diaRNAgnosis (H2020-MSCA-RISE-2020 n. 101007934 – Research Innovation & Staff exchange)

Circulating cell-free ribonucleic acids (ccfRNAs) represent an emerging and important class of molecules, able to provide significant clinical relevance as novel screening, prognostic and therapy monitoring biomarkers in cancer. By analyzing specific ccfRNAs, different cancers can be accurately correlated for type and stage. Unfortunately, despite extensive academic and clinical research identifying and validating in limited clinical trials, ccfRNAs have not yet entered the field of clinical diagnostics. This is amongst others because the current analytical methods remain less than satisfactory, and until now ccfRNA detection remains challenging, costly, and requires elaborate multi-step sample preparations. Prompted by these current analytical limitations, two innovative EU companies, DESTINA Genomica SL (Spain) and OPTOELETTRONICA ITALIA SRL (Italy) have developed the “ODG Platform” for direct quantitative measurement of circulating RNA molecules. The diaRNAgnosis project objective is to complete development of the ODG platform, delivering reliable and robust detection of novel ccfRNA signatures that could be linked to specific cancer types. To ensure timely delivery and success of the diaRNAgnosis project, Cogentech (Italy, Catania) together with a Spanish company NanoGetic SL; three key academic research groups from the Universities of Trento, Catania (Italy), Granada (Spain) and the Princess Máxima Center (The Netherlands) have joined the project. This new pan-European, multidisciplinary and intersectoral team will develop a reliable and innovative method and platform to identify cancer biomarkers in liquid biopsies. The research collaboration will address the need to perform high sensitivity/high specificity analysis of ccfRNAs that are specifically and overexpressed in testicular germ cell tumour (TGCT) and prostate cancer (PCa) respectively.

STRIKE (HORIZON-MSCA-2021-DN-01, n. 101072462 – Doctoral Network)

The project is imbedded in a network of 12 institutions from 8 European countries focusing on the application of innovative magnetic particles to STRIKE cancer in the framework of a Horizon Marie Curie Doctoral Network. The innovative nanotechnological strategy proposed in STRIKE will address the early diagnosis and diagnosis-related therapy in BRCA+ mutation breast cancer (BC). Patients with germline mutations in the BRCA+ genes have unique features such as a high risk of developing tumors and, consequently, the need to be subjected to regular clinical follow-up visits and protocols. These subjects correspond to the ideal cohort of patients to test the feasibility of a “liquid biopsy” approach for an early diagnosis to anticipate the clinical detection of the disease. Innovative magnetic beads (MBs) produced in STRIKE will be exploited to increase operating capture efficiency of DNA or exosome from complex biologic matrix (i.e. blood, urine). Whole Exome Sequencing (WES) and MeDIP seq technology will be performed on the primary tumors (stored in paraffin) using the NGS platform and based on the identified mutational profiles, we will develop customized gene panels and methylation signatures for NGS and/or ddPCR analysis of cfDNA and exosomes from healthy subjects carry BRCA mutations.

BiLiGeCT (PON/FSC 2014-2020 area health, project n. ARS01_00492)

Cogentech received in 2019 as lead partner an important 3 year grant from the Italian Ministry of University and Research (MUR) for the project "BiLiGeCT - Liquid Biopsies for the Clinical Management of Tumors". This project, which involved five other Italian entities of excellence (Carebios srl, Consorzio Interuniversitario Nazionale Metodologie e Processi Innovativi di Sintesi-CINMPIS, Istituto Oncologico del Mediterraneo S.p.a., Istituto Superiore di Sanità and Università degli Studi di Torino), aimed to develop new technologies for the analysis of liquid biopsies, an innovative approach for cancer diagnosis and monitoring and was successfully concluded in 2022. This project had the intention to make significant social, political and economic interventions in areas of Southern Italy with historical and geographical difficulties. Cogentech contributed to the project with its new Operating Unit, located in the Science and Technology Park of Sicily in Catania, creating opportunities for collaboration and transfer of technical-scientific expertise with local realities. As part of the project BiLiGeCT, the Integrated Genomic Unit in Catania developed a "holistic” liquid biopsy protocol. This “holistic” protocol uses cfDNA from a single blood sample to perform four different types of NGS analysis to intercept both epigenetic and genetic tumor-associated signals. The idea behind this approach is that, through the integration of the results using algorithms and AI tools, a sufficient sensitivity to detect cancer in apparently healthy subjects can be obtained. One important step in bringing a new product to the market is the pre-clinical validation. In continuation of the BiLiGeCT project, the Catania Unit is focusing on this critical aspect, validating the technical performance and sensitivity of the protocol.

OncoHRD for personalized therapy with PARP inhibitors

Mutations in BRCA1/2 not only predispose to an increased cancer risk but also provide an opportunity for personalized therapy with PARP inhibitors in ovarian, breast, prostate and pancreatic cancer that harbor a mutation in either of the two genes or in other genes of the homology recombination repair (HRD) pathway. Mutations in any of the genes in fact, leave a scar in the genome that can be detected by NGS sequencing and bioinformatics analysis. In order to respond to a growing demand for somatic analysis of BRCA1/2 combined with HRD analysis for PARP inhibitor prescription we decided to develop a diagnostic approach to fill the gap. We combined somatic analysis of the tumor using OncoPan® with a Low-Pass Whole Genome Sequencing (LP-WGS) integrating and validating the bioinformatics pipeline Ginger from Sophia Genetics for the identification of an HRD scar. OncoHRD combines the advantages of OncoPan (BRCA1/2 analysis expandable to other hereditary tumor genes for SNV and CNV) together with identification of the genomic scar characteristic of HRD to provide the best diagnostic tool to our clients and in the end to the patients.

OncoPed: for pediatric and rare adult cancer syndromes

The underlying cause of most pediatric cancers is currently unknown. However, it has been estimated that germline genetic alterations, responsible for increased susceptibility to tumor development, are present in approximately 8% of pediatric patients, in most cases without significant family history. Although the importance of identifying individuals at genetic risk is recognized, the ability to offer molecular diagnostic insights among pediatric cancer patients is currently limited. We decided thus, as part of our mission as a Benefit Corporation to develop a specific NGS panel (OncoPed) that could assess childhood and rare adult cancer syndromes. In collaboration with the pediatricians of Fondazione IRCCS Istituto Nazionale Tumori of Milan, OncoPed was designed for the analysis of predisposition genes in medulloblastoma, Wilms' tumor, some rare ovarian tumors (Sertoly-Leydig and SCCOTH), retinoblastoma, uveal melanoma/melanoma, renal tumors, some lymphomas, rhabdoid tumors, tumors developing in Li Fraumeni, Cowden, Gorlin, DICER1 and Carney syndromes. Capture probes were designed to cover all exons and the adjacent intronic regions involved in splicing. The panel was designed using Agilent Sure Select technology and validated for MiSeqDX and NextSeq500DX sequencers.

OncoPan®: from cancer risk assessment to personalized therapy

Pancreatic cancer has a high morbidity and mortality with the majority being PC ductal adenocarcinomas (PDAC). A proportion of 5-10% of PDAC have a hereditary origin involving germline variants of homologous recombination genes, such as Mismatch Repair (MMR), STK11 and CDKN2A genes. Very recently, BRCA genes have been demonstrated as a useful biomarker for PARP-inhibitor (PARPi) treatments. In this study, a series of 21 FFPE PDACs were analyzed using OncoPan®, a strategic next-generation sequencing (NGS) panel of 37 genes, useful for identification of therapeutic targets and inherited cancer syndromes. Interestingly, this approach, successful also on minute pancreatic specimens, identified biomarkers for personalized therapy in five PDAC patients, including two cases with HER2 amplification and three cases with mutations in HR genes (BRCA1, BRCA2 and FANCM) and potentially eligible to PARPi therapy. Molecular analysis on normal tissue identified one PDAC patient as a carrier of a germline BRCA1 pathogenetic variant and, noteworthy, this patient was a member of a family affected by inherited breast and ovarian cancer conditions. This study demonstrates that the OncoPan® NGS-based panel constitutes an efficient methodology for the molecular profiling of PDAC, suitable for identifying molecular markers both for therapy and risk assessment. Our data demonstrate the feasibility and utility of these NGS analysis in the routine setting of PDAC molecular characterization (PMID: 35625944).

