostate cancer.
Introduction
Metastatic prostate cancer is a leading cause of cancer-related death for men globally (1). Although androgen receptor (AR)–targeted therapies have significantly improved outcomes for prostate cancer, prolonged and potent suppression of AR signaling has led to evolution of treatment-resistant AR-independent variants of prostate cancer, such as neuroendocrine prostate cancer (NEPC), that rarely arises de novo but can develop in up to 15%–20% of late-stage castration-resistant prostate cancer (CRPC; refs. 2–5). NEPC is clinically aggressive with limited therapeutic options. NEPC is currently diagnosed by its morphologic features that are similar to other poorly differentiated small-cell neuroendocrine (NE) carcinomas, though both pure NE carcinomas and mixed adenocarcinoma/NE histologies may be observed within the spectrum of NEPC (6). There have been very few trials dedicated to NEPC, and therefore treatment is often extrapolated from small-cell lung cancer (SCLC) guidelines based on their clinical and pathologic similarities. On the basis of recent data in SCLC, platinum-based chemotherapy combined with anti-PDL1 immune checkpoint inhibitor therapy (atezolizumab or durvalumab) followed by immunotherapy maintenance is now used in the first-line setting for patients with SCLC (7, 8). Patients with NEPC are now often being managed on the basis of updated SCLC regimens with chemotherapy plus immunotherapy despite a lack of strong supporting data (9). A better understanding of the immune landscape is needed to inform how SCLC regimens are adopted for NEPC.
Prostate cancer generally has an immune “excluded” tumor microenvironment (TME) with limited leukocyte infiltration dominated by immunosuppressive cells that promote tumor proliferation (10–12). Strategies to prime the immune system to recognize tumors in prostate cancer involve combining conventional therapy with immunotherapy, for example, immune checkpoint blockade (ICB; ref. 13). However, success of these treatments has remained limited to rare cases of mismatch repair deficiency and microsatellite instability (14, 15). Recent data from the IMbassador250 trial have suggested that a subset of patients with advanced prostate cancer may still benefit from ICB if they expressed ICB-related immune markers (e.g., PD-L1; ref. 16). Similarly, in a small clinical trial, one patient with NEPC who showed exceptional durable response to avelumab had consistently high levels of Natural Killer (NK) and PD1+ Th cells coupled with increase in CXCR2+ T cells over time (17). Such findings emphasize the potential value of comprehensive TME profiling in advanced prostate cancer.
In this retrospective study, we profiled the immunogenomic landscape of 230 patient biopsies along the prostate cancer continuum. We report an immune-depleted profile of NEPC compared with prostate adenocarcinoma and SCLC pathologies, suggesting an immune-evasive, tumor-promoting TME with potential implications for therapy.
Materials and Methods
Patient cohort
We retrospectively analyzed data from previously published prostate cohorts for this study (2, 18–20). A total of 230 tissue samples from 170 individuals with RNA-sequencing (RNA-seq) data were analyzed, comprised of benign prostate tissue (n = 31), localized prostate cancer (PCa, n = 74), hormone naïve metastatic prostate cancer (mPCa.HN, n = 14), CRPC (n = 75), NEPC (n = 36). Metastatic patient characteristics (n = 93) are reported in Supplementary Table S1. NEPC was defined on the basis of tumor morphology (6). 25 cases were pure small- or large-cell carcinoma and 11 were mixed morphology cases. Survival data were available for 82 patients and matched whole-exome sequencing (WES) was available for 104 tumors.
RNA-seq, data quantification, and analysis
Sequenced reads were aligned to human reference genome hg19/GRC37 using STAR (RRID:SCR_004463; ref. 22) and quantified as gene counts using HTSeq (RRID:SCR_005514; ref. 23) and FPKMs using cufflinks (RRID:SCR_014597; ref. 24) with GENCODE (RRID:SCR_014966; ref. 25) v19 for Gene expression profiles (GEP). Enrichment scores (ES) were calculated using single-sample gene set enrichment analysis (ssGSEA; ref. 26). Cytolytic scores were calculated as geometric mean of genes PRF1 and GZMA (27). Differentially expressed genes (DEG) were identified using gene counts with DESeq2 (RRID:SCR_000154; ref. 28) and pathway analysis of ranked DEGs was done using fgsea in R (FDR, q < 0.25). Top and bottom DEGs selected on the basis of log-fold change threshold of 2 (q < 0.05) were annotated using the collection of “NCI-Nature_2016_pathways” in Enrichr (RRID:SCR_001575; ref. 29; FDR < 0.25). All statistical tests done using Wilcoxon test or Fisher's exact test in R.
WES and data processing
WES data processing and alignment to human reference genome hg19/GRC37 were performed using established protocols described earlier (2, 30, 31). Mutations were identified using Multi-Center Mutation Calling in Multiple Cancers (MC3) pipeline (32). Tumor mutation burden (TMB) was calculated as non-silent mutation counts divided by the total n, modified by including Mutect2 (RRID:SCR_000559) and Strelka2 (RRID:SCR_005109) to existing tools VarScan2 (RRID:SCR_006849), Pindel (RRID:SCR_000560), SomaticSniper (RRID:SCR_005108), RADIA, and MuSE. All mutations calls were combined and only those identified by ≥2 tools were retained. Default MC3 read depth filters were applied and mutations were restricted to HaloPlex regions (number of bases in the HaloPlex captured region per million. Copy-number alterations were identified using CNVkit (RRID:SCR_021917; ref. 33) and CNVSeeqer. The log2 copy ratio threshold of 0.10 was used to calculate copy-number loads. Oncoprints were constructed with ComplexHeatmap in R (RRID:SCR_017270).
T-cell inflammation and deconvolution analysis
GEPs from 230 prostate samples were ordered by their median absolute deviation and top 65% most variable genes across the cohort were selected. The selected genes were median centered and used for unsupervised hierarchical clustering (the wardD2 method), leading to identification of a 361-gene cluster with T-cell marker CD8A (Supplementary Table S2). These 361 genes were used for supervised re-clustering of 230 samples with ConsensusClusterPlus (ref. 34; pam clustering, 200 random selections, 80% re-sampling); 136 depleted and 94 inflamed samples were identified (Fig. 1A). To apply this T-cell signature to independent cancer cohorts, we used the signature for supervised consensus clustering with method pam and 1,000 random selections with 80% re-sampling, followed by removal of misclassified samples as identified by silhouette analysis (R package cluster). ssGSEA (RRID:SCR_003199) was applied to calculate a composite T-cell cluster score per sample for these 361 genes. ssGSEA was also applied to deconvolve GEPs based on 80 published immune and stromal signatures we considered representative of the TME (Supplementary Table S3). Survival analysis was done using Cox Proportional-Hazards models and results visualized as forest plots (survival and forestplot, R). Plots were made with ggpubr or ComplexHeatmap R packages.
Weighted gene co-expression network analysis and module identification
We performed signed weighted gene co-expression network analysis (WGCNA) using the WGCNA R package (RRID:SCR_003302) for automatic network identification and construction (35). The best soft thresholding power of 12 was selected using the pickSoftThreshold function. Module–trait relationships correlation matrix and binomial generalized linear models were used to identify associations between modules and prostate cancer groups. The module eigengenes were gathered, and their ES per sample was calculated using ssGSEA. The modules were annotated for functional enrichment with ReactomePA and clusterProfiler and networks visualized by cnetplot in R.
IHC and pathological review
Tissue studies were conducted in accordance with the Declaration of Helsinki and after institutional review board approval (WCM IRB #1305013903) and with written informed consent from participants. Histopathology examination was performed through routine hematoxylin and eosin stain. NEPC was defined on the basis of tumor morphology (6). Digital image analysis of IHC slides for CD8, CD68, GZMA and PD-L1 was performed using HALO software (Indica Labs; ref. 36). Briefly, IHC slides were scanned at ×40 with the Aperio AT2 slide scanner (Leica Biosystems). Annotation of tumor areas and desired regions of interest were selected through the HALO software, directly. Using an automated algorithm with specific nuclear and membranous characteristics, staining was quantified (0% to 100%), excluding artifacts or nuclear staining (false positive).
Data availability
Prostate cancer datasets used in this study are available within their corresponding publications (2, 18–20) or through dbGAP (phs000909.v.p1, phs001666.v1.p1). The gene expression data (quantified as FPKMs; fragments per kilobase of exon per million mapped fragments) were obtained for the International Stand up to Cancer (SU2C)-Prostate Cancer Foundation (PCF) Dream Team from the cBioPortal (https://github.com/cBioPortal/datahub/tree/master/public/prad_su2c_2019; RRID:SCR_014555) and for The Cancer Genome Atlas (TCGA) cancer cohorts from the Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov; RRID:SCR_014514). Data from SCLC (n = 28) were procured from a controlled access dataset EGAS00001000334 (21).
Results
Identification of T-cell inflamed and depleted phenotypes in prostate cancer
CD8+ T cells are important mediators of antitumor immune responses (37), and their quantification in a sample can provide initial insights into the antitumor potential of the TME. To identify T-cell–enriched samples in our cohort of 230 prostate biopsies, we clustered their GEPs with unsupervised hierarchal clustering and identified a cluster of 361 genes enriched with known markers of T cells and T-cell receptor signaling (Supplementary Fig. S1A and Supplementary Table S2). We re-clustered the samples using consensus clustering (supervised with these 361 genes) resulting in two groups: A T-cell–inflamed group with relatively higher expression of CD8+ T-cell markers and cytolytic scores versus a T-cell–depleted group (Fig. 1A; Supplementary Fig. S1B). Two independent pathologists reviewed a subset of cases to confirm a positive lymphocyte content (10%–20%) in inflamed (n = 3) and a negative lymphocyte content (0%) in depleted cases (n = 3; Supplementary Fig. S1C).
We quantified the immune and stromal-related features to catalog the TME landscape in inflamed versus depleted groups (Supplementary Table S3). In addition to T-cell inflammation (e.g., IFNγ), the inflamed group harbored significantly higher anti-inflammatory M2 macrophages, and exhausted T cells together with elevated levels of stromal cells, for example, cancer-associated fibroblasts (CAF; Fig. 1B). Although most cell types were enriched in the inflamed group, Th2 cells were significantly lower in the inflamed group (q < 0.15; Fig. 1B). Composite T-cell cluster score (quantified from the 361 genes used for clustering) correlated positively not only with genes from the antigen-processing machinery, but also with the negative regulators of T-cell function (FOXP3 and VEGFC) and immune checkpoints (VISTA, TIM-3, TIGIT, PD-L2, and LAG3; Fig. 1C), with strongest positive correlations observed for VISTA (r = 0.90), a gene shown to correlate with poor prostate cancer prognosis and increased in patients treated with ipilimumab likely due to increased M2 macrophage polarization and decreased IFNγ production (38). IHC analysis (CD8, GZMA, CD68, and PD-L1) of a subset of available cases (6 primary PCa, 6 CRPC, and 3 NEPC) where tissue was available confirmed low immune infiltration in the deserted subtype (Supplementary Fig. S1D).
To identify genomic alterations associated with T-cell inflammation, we used available WES data from 104 tumors. We did not find differences in median mutational loads between the inflamed and depleted groups (median TMB: inflamed = 1.46; depleted = 1.14 Mut/Mb; Fig. 1D). Total copy-number loads were also not different between the groups (Fig. 1D), but we noted significantly higher copy-number gains in depleted versus inflamed tumors (Wilcox P value = 0.007; Supplementary Fig. S1E). The frequency of known prostate cancer mutations (2) did not differ significantly between the two groups, but we observed trends; TP53 (33% vs. 24%) and BRCA2 (8% vs. 1%) mutations were higher in inflamed, whereas FOXA1 (11% vs. 21%), PIK3CA (0% vs. 6%), ATM (3% vs. 7%), and AR (6% vs. 12%) mutations were higher in depleted tumors (Fig. 1E; Supplementary Fig. S1F). For copy-number changes (including those in genes related to antigen presentation and immune checkpoints), only CDKN1B and ATM had significantly higher copy-number losses in the depleted compared with the inflamed groups [Fisher's P = 0.012; (CDKN1B); P = 0.038 (ATM)] but did not remain significant after multiple hypothesis correction (q > 0.1; Fig. 1F; Supplementary Fig. S1G).
In many solid tumors, tumor immune infiltration correlates with clinical outcomes (39), but such correlations are not well established in prostate cancer (10, 40, 41). In our cohort, 82 patients had overall survival (OS) data available. In this exploratory analysis, patients from the inflamed group associated with shorter OS compared with those from the depleted group (HR, 2.62; P < 0.05; Fig. 1G). We reasoned that perhaps the elevated co-presence of pro-tumor factors (e.g., VISTA, M2 macrophages, CAFs, T-cell) with cytotoxic T cells in the T-cell inflamed group antagonized antitumor T-cell function (Fig. 1B and C). Further validation is required.
NEPC is predominantly T-cell depleted
Tumors in the T-cell inflamed, and depleted groups differed by their localization and aggressiveness (X2 = 20.48, P = 0.0004; Fig. 2A; Supplementary Fig. S2A). We found that prostate tumors were relatively immune depleted (n = 127/199; 64%) compared with the benign prostate tissues (n = 9/31; 29%; Fig. 2A). To understand how this classification compared with other prostate cancers, we applied our signature to the SU2C-PCF metastatic CRPC and TCGA localized prostate cancer cohorts (Supplementary Fig. S2B and S2C). Overall, we found that the majority of samples classified as T-cell depleted (56%) in the SU2C-PCF cohort (Supplementary Fig. S2B). Similarly, 78% of the samples classified as depleted in the primary prostate cohort of the TCGA (Supplementary Fig. S2C). Analysis of other cancer types using the same method identified 59% primary lung adenocarcinomas and 51% of metastatic melanomas as T-cell inflamed (Supplementary Fig. S2C).
In our cohort, metastatic prostate cancers were mostly T-cell depleted, apart from mHN.PCa (Fig. 2A). Among the prostate cancers (primary PCa, mPCa.HN, CRPC, and NEPC), we found the highest frequency of T-cell–depleted samples in the most aggressive NEPCs (n = 27/36, 75%), with only 9 NEPC tumors classified as T-cell inflamed (iNEPC; Fig. 2A). Analysis of the SU2C-PCF dataset (5) also confirmed T-cell depletion in NEPC cases (Supplementary Fig. S1D). In our cohort, iNEPC cases had the lowest median expression of AR and were of both pure and mixed morphology (P < 0.05; Supplementary Fig. S3A; Fig. 2B). These observations revealed a minority of NEPC cases with inflammation in the otherwise T-cell-depleted NEPC.
We sought to identify additional biological processes associated with inflammation in the subset of iNEPC. We applied WGCNA to NEPC GEPs and found a gene module (MEmagenta) with strongest positive correlation with iNEPC compared with non-inflamed NEPCs (r = 0.68). This module enriched in genes of IFNγ signaling, MHC Class II antigen presentation, and PD-1 signaling (Fig. 2C; Supplementary Fig. S3B). Similarly, we identified that the extracellular matrix (ECM) organization and collagen degradation processes also correlated positively with iNEPC (r = 0.59; Supplementary Fig. S3C). Conversely, a gene module (MEpurple) with strongest negative correlation with iNEPC (r = −0.52) was enriched in pathways associated with cell cycle (e.g., M phase, G2–M transition; Fig. 2D).
In addition to the biological processes differentially enriched among the NEPC subtypes, we found that the CDKN1B loss was exclusive to non-inflamed NEPC (Fisher's P = 0.0025), as was PD-1 loss (Fisher's exact P = 0.03; Fig. 2E). The expression of PD-1 was significantly higher in iNEPC (P = 0.03) whereas expression of its ligand PD-L1 was comparable with other NEPC cases (Fig. 2F). Taken together, higher CD8+ T cells, IFNγ signaling, and higher expression of PD-1 in a subset of NEPC suggest a potential window of opportunity for ICB (42).
NEPC has higher PD-L1 expression and cell proliferation compared with other prostate cancers
We surveyed shifts in TME profiles of samples (comprised of 80 immune and stromal features) along the prostate cancer continuum. A principal component analysis (PCA) of the TME profiles showed that advanced tumors (e.g., CRPC, NEPC) had higher heterogeneity in their TME profiles whereas the localized prostate cancer and benign tissues clustered more tightly (Fig. 3A). We identified distinctions within the TME profiles of NEPC by T-cell immune class. Clustering of TME profiles averaged by cancer diagnosis showed that immune-depleted (dNEPC) clustered independently whereas iNEPC clustered with the prostate cancer tumors and benign prostate tissues (Fig. 3A).
The TME profiles averaged by cancer type showed an overall decrease in immune and stromal features from benign prostate tissues toward primary PCa, CRPC, and finally NEPC, especially in dNEPC (Fig. 3B). The only cell types enriched in dNEPC compared with other cancers were NK, T follicular helper (Tfh), and Th2 cells (Fig. 3B; Supplementary Fig. S4A). Median levels of Th2 cells increased progressively with cancer aggressiveness (Supplementary Fig. S4A). Consistent with our WGCNA, we found significantly higher expression of the MHC presentation pathway and antigen-presenting machinery genes in iNEPC compared with dNEPC (Fig. 3B).
In a survival analysis, we found that higher levels of individual TME features correlated with poor OS, a finding consistent with the outcomes observed in the T-cell inflamed versus depleted groups (Figs. 1G and 3C). Top features with negative associations with OS included ICB resistance signature (HR, 1.9) and hypoxia (HR, 1.6). Features with significantly positive associations with OS were T gamma delta (Tgd) and the NK CD56-dim cells (Fig. 3C).
In context of immunotherapy options for patients in our cohort, we reviewed the expression of prominent checkpoint genes such as the PD1–PD-L1 axis for cytotoxic T-cell regulation (43). We identified PD-L1 as the only checkpoint gene that had a significantly higher expression in NEPC compared with other prostate cancers (P < 0.05; Fig. 3D; Supplementary Fig. S4B). PD-L1 expression was higher in tumors that metastasized to lymph nodes or visceral organs compared with localized or other metastatic sites (Supplementary Fig. S4C). Despite high PD-L1, NEPCs had significantly lower expression of B2M (key for neoantigen presentation) compared with other cancers, possibly indicative of immune evasion in NEPC (Supplementary Fig. S4B).
Because NEPC tumors were broadly immune and stroma depleted compared with other prostate cancers, we sought to identify pathways that were specifically upregulated in NEPC. We quantified 50 cancer hallmark pathways (MSigDB RRID:SCR_016863; ref. 44) from GEPs using ssGSEA (Supplementary Fig. S4D). Pathways significantly upregulated in NEPC (compared with all other prostate cancers) were functionally related to cell proliferation and cancer invasiveness, that is, cell cycle G2–M checkpoint, E2F targets, Hedgehog signaling (q < 0.1; Fig. 3E; Supplementary Fig. S4D). These findings concluded that NEPC, the most aggressive variant of prostate cancer, has actively proliferating tumor cells coupled with an immune desert.
NEPC has distinct immune profile compared with SCLC
NEPC shares histological and molecular features with SCLC and because patients with SCLC are treated with ICB (7, 8, 45), we compared the immune features of NEPC and SCLC. A PCA plot of GEPs showed that NEPC tumors were clearly distinguishable from SCLC and were more heterogenous among themselves compared with prostate cancer and SCLC tumors (Fig. 4A; Supplementary Fig. S5A). This may be, in part, a reflection of the heterogeneity observed within the spectrum of NEPC, with both small-cell carcinoma and mixed small cell/adenocarcinoma histologies (46). Globally, SCLC had significantly higher expression of most immune features tested compared with NEPC, except the expression of two checkpoints PD-L1 (proposed predictor of response to pembrolizumab in SCLC; ref. 47) and CTLA-4 that was not distinguishable between SCLC and NEPC tumors (PD-L1 P = 0.93, CTLA-4 P = 0.11; Fig. 4B; Supplementary Fig. S5B–S5D).
Differential gene expression analysis between SCLC and NEPC tumors revealed that the cancer hallmark pathways associated with immune processes (e.g., IFNγ, inflammatory response, allograft rejection) were significantly upregulated in SCLC tumors (q < 0.25) compared with immune depleted NEPC (Fig. 4C; Supplementary Table S4 and Supplementary Fig. S5E). In addition, cell-cycle processes were upregulated whereas angiogenesis and hedgehog signaling genes were downregulated in SCLC compared with NEPC (Fig. 4C). The top DEGs in SCLC versus NEPC (LFC > 2, q < 0.05) contained canonical T-cell signaling markers (CD8A, CD8B, CD3D, and GZMA), whereas the bottom DEGs (LFC < −2, q < 0.05) were enriched with integrin and collagen genes, and uPAR-mediated signaling (which regulates ECM degradation and cancer cell migration and metastasis; Supplementary Tables S4 and S5; Fig. 4D).
Higher mutation burden is a predictor of ICB response (48), and in that context we found that the median mutation load in SCLC was significantly higher than NEPC as well as other prostate cancers (Fig. 4E, Supplementary Fig. S5F). Taken together, these findings suggest that despite similarities in tumor morphology and comparable PD-L1 expression, highly immune-depleted TME and relatively lower mutation load in NEPC could make it challenging to extend the clinical success of immunotherapies in SCLC to patients diagnosed with NEPC.
Discussion
Transcriptome analysis of a large cohort of patients with prostate cancer comprising localized prostate cancer, mHN.PCa, CRPC, and NEPC for signatures of T-cell infiltration delineated a significant lack of immune infiltration in prostate cancer. Although immune infiltration was limited across the different of stages of prostate cancer progression, mHN.PCa was an outlier with more than half displaying an inflamed phenotype. Further analysis of TME profiles across our cohort demonstrated a loss of reactive immune cells across the malignant castration-resistant subgroups, indicative of a poor immune response.
Of specific interest was the observation that a small subset of NEPC had increased immune infiltration. When interrogated independently, NEPC represents the most immune-depleted TME, with an absence of antigen presenting machinery or cytotoxic T cells. The immunosuppressive regulatory T-cells observed in NEPC cases are perhaps indicative of prior therapeutic interventions. Cases of iNEPC display a significantly higher expression of PD1, PDL-2, and cytolytic activity compared with other NEPC subtypes and upregulation of antigen presentation pathways. Although the number of cases was small and validation is required, this finding indicates that there might be a subset of NEPC cases that respond to immunotherapy that warrants further study.
We further characterized our NEPC cohort by comparing the immunogenomic landscape with another small-cell NE carcinoma, SCLC. Clinically, NEPC is often treated with SCLC therapies given a lack of prospective trials showing efficacy to support NEPC-specific therapeutics, and this approach is supported by National Comprehensive Cancer Network guidelines. We discovered NEPC to be significantly less immunogenic than SCLC, an observation that may be due, in part, to the higher mutation load associated with SCLC and/or a higher MHC 1 expression in a subset. When we examine the checkpoint marker PD-L1 and CTLA-4, NEPC tumors had expression levels comparable with SCLC. The implication of checkpoint inhibition in this context is significant and currently being tested in ongoing clinical trials (NCT03179410, NCT04848337, NCT04709276, and NCT03582475). Our data suggest that although NEPC is an immune-depleted tumor subtype, the expression profile of a limited subset of clinically relevant immune-related genes is closely associated with other small-cell malignancies.
There are several limitations to this study. Our findings are based on GEPs, and further studies that include multiplexed IHC or spatial-omics analyses are needed to confirm expression of identified immune markers within the tumor and stromal milieu as well as distinguish those expressed by tumor and immune cells. NEPC is a heterogenous disease, including pure small-cell carcinoma and mixed features (6); moreover, there are other emerging phenotypes within CRPC (e.g., AR-negative, NE-negative double-negative CRPC, and amphicrine CRPC) that have not been addressed here (6). The study is retrospective and furthermore, treatment response data for patients treated with ICB were not available. Finally, these analyses were not stratified by race or ethnicity, and it is possible that the TME and immune milieu differs across diverse patient populations.
By assessing the expression profiles of NEPC within the paradigms of prostate malignancies and small-cell lung carcinoma, we have revealed distinct immune-related GEPs related to NEPC. These findings may have implications for future immunotherapy strategies and for biomarker development.
Authors' Disclosures
V. Conteduca reports personal fees from Janssen, Astellas, Merck, AstraZeneca, Amgen, Eisai, Ipsen, Bristol-Myers Squibb, and Bayer outside the submitted work. O. Elemento reports other support from Owkin and Freenome; personal fees and other support from Pionyr; grants, personal fees, and other support from Volastra Therapeutics and OneThree Biotech; and personal fees from Champions Oncology during the conduct of the study. H. Beltran reports grants from Janssen, Novartis, Bristol Myers Squibb, and Circle Pharma; personal fees from Merck, Pfizer, Foundation Medicine, Blue Earth Diagnostics, Amgen, Bayer, Oncorus, Loxo, Sanofi, Curie Therapeutics, and AstraZeneca; and grants and personal fees from Daiichi Sankyo outside the submitted work. No disclosures were reported by the other authors.
Authors' Contributions
B. Bhinder: Conceptualization, data curation, formal analysis, investigation, writing–original draft, writing–review and editing. A. Ferguson: Conceptualization, data curation, formal analysis, investigation, writing–original draft, writing–review and editing. M. Sigouros: Project administration, writing–review and editing. M. Uppal: Investigation, writing–review and editing. A.G. Elsaeed: Validation, writing–review and editing. R. Bareja: Investigation. H. Alnajar: Investigation. K.W. Eng: Investigation. V. Conteduca: Investigation. A. Sboner: Investigation. J.M. Mosquera: Investigation. O. Elemento: Conceptualization, resources, supervision, funding acquisition, investigation, writing–original draft, project administration, writing–review and editing. H. Beltran: Conceptualization, resources, supervision, funding acquisition, investigation, writing–original draft, project administration, writing–review and editing.
Acknowledgments
H. Beltran is supported by the Prostate Cancer Foundation, Department of Defense (W81XWH-17–1-0653), and NIH/NCI (R37CA241486, P50-CA211024).
The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).