Ricerc@Sapienza

Prostate cancer has become, in many Western countries, the most frequent cancer in the male population. The etiology of prostate cancer is represented by a complex interaction of genetic and environmental factors. As for family history, it is estimated that the risk is at least doubled in the event that a first degree family member is affected by this neoplasm with a risk that increases by 5-11 times if two or more first degree relatives are affected.Prostate cancer may, in fact, be associated with Hereditary Breast and Ovarian Cancer (HBOC) and Lynch syndrome, both of which are linked to germline mutations of genes involved in DNA repair. Among the pathogenetic variants (PV) found in one of the 16 genes involved in DNA repair deficiencies (DNA Damage Response-DDR) analyzed, those in BRCA2 were found more frequently, with a progressively higher percentage depending on the stage metastatic disease. The other PVs mainly related to an increased risk of prostate cancer involve the ATM, PALB2 and CHEK2 genes. Pathogenetic variants (VP) of the BRCA 1 and 2 genes are mainly implicated in the predisposition of breast and ovarian cancers but recent international studies show that they are also involved in the predisposition to cancers of the prostate, pancreas, stomach, male breast. At the biological level, BRCA1 and BRCA2 play a crucial role in the repair of double-stranded DNA breaks by homologous recombination (HR). DNA repair for HR is a highly conserved mechanism that occurs mainly during the later S and G2 phases of the cell cycle. BRCA1 and BRCA2 are key components of this pathway and interact with different proteins involved in DNA repair such as: ATM , the MRN complex (MRE11, NBN, RAD50) ,CHEK2, BARD1, BRIP1, RAD51C, RAD51D and PALB2. Recommendations are hypothesized for the implementation of the BRCA test in patients with adenocarcinoma of the prostate, with a possible double application: a) the identification in patients with metastatic castration-resistant prostate cancer (mCRPC), progressing after therapy including a second generation hormonal agent, of subjects susceptible to systemic anticancer therapy with poly ADP-ribose polymerase (PARP) enzyme inhibitors . b) the identification of subjects with constitutional (germinal) PVs in the BRCA1 / 2- genes associated with high risk of various cancers (breast, ovary,pancreas, prostate) for the purposes of one prevention (primary and / or secondary) oncology in the family. In 2015, The Cancer Genome Atlas (TCGA) published a molecular analysis of 333 primary prostate cancers showing a prevalence of alterations of 19% for different DNA-repair genes, including BRCA2, BRCA1, ATM, CDK12, FANCD2 or RAD51C. Additionally, a report from the International Stand Up to Cancer / American Association for Cancer Research Prostate Cancer / Prostate Cancer Foundation Team (SU2C-PCF) identified genomic alterations involving DDR genes in 23% of metastatic biopsies analyzed in prostate cancer patients. BRCA2 was altered in 13% of cases, followed by TMJ (7.3%), MSH2 (2%) and BRCA1, FANCA, MLH1, RAD51B and RAD51C (all with a prevalence of 0.3%). The recent PROfound study evaluated the efficacy of the PARP inhibitor olaparib in patients with metastatic castration resistant prostate cancer (mCRPC), evaluated 2792 biopsies for the presence of aberrations in 15 genes involved in DNA repair with the finding of such alterations in 28% of the analyzed samples, with a similar frequency in the primary tumor (27%) and in the metastatic tissue (32%). The importance of the BRCA1 / 2 test in patients with prostate cancer is justified by the following reasons: 1. It has been shown that the PVs of the BRCA genes, whether of a germinal or somatic nature, represent a predictive biomarker of greater sensitivity to treatment with inhibitors of the enzyme Poly (ADP-ribose) Polymerase (PARP), in patients with hormone-resistant metastatic prostate cancer. 2. For patients with prostate cancer and positive for BRCA germline PVs, appropriate surveillance programs should be indicated to manage the risk of developing second cancers associated with BRCA PVs. 3. The relevant implications on cancer prevention in family members, especially in the case of a positive outcome of the germinal BRCA test. Currently, the BRCA test on peripheral blood ("constitutional or germline test") for the search for constitutional pathogenetic variants is carried out using widely validated methodologies, in particular next-generation sequencing (NGS) and are generally confirmed by methods such as Multiplex Ligation Probe Dependent Amplification (MLPA) or Multiplex Amplicon Quantification (MAQ).Although there are numerous ways of classifying the BRCA constitutional variants, it is appropriate to adopt the criteria developed by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA).

Primary objective: 

- evaluate and compare the tissue expression of pathogenetic variants (PV) of different genes involved in homologous recombination (HRDR) in patients with the first diagnosis of prostate adenocarcinoma (de novo) in relation to tumor aggressiveness and early risk of progression.

Secondary objectives:

- Search for the presence at the germline level of PVs of DDR genes found on tumor tissue after oncological genetic counseling in the affected person -

-Extend the germline analysis of segregation on family members of patients in whom PV has been found and inclusion in the surveillance protocol of subjects with these PVs -

-analyze the genotype-phenotype correlation by analyzing the neoplasms (breast, ovary, prostate, colon, pancreas) present in the family of the patients in which PV is found for the various genes examined

Study protocol :

Multicenter and multidisciplinary longitudinal prospective study in normal clinical practice with family genetic analysis

Population: histological diagnosis of de novo prostatic adenocarcinoma Inclusion criteria: patients with newly diagnosed histologically confirmed prostate adenocarcinoma at biopsy (de novo), intermediate or high risk (ISUP score 2-5, presence of Gleason Grades 4-5).

Exclusion criteria: current or previous androgen deprivation therapies, chemotherapy, radiotherapy or other therapies capable of influencing the growth and progression of prostate cancer.

Protocol 1st phase: patients with de novo histological diagnosis of prostate adenocarcinoma on biopsy 

- Patient selection based on inclusion and exclusion criteria

- Informed consent

- Definition of the TNM stage (total PSA, multiparametric magnetic resonance imaging of the prostate, PET CT with choline or PSMA) and grading on histological samples obtained at biopsy (Gleason grading and ISUP grading) of prostate cancer or radical prostatectomy (in non-metastatic patients selected for intervention as a therapeutic option)

- Stratification of patients in metastatic (oligo (up to 3 metastases) or poly (more than 3 metastases or visceral metastases) and non-metastatic

- Analysis of the most representative prostate adenocarcinoma tissue with Grades 4-5 at biopsy for metastatic patients and radical prostatectomy for non-metastatic patients (somatic line) of pathogenetic variants of the DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2, CDK12)

- Analysis of blood sampling (germline) of pathogenetic variants of the DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2, CDK12)

- Assessment of early (within 12 months) biochemical (total PSA) and / or radiological progression after or during treatment for prostate cancer according to clinical practice and guidelines (UAE guidelines)

2nd phase in patients with prostate cancer positive for pathogenetic variants of the DDR (somatic) genes

- Family analysis up to the third generation with: 1. Serum (germline) expression of pathogenetic variants of DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2, CDK12)

- Anamnestic evaluation of prostate, breast, ovary, colon, pancreas, stomach and their staging and grading characteristics

- In subjects tested positive for PV of DDR genes, clinical and radiological evaluation for new incidental diagnosis of: a. prostate cancer (examination with rectal examination, total PSA, multiparametric magnetic resonance imaging of the prostate, prostate biopsy); b. breast cancer (examination, MRI, biopsy); c. ovary cancer (examination, transvaginal ultrasound, CA-125, HE-4); d. pancreas cancer (MRI of the abdomen, biopsy if necessary); e. colon (examination, colonoscopy, biopsy)

MATERIALS AND METHODS

Population: Initial diagnosis of prostate adenocarcinoma As normal clinical practice and guidelines, the de novo diagnosis of prostate adenocarcinoma will be performed on the clinical suspect at the visit with rectal exploration, total PSA determination and multiparametric magnetic resonance imaging of the prostate, through the execution of a targeted biopsy (two samples on each PIRADS 3-5 area on MRI) and random (on the two prostatic lobes peripherally for a further 6 samples: basal, intermediate and apex) with MRI-transrectal ultrasound image fusion method

Staging of prostate cancer As normal clinical practice and guidelines, local and systemic staging will be performed through multiparametric magnetic resonance imaging of the prostate, PET CT with choline or PSMA total body characterizing in non-metastatic and metastatic form (oligo or poly)

Histological analysis At the prostate biopsy performed to establish the initial diagnosis of prostate adenocarcinoma, the Gleason grade, its score and ISUP grading will be performed. Determination of Grade 4-5 percentages in the tumor tissue. Determination of the presence of perineural invasion (PNI) or ductal carcinoma.

Tumor progression analysis As  normal clinical practice and guidelines, the presence of an early (within 12 months) progression after primary therapy will be analyzed by measuring the total PSA (biochemical progression) with progressive increases> 0.2 ng / ml and through local  ( Multiparameter MRI) and systemic (PET CT with choline or PSMA) radiologic examinations

Methods for the determination of pathogenetic variants (VP) of DDR genes -DDR genes included in the analysis VPs related to the following DDR genes will be evaluated: BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2, CDK12

Somatic line analysis Analysis of prostate cancer samples obtained at biopsy or / and radical prostatectomy with histologically confirmed diagnosis. In the area described with the highest concentration and representation of Grade 4-5 prostate adenocarcinoma cells for prostate cancer, the determination by PV of the DDR genes will be performed

The mutation analysis by Whole-exome Sequencing (WES) targeted NGS and the analysis of genetic data. Specifically whole exome sequencing of PCa enrolled patients  will be uniformly performed using the Illumina NovaSeq™ 6000 platform (Illumina, San Diego, CA, USA)  DNA fragments will be hybridized and captured by Illumina Exome Panel (Illumina) according to the manufacturer's protocol. The libraries will be tested for enrichment by qPCR, and the size distribution and concentration will be determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). 

Germline analysis On a blood sample, the PV search for DDR genes will be performed with next generation sequencing (NGS) using methods such as Multiplex Ligation Probe Dependent Amplification (MLPDA) or Multiplex Amplicon Quantification (MAQ). The classification of pathogenic variants will be performed according to the criteria of the Evidence Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) (classification in: benign, probably benign, uncertain, probably pathogenetic, pathogenetic). 

Germline DNA will be  extracted from peripheral-blood samples. The cod- ing region of the  DDR  genes will be  screened using multiplex fluorescent heteroduplex detection, Sanger sequencing, and multiplex ligation-dependent probe amplification. (Thermo Fisher Scientific, Waltham, MA, USA) 

End points and statistical analysis 

Primary - Evaluate expression of different PVs of the DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2, CDK12) in correlation with the stage (localized (T2), locally advanced (T3), metastatic (M +) oligo and poly, and aggressiveness (ISUP / Gleason score / Grade 4-5 percentage at biopsy and / or radical prostatectomy) in de novo diagnosis of intermediate and high risk prostate cancers

Secodary- To evaluate the expression of different PVs of the DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2, CDK12) in correlation to an early progression (less than 12 months after treatment) of the primary neoplasm - To evaluate the correlation between somatic and germline expression of PV of the DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2, CDK12) in patients with prostatic neoplasia. - Family analysis up to third generation in subjects with PV positive primary tumor of DDR genes: 1. PV expression in the germline of DDR genes 2. Family history of prostate cancer, breast, ovary, colon, pancreas, stomach and their stage and aggression characteristics in relation to the presence of DDR mutations 3. New diagnosis of prostate, breast, ovary, colon, pancreas, stomach and their stage and aggression characteristics in relation to the presence of DDR mutations.

Statistic analysis Patients diagnosed with prostatic neoplasia enrolled in the present study will be stratified according to the presence of PV in the analyzed DDR genes. The descriptive analysis will be reported as the number of cases (%), mean +/- SD, median and interquartile range. For comparative evaluations of quantitative data the Mann – Whitney – Wilcoxon test will be used, while for qualitative data the Fisher and Chi-square tests will be used.Logistic regression multivariate models will be constructed to establish an association between the presence of PV of the DDR genes and the binary endpoints after adjustments for known confounders. In addition, Kaplan-Meier curves and multivariate Cox regression models will be used to evaluate the association between the presence of VP of the DDR genes and tumor progression after adjustments for known confounders.The STATA 1.7 software will be used to establish the statistical significance of the various analyzes, set at p <0.05.

 The calculation of the sample size for the primary endpoint is based on data from previous scientific research. The minimum sample size of 126 patients was estimated based on a prevalence of PV of the DDR genes of 20% and will serve to ensure a statistical power of 80% in identifying a difference in the prevalence of ISUP 4-5 among PV carriers (60 %) and non-carriers (30%) with a significance level set at 0.05. The National Institutes of Health online tool was used for the calculation of the sample size (https://sample-size.net/sample-size-proportions/).

Prostate cancer (PCa) is a very common neoplasm in male population able to affect survival and quality of life with a significant impact on social life. It has been the focus of much research in recent years,however, poor levels of genetic PCa risk awareness were found and there is an un-met medical need to design acceptable and appropriate health promotion interventions and screening in health young men with gene mutations linked to high-risk of developing PCa. At the present, there is no international consensus on the optimal management to perform in men with a genetic risk for PCa. Several points remain undefined and relevant unmet need must be addressed. The innovation of our research is based on different points: - it may better define clinical and pathological characteristics of newly diagnosed prostate cancer associated to DDR genes defects - it may offer a platform for approaching personalized medicine based on genetic assessment of PV in DDR genes - it may be able to answer to different undefined points: - is the expression of PV of DDR genes relevant also in non -metastatic prostate cancer at intermediate or high risk ? - is the somatic determination of PV for DDR genes on tissue samples obtained at prostatic biopsy a reliable method for this analysis when compared to the whole specimen analysis obtained at radical prostatectomy ? - is the germline determination of PV for different DDR genes able to characterize prostate cancer cases or a somatic analysis on tissue is always necessary ? -is the rate of germline and somatic expression for PV of DDR genes similar and related in prostate cancer cases? - is BRCA2 the main PV expressed in prostate cancer cases or other PV for different DDR genes are similarly expressed and useful ? - is there a different correlation between germline or somatic expression of PV for DDR genes and unfavorable pathologic features or risk for early progression of prostate cancer ? - is there a different correlation between the PV of the different DDR genes and unfavorable pathologic features or risk for early progression of prostate cancer ? - is a familial analysis and genetic counseling for germline PV of DDR genes useful in patients with prostate cancer with somatic and/or germline PV of DDR genes ? - is a familial analysis and genetic counseling for germline PV of DDR genes in patients with prostate cancer useful to obtain early diagnosis for ovarian, breast and gastro-intestinal neoplasms ? This study allows an analysis on the expression and role of different pathogenetic variants related to genes of the DDR family in prostate adenocarcinoma, comparing the more well-known BRCA2 with other DDR variants. The analysis of these variants has not only prognostic but also possibly therapeutic significance considering the current interest in new classes of systemic therapies such as PARP-inhibitors, related to the expression of these DDR pathogenic variants and in particular to BRAC2. Our analysis can improve the knowledge on the role of a somatic determination on tissue, with all the possible difficulties deriving from histological preparations, and the germinal one from blood sampling, establishing the correlation in the two expressions for each single pathogenetic variant DDR in patients with prostate cancer. In the family and multidisciplinary analysis phase, our study can allow us to define how much, in patients with prostate cancer positive for DDR pathogenic variants, these variants exist in the family and how much they involve the presence of multiple tumors associated with these variants (prostate, breast, ovary, stomach, colon, pancreas). This type of analysis can allow to define the relevance of a widespread family study in patients with initial detection of PV for DDR genes, in particular in prostate cancer.The development of greater knowledge on the correlations between DDR variants and characteristics of first diagnosed prostate adenocarcinoma is strongly supported by various scientific societies in the urological and oncological fields, so as to bridge the knowledge gap with respect to the field of breast cancer and open to new therapeutic lines (PARP-inhibitors) in various stages of cancer. This type of analysis can help to define the importance of a multidisciplinary collaboration within highly specialized centers, with development centered on the analysis of pathogenetic variations of DDR genes in family situations with possible multiple neoplasms.It will allow the development of publications in high impact international journals (eg Lancet Journals), presentations in national and international congresses, sending to national and international urological and oncological societies in support of new guidelines.

1. Urologic team (Prof Alessandro Sciarra, Prof Stefano Salciccia,Prof Giampiero Ricciuti): - identification of the population with prostate cancer and selection on the basis of inclusione and exclusion criteria - initial diagnosis with prostatic biopsy - staging of the disease and identification of the risk class - performance of radical prostatectomy - follow-up of the patients defining biochemical and radiological progression 2. Breast Unit (Prof Laura De Marchis): -familial analysis related to patients with prostate cancer and Pathogenetic Variant (PV) of DDR genes - familial germline analysis of PV of DDR genes - familial germline analysis of PV of DDR genes related to ovarian and breast cancer diagnosis 3: Genetic Unit (Prof Paola Grammatico): - next generation sequencing (NGS) using methods such as Multiplex Ligation Probe Dependent Amplification (MLPDA) or Multiplex Amplicon Quantification (MAQ). - classification of pathogenic variants according to the criteria of the Evidence Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) - analysis of PV of a series of DDR genes: BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2,CDK12 - analysis of germline expression for PV of the different DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2,CDK12) in patients and in families - analysis of somatic expression for PV of the different DDR genes ( BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2,CDK12) in prostatic adenocarcinoma tissue specimens from biopsy and radical prostatectomy - genetic evaluation and comparison between germline and somatic expression of the different PV - comparative analysis of the PV of the different DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2,CDK124) in relation with prostate cancer characteristics 4. Pathology Unit (Prof Fabio Magliocca): - pathologic analysis of prostate tissue specimens from biopsy and radical prostatectomy for the diagnosis of prostatic adenocarcinoma, histological grading according to ISUP system, pathologic stage, perineurial invasion, ductal form. - preparation of tissue samples representative of the higher grading of prostate cancer for the analysis of PV of the DDR genes 5. Statistical Team (Dr Francesco DEL GIUDICE, Dr Giulio Bevilacqua): - descriptive analysis of the population in terms of clinical and pathologic characteristics - correlation between the expression of PV of the different DDR genes and clinical and pathologic prognostic variables for prostate cancer - determination of the predictive value of PV of DDR genes in terms of unfavourable pathologic features and risk of early progression of prostate cancer - comparative analysis of germline and somatic PV determination comparative analysis on the different DDR genes (BRCA1, BRCA2, ATM, RAD51, CHEK2, MSH2,CDK12) examined