1. Background:

Cancers of the prostate are the second most commonly occurring cancer in men and the fourth most commonly occurring cancer. Risk factors for prostate cancers include age, hereditary factors (e.g., germline mutations in BRCA gene), diet, obesity and inflammation of the prostate. The majority (~95%) of prostate malignancies are adenocarcinomas whereas the remainder comprise of urothelial carcinoma, basal cell carcinoma, small cell carcinoma, lymphoma and sarcomas. Globally, there were ~1,300,000 new cases in 2018 accounting for 7.1% of all cancers¹. Prostate cancers also contributed to 360,000 deaths^2,3, accounting for 3.8% of all annual cancer related mortalities. While the global age standardized incidence rates (ASR) for prostate cancers were around 30%¹, the same for India was <10.6%¹; however, projections indicate a potential doubling in number of cases by 2020¹. It is also hypothesized that the lower incidences in India, especially among non-urban population, may be due to lower rate of detection rather than a low incidence rate¹. Prostate cancers remain undetected for extended periods since they are generally slow-growing and asymptomatic until advanced stages. Though advances in modalities for early screening, as well as an increased awareness, have led to an increase in global ASR for incidence, developing regions continue to exhibit the disparity of relatively low ASR for incidence.

2.        Study Rationale

Limitations of the Current Gold Standard in Diagnosis and Monitoring

Current modalities for screening and diagnosis of prostate cancers include digital rectal examination (DRE), prostate biopsies, serological markers as well as radiological imaging. The DRE⁴ checks for palpable growths in or enlargement of the prostate gland via the rectum. However, the absence of a palpable hard mass or the failure of DRE to detect the same does not entirely rule out prostate malignancies. While the DRE is a non-invasive procedure, it may be associated with moderate risks of pain, discomfort and bleeding. Prostate biopsies are obtained surgically, e.g., ultrasound or magnetic resonance imaging (MRI)-guided biopsy⁵, or via fine needle-aspiration cytology⁶ (FNAC), where the needle is inserted via the rectum (transrectal), urethra (transurethral) or perineum (transperineal), to obtain tissue samples, for histopathological analysis and determination of the Gleason Score⁷ based on visual assessment of tumor cell differentiation. However prostate biopsies, especially FNAC, may be prone to false negatives and multiple sampling may be required for unambiguous establishment of presence or absence of malignancy. Biopsies are associated with risks of pain, bleeding and infection at the site of puncture. It has also been hypothesized that viable tumor cells may be mechanically displaced into the vasculature during biopsies (or FNAC), and dissemination of these circulating tumor cells (CTCs) may contribute to development of distal metastatic lesions. Radiological methods such as computed tomography (CT) and positron emission tomography-computed tomography (PET-CT) scans have been considered the de facto gold standard for detection and determination of extent of various solid organ malignancies. However, radiological methods of detection are associated with risks of exposure to ionizing radiation from oral / IV contrast as well as non-ionizing radiation from the instrument, as well as an inability to detect tumors less than 4 mm in diameter^8,9. PET-CT with ¹⁸F-fluorodeoxyglucose (FDG) tracer frequently encounters tracer artifacts due to higher FDG uptake in non-malignant tissue (e.g., inflammatory tissue / surgical scars), as well as due to retention in visceral organs such as the kidney and urinary bladder – these artifacts pose a challenge to discerning true metastases from background uptake. Additionally, in case of prostate malignancies, the accumulation of excreted tracer in the urinary bladder prevents analysis of the prostate-specific uptake due to anatomical proximity. While Prostate-Specific Membrane Antigen (PSMA)-PET¹⁰ scan appears to offer improved specificity over FDG-PET-CT, clinical studies have not yet unambiguously established immunity of PSMA-PET from the same confounding factors that are routinely encountered in other radiological methods.

The identification of the serum Prostate Specific Antigen (PSA) and its association with prostate malignancies led to the development of blood-based rapid in vitro diagnostic tests for determination of malignancy. This non-invasive diagnostic procedure, based on analysis of 1-2 mL of peripheral blood, offered obvious advantages (and reduced risks) as compared to invasive procedures such as biopsies / FNAC. However, elevated PSA levels were also found to be associated with several noncancerous conditions, including age-related Benign Prostatic Hyperplasia (BPH) contributing to a reduced specificity¹¹.

Distinct Advantages of Liquid Biopsy-based Approaches

Liquid biopsies open multiple opportunities in diagnosis of prostate malignancies due its non-invasive sampling method. It enables longitudinal monitoring of disease evolution and treatment response. Further, it helps to address tumor heterogeneity as the nucleic acids analysed in liquid biopsy are released from all areas of tumor. Additionally, due to advances in the sequencing technology, the scope of molecular analysis conducted in liquid biopsy as increased and has potential to reveal all diagnostic, prognostic and therapy relevant molecular alterations. Like other malignant neoplasms, prostate tumor cells release intracellular components into circulation. Tumor-derived biomarkers include circulating tumor cells (CTCs), extracellular vesicles (e.g., exosomes) and cell-free nucleic acids which accumulate in plasma, serum and/or cerebrospinal fluid. Circulating cell-free tumor DNA (ctDNA) are fragments of DNA, released from dying tumor cells. The presence of ctDNA has been correlated with overall tumor burden and disease activity. Presence of certain unique molecular signatures can help to distinguish between malignant / non-malignant disorders and has potential to indicate morphological type of malignant neoplasm. Much like non-malignant cells, viable tumor cells secrete extracellular vesicles called exosomes tumor cell derived mRNA, miRNA, other nucleic acids and proteins. These circulating biomarkers may be useful as easily accessible diagnostic, prognostic and/or predictive biomarkers to guide patient management. Thereby, this approach may help to circumvent problems related to tumor heterogeneity and sampling error at the time of diagnosis. Liquid biopsies allow for serial monitoring of treatment responses and of changes in the molecular characteristics of the prostate malignancy over time. Liquid biopsy provides dynamic information not only regarding tumor burden to monitor disease progression and treatment response, but also regarding genetic profile to enable changes in management to match a constantly evolving tumor.

ProState^TM – an Integrative Platform for Prostate Malignancies

ProStateâ„¢ is a liquid biopsy platform that detects prostate cancer derived molecular abnormalities in blood. Molecular characterization of the tumor via blood-derived biomarkers bypasses the painful and potentially debilitating procedures of obtaining tumor tissue via needle or surgical biopsies. ProStateâ„¢ incorporates characterization of CTCs and ctDNA. ProStateâ„¢ is a multi-coordinate platform which evaluates >4000 mutations from over 50 genes and CTCs to predict presence of prostate cancer. This integrative assessment of the tumor interactome translates into a more meaningful clinical decision for the patient, as compared to conventional means based on univariate analytics.

The performance evaluation and characterization of ProStateâ„¢ has been established and validated by Datar Cancer Genetics Limited in compliance with the requirements of CLIA as applicable for Single Laboratory Developed Tests. Reference cell lines obtained from ATCC were used as reference materials for determining the lower limit of variant detection, linearity, analytical specificity and sensitivity. Patient samples were collected after Institutional Ethics Committee approval at the participating centers and the Ethics Committee of Datar Cancer Genetics Limited. The protocols were conducted in accordance with the Helsinki Declaration. 20 mL whole blood was collected from subjects with enlarged prostate, elevated PSA or suspicious findings on DRE. CTCs as well as ctDNA obtained from subjects’ peripheral blood were used for analysis. Clinical sensitivity for CTCs and ctDNA was 74.5% and 75%, respectively.   

 3. OBJECTIVES AND OUTCOME MEASURES

3.1         Primary Objective:

To evaluate utility of ProState^TM in distinguishing prostate malignancies from Benign Prostatic Hyperplasia.

Outcome Measure: Positive predictive value of ProState^TM to differentiate prostate malignancies from Benign Prostatic Hyperplasia.

Evaluation Timepoint: Day 0 (Blood sampling)

3.2         Secondary Objectives:

To determine the grade (Gleason Score) of confirmed Ca Prostate.

Outcome Measure: Comparison of Gleason Score with molecular profiling of blood sample.

Evaluation Timepoint: Day 0 (Blood sampling).

4.             STUDY DESIGN

Study type: Observational.

Observational model: Cohort.

Time perspective: Prospective.

Estimated enrollment: 300 ± 15 cases.

Start Date: After clearance of ethics committee/IRB – tentatively January 2019.

Estimated Primary Completion Date: Six months from study initiation – tentatively July 2019 (which may be extended in case of insufficiency of volunteers / other contingencies to be evaluated by PI and sponsor).

Estimated Study Completion Date: Two months from completion of study cohort – tentatively September 2019.

Number of study groups/arms:  One.

Subjects: Therapy-naïve subjects with enlarged prostate or elevated serum Prostate Specific Antigen or suspicious findings on Digital Rectal Examination.

Name of study intervention(s): None.

Study Procedures: Collection of peripheral blood.

Study outline: