Protocol for a prospective study evaluating circulating tumour cells status to predict radical prostatectomy treatment failure in localised prostate cancer patients (C-ProMeta-1).

BACKGROUND: Treatment decisions in prostate cancer (PCa) rely on disease stratification between localised and metastatic stages, but current imaging staging technologies are not sensitive to micro-metastatic disease. Circulating tumour cells (CTCs) status is a promising tool in this regard. The Parsortix® CTC isolation system employs an epitope-independent approach based on cell size and deformability to increase the capture rate of CTCs. Here, we present a protocol for prospective evaluation of this method to predict post radical prostatectomy (RP) PCa cancer recurrence. METHODS: We plan to recruit 294 patients diagnosed with unfavourable intermediate, to high and very high-risk localised PCa. Exclusion criteria include synchronous cancer diagnosis or prior PCa treatment, including hormone therapy. RP is performed according to the standard of care. Two blood samples (20 ml) are collected before and again 3-months after RP. The clinical team are blinded to CTC results and the laboratory researchers are blinded to clinical information. Treatment failure is defined as a PSA ≥ 0.2 mg/ml, start of salvage treatment or imaging-proven metastatic lesions. The CTC analysis entails enumeration and RNA analysis of gene expression in captured CTCs. The primary outcome is the accuracy of CTC status to predict post-RP treatment failure at 4.5 years. Observed sensitivity, positive and negative predictive values will be reported. Specificity will be presented over time. DISCUSSION: CTC status may reflect the true potential for PCa metastasis and may predict clinical outcomes better than the current PCa progression risk grading systems. Therefore establishing a robust biomarker for predicting treatment failure in localized high-risk PCa would significantly enhance guidance in treatment decision-making, optimizing cure rates while minimizing unnecessary harm from overtreatment. TRIAL REGISTRATION: ISRCTN17332543.

The interaction of p53 and DNA repair gene mutations and their impact on tumor mutation burden and immune response in human malignancies.

P53 suppresses tumorigenesis through multiple cellular functions/mechanisms, including genomic stability surveillance. Recently, it has also be reported for its role in cancer immune response modulation. Deficiency in DNA repair pathways lead to the accumulation of genomic alterations and tumor mutation burden and in consequence resulting in the activation of immune response. We investigated the interaction of p53 and DNA repair gene mutations and their impact on tumor mutation burden and immune response in human malignancies by mining cBioPortal data of a range of human cancers. We found that in the majority of human cancers, p53 mutations are equally distributed between DNA repair gene mutation positive and negative cases and in a number of human cancers, p53 and DNA repair gene mutations have a tendency of co-occurrence. Only in colorectal cancer, there is a tendency of 'mutual exclusivity' of mutations in p53 and DNA repair genes. In most tumors, p53 and DNA repair gene mutations have synergistic/additive effect in increasing tumor mutation burden, but not in colorectal cancer where they are mutually exclusive. The impact of p53 and DNA repair gene mutations and their interaction on tumor microenvironment immune cells are complex and tumor type specific and not always correlated with tumor mutation burden. In colorectal cancers, these two types of mutations resulted in similar immune cell subpopulation changes and in tumors where the mutations have a tendency of co-occurrence, p53 showed dominant roles on immune response, although they can also counter-act each other for their effect on certain immune cell subtypes.

The potential of using circulating tumour cells and their gene expression to predict docetaxel response in metastatic prostate cancer.

Background: Docetaxel improves overall survival (OS) in castration-resistant prostate cancer (PCa) (CRPC) and metastatic hormone-sensitive PCa (mHSPC). However, not all patients respond due to inherent and/or acquired resistance. There remains an unmet clinical need for a robust predictive test to stratify patients for treatment. Liquid biopsy of circulating tumour cell (CTCs) is minimally invasive, can provide real-time information of the heterogeneous tumour and therefore may be a potentially ideal docetaxel response prediction biomarker. Objective: In this study we investigate the potential of using CTCs and their gene expression to predict post-docetaxel tumour response, OS and progression free survival (PFS). Methods: Peripheral blood was sampled from 18 mCRPC and 43 mHSPC patients, pre-docetaxel treatment, for CTC investigation. CTCs were isolated using the epitope independent Parsortix® system and gene expression was determined by multiplex RT-qPCR. We evaluated CTC measurements for post-docetaxel outcome prediction using receiver operating characteristics and Kaplan Meier analysis. Results: Detection of CTCs pre-docetaxel was associated with poor patient outcome post-docetaxel treatment. Combining total-CTC number with PSA and ALP predicted lack of partial response (PR) with an AUC of 0.90, p= 0.037 in mCRPC. A significantly shorter median OS was seen in mCRPC patients with positive CTC-score (12.80 vs. 37.33 months, HR= 5.08, p= 0.0005), ≥3 total-CTCs/7.5mL (12.80 vs. 37.33 months, HR= 3.84, p= 0.0053), ≥1 epithelial-CTCs/7.5mL (14.30 vs. 37.33 months, HR= 3.89, p= 0.0041) or epithelial to mesenchymal transitioning (EMTing)-CTCs/7.5mL (11.32 vs. 32.37 months, HR= 6.73, p= 0.0001). Significantly shorter PFS was observed in patients with ≥2 epithelial-CTCs/7.5mL (7.52 vs. 18.83 months, HR= 3.93, p= 0.0058). mHSPC patients with ≥5 CTCs/7.5mL had significantly shorter median OS (24.57 vs undefined months, HR= 4.14, p= 0.0097). In mHSPC patients, expression of KLK2, KLK4, ADAMTS1, ZEB1 and SNAI1 was significantly associated with shorter OS and/or PFS. Importantly, combining CTC measurements with clinical biomarkers increased sensitivity and specificity for prediction of patient outcome. Conclusion: While it is clear that CTC numbers and gene expression were prognostic for PCa post-docetaxel treatment, and CTC subtype analysis may have additional value, their potential predictive value for docetaxel chemotherapy response needs to be further investigated in large patient cohorts.

Noninvasive Detection of Clinically Significant Prostate Cancer Using Circulating Tumor Cells.

PURPOSE: Prostate specific antigen testing results in unnecessary biopsy and over diagnosis with consequent overtreatment. Tissue biopsy is an invasive procedure associated with significant morbidity. More accurate noninvasive or minimally invasive diagnostic approaches should be developed to avoid unnecessary prostate biopsy and over diagnosis. We investigated the potential of using circulating tumor cell analysis in cancer diagnosis, particularly to predict clinically significant prostate cancer in prebiopsy cases. MATERIALS AND METHODS: We enrolled 155 treatment naïve patients with prostate cancer and 98 before biopsy for circulating tumor cell enumeration. RNA was extracted from circulating tumor cells of 184 patients for gene expression analysis. The Kruskal-Wallis and Spearman rank tests, multivariate logistic regression and the random forest method were applied to assess the association of circulating tumor cells with aggressive prostate cancer. RESULTS: Of patients with localized prostate cancer 54% were scored as having positive circulating tumor cells, which was associated with a higher Gleason score (p=0.0003), risk group (p <0.0001) and clinically significant prostate cancer (p <0.0001). In the prebiopsy group a positive circulating tumor cell score combined with prostate specific antigen predicted clinically significant prostate cancer (AUC 0.869). A 12-gene panel prognostic for clinically significant prostate cancer was also identified. When combining the prostate specific antigen level, the circulating tumor cell score and the 12-gene panel, the AUC of clinically significant prostate cancer prediction was 0.927. Adding those data to cases with available multiparametric magnetic resonance imaging data significantly increased prediction accuracy (AUC 0.936 vs 0.629). CONCLUSIONS: Circulating tumor cell analysis has the potential to significantly improve patient stratification by prostate specific antigen and/or multiparametric magnetic resonance imaging for biopsy and treatment.

The Identification of Plasma Exosomal miR-423-3p as a Potential Predictive Biomarker for Prostate Cancer Castration-Resistance Development by Plasma Exosomal miRNA Sequencing.

Castration-resistant prostate cancer (CRPC) is the major cause of death from prostate cancer. Biomarkers to improve early detection and prediction of CRPC especially using non-invasive liquid biopsies could improve outcomes. Therefore, we investigated the plasma exosomal miRNAs associated with CRPC and their potential for development into non-invasive early detection biomarkers for resistance to treatment. RNA-sequencing, which generated approximately five million reads per patient, was performed to identify differentially expressed plasma exosomal miRNAs in 24 treatment-naive prostate cancer and 24 CRPC patients. RT-qPCR was used to confirm the differential expressions of six exosomal miRNAs, miR-423-3p, miR-320a, miR-99a-5p, miR-320d, miR-320b, and miR-150-5p (p = 7.3 × 10-8, 0.0020, 0.018, 0.0028, 0.0013, and 0.0058, respectively) firstly in a validation cohort of 108 treatment-naive prostate cancer and 42 CRPC patients. The most significant differentially expressed miRNA, miR-423-3p, was shown to be associated with CRPC with area under the ROC curve (AUC) = 0.784. Combining miR-423-3p with prostate-specific antigen (PSA) enhanced the prediction of CRPC (AUC = 0.908). A separate research center validation with 30 treatment-naive and 30 CRPC patients also confirmed the differential expression of miR-423-3p (p = 0.016). Finally, plasma exosomal miR-423-3p expression in CRPC patients was compared to 36 non-CRPC patients under androgen depletion therapy, which showed significantly higher expression in CRPC than treated non-CRPC patients (p < 0.0001) with AUC = 0.879 to predict CRPC with no difference between treatment-naive and treated non-CRPC patients. Therefore, our findings demonstrate that a number of plasma exosomal miRNAs are associated with CRPC and miR-423-3p may serve as a biomarker for early detection/prediction of castration-resistance.