Immune profiling of the tumour microenvironment in prostate cancer

Prostate cancer is the most common cancer among men in the UK and is characterised by large biological and clinical heterogeneity. There is an urgent need for better-personalised patient stratification, for example in accurately identifying patients with regional lymph node metastasis. Nodal involvement negatively impacts on patient survival outcomes and the current pre-operative staging tools to determine the need for extended pelvic lymph node dissection at time of radical prostatectomy are far from precise. The primary tumour immune microenvironment influences tumour immune editing and therefore disease progression. The primary aim of this research was to investigate the in situ phenotype of prostate cancer tumour infiltrating immune cells and determine their potential as biomarkers for regional lymph node invovlement and further explore possible underlying mechanisms for their distribution. The discovery tissue microarray comprised of index lesions from 94 patients undergoing radical prostatectomy and pelvic node dissection (50 with and 44 without histologic evidence of pelvic nodal disease respectively, referred to as LN+ and LN- thereafter). Two multiplex immunofluorescence panels were optimised to comprehensively characterise the immune microenvironment: (1) The macrophage and B cell panel includes CD68, CD163, CD20, AE1/3 (PanCK) and DAPI and (2) The T lymphocytic panel assays for CD4, CD8, FoxP3, PD-1, AE1/3 and DAPI. The macrophage (CD68, CD163+), T (CD8+, CD4+) and B (CD20+) cell immune cell subpopulations within the malignant epithelium and associated stroma were measured and correlated to the nodal status. Stromal infiltration by M1-like macrophages (CD68+CD163-) (p=0.047), CD8 effector (CD8+FoxP3-PD-1-) (p=0.008) and CD4 effector (CD4+FoxP3-PD-1-) T cells (p=0.0003, Mann Whitney test) were lower in LN+ patients. Stromal CD4 effector immune cell density remained a statistically significant independent predictor of lymph node spread in multivariate regression analysis (OR= 0.15, p=0.004). Additionally, in an independent validation cohort of 184 radical prostatectomy specimens, stromal CD4 effector immune cell density predicted the presence of nodal metastasis (OR=0.26, p=0.0004). Addition of stromal CD4 effector T cell density to currently used clinicopathological factors, namely T stage, PSA level, Gleason score and percentage of tumour positive cores, improved the predictive accuracy of current nomograms (from 63.5% to 76.8%, p<0.0001). Tumour infiltrating immune cells did not however correlate with common molecular alterations of prostate cancer such as ERG overexpression and PTEN deletion. Transcriptomic analysis (by HTG EdgeSeq) of the tumour microenvironment was performed to assay 1,041 host immune response related genes. Surprisingly, I did not observe significant differences in the expression levels of adhesion molecules or chemokines (common regulators of immune cell migration) between LN+ and LN- cases. Instead, there was significant upregulation (FC>1.5, adj p value <0.05) of extracellular matrix components (collagen I, collagen III, fibronectin 1) in LN+ tumours, suggesting increased extracellular matrix fibrosis to be associated with reduced T lymphocytic infiltration and tumour immune evasion. Increased collagen III and fibronectin 1 protein expression were confirmed in LN+ patients. Collagen I had increased density score (by second generation harmonic), but not overall abundance, in LN+ patients, eluding to a disorganised stroma with increased cross-linking and elongated fibres. B7-H3 is a newly discovered member of the B7 family of immune checkpoint molecules with both immune and non-immune functions. I investigated the relationship of B7-H3 to the tumour microenvironment as well as its non-immune functions in prostate cancer. Contrast to PD-1, high B7-H3 expression correlated with worse clinicopathological patient features: higher T stage (p<0.0001), perineural invasion (p=0.01) and lymph node spread (p=0.0006). Furthermore, there was significant decrease in migration and invasion in vitro following suppressed B7-H3 expression in multiple human prostate cancer cell lines. RNA sequencing identified extracellular space chemotactic cytokines and their receptors to be highly downregulated genes in PC3M cells with B7-H3 knocked out. Future experiments will investigate the mechanistic downstream pathways of this phenotype and further evaluate the role of B7-H3 in metastasis in vivo. Data presented in this thesis reveal differences in the immune infiltrates, particularly CD4 effector (CD4+FoxP3-PD-1-) T cells between LN+ and LN- patients. Prospective clinical studies are needed to test the predictive value of stromal CD4 effector T cell density in diagnostic prostatic biopsies for regional nodal disease. The role of increased extracellular matrix components in determining tumour immune infiltrates also warrants additional research

Analysis of Prostate Cancer Tumor Microenvironment Identifies Reduced Stromal CD4 Effector T-cell Infiltration in Tumors with Pelvic Nodal Metastasis.

BACKGROUND: Pelvic nodal metastasis in prostate cancer impacts patient outcome negatively. OBJECTIVE: To explore tumor-infiltrating immune cells as a potential predictive tool for regional lymph node (LN) metastasis. DESIGN SETTING AND PARTICIPANTS: We applied multiplex immunofluorescence and targeted transcriptomic analysis on 94 radical prostatectomy specimens in patients with (LN+) or without (LN-) pelvic nodal metastases. Both intraepithelial and stromal infiltrations of immune cells and differentially expressed genes (mRNA and protein levels) were correlated with the nodal status. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The identified CD4 effector cell signature of nodal metastasis was validated in a comparable independent patient cohort of 184 informative cases. Patient outcome analysis and decision curve analysis were performed with the CD4 effector cell density-based signature. RESULTS AND LIMITATIONS: In the discovery cohort, both tumor epithelium and stroma from patients with nodal metastasis had significantly lower infiltration of multiple immune cell types, with stromal CD4 effector cells highlighted as the top candidate marker. Targeted gene expression analysis and confirmatory protein analysis revealed key alteration of extracellular matrix components in tumors with nodal metastasis. Of note, stromal CD4 immune cell density was a significant independent predictor of LN metastasis (odds ratio [OR] = 0.15, p = 0.004), and was further validated as a significant predictor of nodal metastasis in the validation cohort (OR = 0.26, p < 0.001). CONCLUSIONS: Decreased T-cell infiltrates in the primary tumor (particularly CD4 effector cells) are associated with a higher risk of LN metastasis. Future evaluation of CD4-based assays on prostate cancer diagnostic biopsy materials may improve selection of at-risk patients for the treatment of LN metastasis. PATIENT SUMMARY: In this report, we found that cancer showing evidence of cancer metastasis to the lymph nodes tends to have less immune cells present within the tumor. We conclude that the extent of immune cells present within a prostate tumor can help doctors determine the most appropriate treatment plan for individual patients

BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (Pten BRF1 ) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In Pten BRF1 tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis

Demographic, clinical, and pathological features of early onset pancreatic cancer patients.

BACKGROUND: Early onset pancreatic cancer (EOPC), i.e. pancreatic ductal adenocarcinoma (PDAC) occurring in patients below 50 years of age, is rare and there is limited information regarding risk factors, molecular basis and outcome. This study aimed to determine the demographic and clinicopathological features and survival figures for EOPC. METHODS: A retrospective analysis of patients treated at the Royal London Hospital for PDAC between September 2004 and September 2015 was performed. Data on demographics, risk factors, presentation, pathological features, treatment and survival outcome were compared in EOPC and older PDAC patients. RESULTS: Of 369 PDAC cases identified, 35 (9.5%) were EOPC. Compared to older patients, EOPC patients were more frequently male (71% vs 54%, p = 0.043) and less commonly of British origin (37% vs 70%, p = 0.002). There was no significant difference regarding the prevalence of any of the risk factors known to be associated with older PDAC patients. Fewer EOPC patients presented with resectable disease (23% vs 44%, p = 0.015) and more received adjuvant chemo/radiotherapy (60% vs 46%, p = 0.008). The overall median survival and stage specific survival did not differ significantly between the two groups, although a longer survival for localized disease was seen in EOPC patients (25 months (12.9-37, 95%CI) vs 13 months (10.5-15.5 95%CI) for older PDAC patients). CONCLUSIONS: The EOPC patients had different demographics and were more likely than their older PDAC counterparts to be male. Typically they presented with more advanced disease, received more aggressive treatment, and had on overall similar survival outcome

Molecular characteristics of early‐onset pancreatic ductal adenocarcinoma

The median age of patients with pancreatic ductal adenocarcinoma (PDAC) at diagnosis is 71 years; however, around 10% present with early‐onset pancreatic cancer (EOPC), i.e., before age 50. The molecular mechanisms underlying such an early onset are unknown. We assessed the role of common PDAC drivers (KRAS, TP53, CDKN2A and SMAD4) and determined their mutational status and protein expression in 90 formalin‐fixed, paraffin‐embedded tissues, including multiple primary and matched metastases, from 37 EOPC patients. KRAS was mutated in 88% of patients; p53 was altered in 94%, and p16 and SMAD4 were lost in 86% and 71% of patients, respectively. Meta‐synthesis showed a higher rate of p53 alterations in EOPC than in late‐onset PDAC (94% vs. 69%, P = 0.0009) and significantly higher loss of SMAD4 (71% vs. 44%, P = 0.0025). The majority of EOPC patients accumulated aberrations in all four drivers; in addition, high tumour heterogeneity was observed across all tissues. The cumulative effect of an exceptionally high rate of alterations in all common PDAC driver genes combined with high tumour heterogeneity suggests an important mechanism underlying the early onset of PDAC

SLFN5 regulates LAT1-mediated mTOR activation in castration-resistant prostate cancer

Androgen-deprivation therapy (ADT) is the standard of care for treatment of non-resectable prostate cancer (PCa). Despite high treatment efficiency, most patients ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we performed a comparative proteomic analysis of three in vivo, androgen receptor (AR)-responsive orthograft models of matched hormone-naive PCa and CRPC. Differential proteomic analysis revealed that distinct molecular mechanisms, including amino acid (AA) and fatty acid (FA) metabolism, are involved in the response to ADT in the different models. Despite this heterogeneity, Schlafen family member 5 (SLFN5) was identified as an AR-regulated protein in CRPC. SLFN5 expression was high in CRPC tumors and correlated with poor patient outcome. In vivo, SLFN5 depletion strongly impaired tumor growth in castrated conditions. Mechanistically, SLFN5 interacted with ATF4 and regulated the expression of LAT1, an essential AA transporter. Consequently, SLFN5 depletion in CRPC cells decreased intracellular levels of essential AA and impaired mTORC1 signalling in a LAT1-dependent manner. These results confirm that these orthograft models recapitulate the high degree of heterogeneity observed in CRPC patients and further highlight SLFN5 as a clinically relevant target for CRPC

Activation of β-catenin cooperates with loss of Pten to drive AR-independent castration-resistant prostate cancer

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Prostate cancer patients with alterations in canonical WNT pathway genes, which lead to β-catenin activation, are refractory to AR-targeted therapies. Here using clinically relevant murine prostate cancer models, we investigated the significance of β-catenin activation in prostate cancer progression and treatment resistance. β-catenin activation, independent of the cell-of-origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumours with β-catenin activation relied on the non-canonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of β-catenin activation, by mediating nuclear localization of NFƙBp65 and β-catenin. Overall, WNT/β-catenin and AR signalling are reciprocally inhibited. Therefore, inhibiting WNT/β-catenin signalling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes