CD38 in Advanced Prostate Cancers

Abstract

Background: CD38, a druggable ectoenzyme, is involved in the generation of adenosine, which is implicated in tumour immune evasion. Its expression and role in prostate tumour-infiltrating immune cells (TIICs) have not been elucidated.

Objective: To characterise CD38 expression on prostate cancer (PC) epithelial cells and TIICs, and to associate this expression with clinical outcomes.

Design, setting, and participants: RNAseq from 159 patients with metastatic castration-resistant prostate cancer (mCRPC) in the International Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF) cohort and 171 mCRPC samples taken from 63 patients in the Fred Hutchinson Cancer Research Centre cohort were analysed. CD38 expression was immunohistochemically scored by a validated assay on 51 castration-resistant PC (CRPC) and matching, same-patient castration-sensitive PC (CSPC) biopsies obtained between 2016 and 2018, and was associated with retrospectively collected clinical data. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: mCRPC transcriptomes were analysed for associations between CD38 expression and gene expression signatures. Multiplex immunofluorescence determined CD38 expression in PC biopsies. Differences in CD38⁺ TIIC densities between CSPC and CRPC biopsies were analysed using a negative binomial mixed model. Differences in the proportions of CD38⁺ epithelial cells between non-matched benign prostatic epithelium and PC were compared using Fisher's exact test. Differences in the proportions of biopsies containing CD38⁺ tumour epithelial cells between matched CSPC and CRPC biopsies were compared by McNemar's test. Univariable and multivariable survival analyses were performed using Cox regression models.

Results and limitations: CD38 mRNA expression in mCRPC was most significantly associated with upregulated immune signalling pathways. CD38 mRNA expression was associated with interleukin (IL)-12, IL-23, and IL-27 signalling signatures as well as immunosuppressive adenosine signalling and T cell exhaustion signatures. CD38 protein was frequently expressed on phenotypically diverse TIICs including B cells and myeloid cells, but largely absent from tumour epithelial cells. CD38⁺ TIIC density increased with progression to CRPC and was independently associated with worse overall survival. Future studies are required to dissect TIIC CD38 function.

Conclusions: CD38⁺ prostate TIICs associate with worse survival and immunosuppressive mechanisms. The role of CD38 in PC progression warrants investigation as insights into its functions may provide rationale for CD38 targeting in lethal PC.

Patient summary: CD38 is expressed on the surface of white blood cells surrounding PC cells. These cells may impact PC growth and treatment resistance. Patients with PC with more CD38-expressing white blood cells are more likely to die earlier.

Keywords: Adenosine pathway; B lymphocyte; CD38; Castration-resistant prostate cancer; Inflammation; Myeloid cell; Plasmacyte; Prostate cancer; T cell exhaustion; Tumour microenvironment.

Fig. 1

Overview of the clinical cohort. The patient cohort studied included 51 treatment-naïve, castration-sensitive prostate cancer biopsies and 51 same-patient, castration-resistant prostate cancer biopsies. CRPC = castration-resistant prostate cancer; CSPC = castration-sensitive prostate cancer; TURP = transurethral resection of the prostate.

Fig. 2

Transcriptome analyses associating CD38 mRNA expression with immunoregulatory signalling pathway transcripts. (A and B) Unbiased transcriptome analyses of RNA-sequencing data from 159 patient biopsies in the SU2C/PCF mCRPC cohort for the association between CD38 and 200 PID signalling pathways. Red dots indicate immune signalling pathways and green dots nonimmunoregulatory pathways. CD38 was most significantly associated with the expression of multiple immune signalling pathway genes, and the ten most significant associations are shown (B). SU2C/PCF mCRPC transcriptome analyses of RNA-sequencing data from 159 patient biopsies for associations between CD38 and (C) a 14-gene adenosine signature, (D) an 18-gene adenosine signature, genes associated with CD8⁺ T cell exhaustion, (F) progenitor exhausted CD8⁺ T cell signature, and (G) terminally exhausted CD8⁺ T cell signature. The p values were calculated using linear regression analysis. mCRPC = metastatic castration-resistant prostate cancer; SU2C/PCF = International Stand Up To Cancer/Prostate Cancer Foundation.

Fig. 3

CD38 expression on prostate cancer epithelial cells and benign epithelial cells. (A) Representative immunofluorescence image showing CD38 expression in benign prostatic epithelium (× 200 magnification, scale bar 100 µm). (B) Representative images showing CD38 expression in prostate cancer epithelial cells. Top row shows examples of CD38⁺ tumours and bottom row shows CD38⁻ tumours (200× magnification, scale bar 50 µm). (C) CD38 expression was more frequent on benign prostatic epithelial cells (35.8%) than on malignant epithelial cells (7.7%). Fisher’s exact test, p < 0.001. (D) Tumour epithelial cell CD38 expression was more frequent in CSPC biopsies (9.8%; 5/51) than in CRPC biopsies (3.9%; 2/51). McNemar’s test, p = 0.45, not significant. BMT = bone marrow trephine; CRPC = castration-resistant prostate cancer; CSPC = castration-sensitive prostate cancer; LN = lymph node; mCRPC = metastatic castration-resistant prostate cancer; Prostate Bx = prostate biopsy.

Fig. 4

CD38 is expressed on a heterogeneous population of TIICs coexpressing myeloid and lymphoid markers. (A) Representative immunofluorescence images showing subpopulations of CD38⁺ tumour-infiltrating immune cells and PMN-MDSC. White arrows point to a cell with the leucocyte’s phenotype specified on each row (200× magnification, scale bar 100 µm). (B) Representative multispectral six-colour immunofluorescence image of an mCRPC lymph node biopsy stained with DAPI (blue), CD38 (cyan), CD11b (magenta), CD15 (green), CD33 (red), and EpCAM (yellow; 200× magnification, scale bar 100 µm). (C) Proportion of immune cell subpopulations expressing CD38 in CSPC and CRPC biopsies (n = 51). (D) Representative immunofluorescence images of mCRPC biopsy showing subpopulations of CD38⁺ coexpressing CD79a, both CD33 and CD79a, or CD3. White arrows point to a cell with the leucocyte’s phenotype specified on each row (200× magnification, scale bar 100 µm). (E) Five-colour immunofluorescence micrograph of mCRPC biopsy, with areas of stroma and tumour demarcated by white lines. Image staining for DAPI (grey), CD38 (green), CD79a (red), CD33 (yellow), and CD3 (blue). White arrows indicating representative phenotypes expressing CD38 (200× magnification, scale bar 100 μm. (F, G) Proportion of CD38⁺ tumour-infiltrating immune cells expressing other immune cell surface markers in 4 CSPC biopsies. CRPC = castration-resistant prostate cancer; CSPC = castration-sensitive prostate cancer; mCRPC = metastatic castration-resistant prostate cancer; PMN-MDSC = polymorphonuclear myeloid-derived suppressor cells; TIIC = tumour-infiltrating immune cell.

Fig. 5

CD38⁺ TIICs increased with the emergence of castration resistance and is associated with worse overall survival. (A) Representative “pseudo-DAB” images showing an increase in CD38⁺ tumour-infiltrating immune cells as tumours progressed from CSPC to CRPC in matched, same-patient biopsies. Dark-field immunofluorescence images were transformed to pseudo bright-field immunohistochemistry images to facilitate visualisation (200× magnification, scale bar 100 µm). CD38⁺ tumour-infiltrating immune cell density in the (B) tumour compartment and (C) stromal compartment of 51 matched, same-patient CSPC and CRPC biopsies. The median and interquartile ranges (IQRs) for intratumoural CD38⁺ immune cell density (cell/mm²) for CSPC (median; IQR: 0.77; 0.00–7.80) and CRPC (median; IQR: 3.49; 0.00–18.33) are shown. The median and IQRs for stromal CD38⁺ tumour-infiltrating immune cell density (cell/mm²) for CSPC (median; IQR: 11.66; 2.53–44.60) and CRPC (median: 31.13; 3.32–111.06) are shown. The p values were calculated using negative binomial mixed model (CD38⁺ tumour-infiltrating immune cell density, intratumoural p = 0.03; stromal p = 0.03. (D) Kaplan-Meier curves of overall survival from the time of initial diagnosis for CD38⁺ tumour-infiltrating immune cell density in CSPC samples. (E) Kaplan-Meier curves of overall survival from time of CRPC diagnosis for CD38⁺ tumour-infiltrating immune cell density at CRPC biopsy. Hazard ratio (HR) with 95% confidence intervals (CIs) and p values for the univariate Cox survival model are shown. (F) Multivariable analysis of overall survival (n = 36) from the time of initial diagnosis for log-transformed CD38⁺ immune cell density, log-transformed PSA at the time of diagnosis, Gleason score, and the presence of metastatic disease at the time of diagnosis. HR with 95% CI and p values for multivariable Cox survival models are shown. (G) Kaplan-Meier curves of overall survival from the time of CRPC biopsy for CD38 mRNA expression (n = 159; dichotomised by the median: 0.64 FPKM). HR with 95% CIs and p value for the univariate Cox survival model are shown. BMT = bone marrow trephine; CRPC = castration-resistant prostate cancer; CSPC = castration-sensitive prostate cancer; FPKM = fragments per kilobase of transcript per million mapped reads; LN = lymph node; Prostate Bx = prostate biopsy; PSA = prostate-specific antigen; TIIC = tumour-infiltrating immune cell; TURP = transurethral resection of the prostate.