Polygenic Risk Score

Polygenic Risk Score (PRS) are an important component of breast cancer risk prediction and explain over 30% of heritable breast cancer. PRS are based on the combined result of common genetic variants. Oncopan® has incorporated 313 SNP (single nucleotide polymorphism) for the calculation of the PRS Z-score with a dedicated algorithm that Cogentech has implemented. With a dedicated software (www.canrisk.org) a medical professional can use the PRS to better calculate the risks of developing breast and ovarian cancer in women by integrating also other risk factors such as lifestyle, genetic test results and family history. Currently, the PRS for breast and ovarian cancer has been validated in European cohorts only and further testing for different ancestries is needed and ongoing. In order to assess how much the PRS might influence the cancer risk in subjects carrying variants in BRCA1, a small pilot study involving a limited number of subjects (17/20) is being carried out in collaboration with Fondazione IRCCS Istituto Nazionale Tumori of Milan The study participants carrying a specific variant of unknown significance (VUS) show significant phenotypic variability. The outcome of this pilot study will be the basis for promoting evaluation of the clinical utility of PRS for genetic risk personalization and subsequently proposing to specific entities a more extensive, retrospective or new-case, consortium-type research project, regarding a specific geographic area.

Case report: de nuovo mutation in Lynch Syndrome

Colon cancers occur in about 5-6% of individuals in the general population. In some rare cases (5-10%) these cancers are due to the presence of an inherited genetic predisposition. One such form of predisposition is Lynch syndrome, due to alterations in the MLH1, MSH2, MSH6, PMS2, and EPCAM genes. The identification of individuals at high risk for colorectal carcinomas has significant importance for prevention purposes, with the primary goals of reducing mortality and morbidity, both in affected patients and in healthy relatives carrying pathogenic variants in a predisposition gene; precise recommendations are available for selection criteria at genetic testing and for the management of affected patients and their relatives (AIOM Recommendations: Hereditary Cancers of the Stomach and Colorectum. Ed. January 2022). The literature describes thousands of cases with pathogenic variants in Lynch syndrome susceptibility genes, but very few de novo alterations are known, i.e., not inherited from parents but arising in the zygote or very early in embryonic life. The study, in collaboration with the U.O Hereditary Tumors of the Digestive System of the Fondazione IRCCS Istituto Nazionale dei Tumori (Milan, Italy), investigated a young patient with colon cancer, with no apparent familiarity, but presenting with de novo deletion of exon 6 of the MLH1 gene, identified by Next Generation Sequencing (NGS) and confirmed by Multiplex Ligation-dependent Probe Amplification (MLPA). The patient's tumor was found to be defective for MLH1 and PMS2 by immunohistochemistry. The description of this case, which has been published (PMID: 37691472), emphasizes the importance of genetic testing for Lynch Syndrome in the absence of familiarity, if one or more Mismatch Repair genes are found to be defective by immunohistochemistry.

Case Report: Male Lobular Breast Cancer in Hereditary Cancer Syndromes.

Lobular breast carcinoma (LBC) is considered an exceptionally rare disease in men, representing only 1% of all male breast malignancies. It is well recognized that LBC in women could be involved in both hereditary breast and ovarian cancer (HBOC) and hereditary diffuse gastric cancer (HDGC) syndromes. However, there are no data present in literature about the involvement of male LBC in these inherited conditions. To investigate whether germline mutations in cancer syndromes might be present also in men that present with LBC, DNA from 2 affected men was analyzed using OncoPan®. Both men were identified to be carriers of a pathogenic variant in BRCA2 and CDH1 genes, respectively. Male LBC could represent a sentinel cancer for inherited syndrome identification, and early identification of cancer susceptibility could improve cancer prevention both for men and women in these families. The history of the LBC patient carrier of the CDH1 variant suggests to include male LBC genetic testing criteria and male breast surveillance in HDGC guidelines (PMID: 35686104). This study was carried out in collaboration with the Research Center for the Study of Hereditary and Familial Tumors, University of Insubria.

Identification of germ line alterations predisposing to the pathogenesis of large cell anaplastic lymphoma associated with mammary prostetic implants (BI-ALCL) 2021 -2022

Although rarely, it has been observed, that breast cancer patients that received a breast implant after total mastectomy, develop anaplastic large cell lymphoma (ALCL) similarly to healthy people that do received a breast implant for aesthetic reasons.. Thus, it appears that breast implants could promote ALCA in a sub group of patients. The development of ALCL in only a sub group of patients might be due either to environmental (e.g., implant texture, chronic inflammation) or genetic factors. To address the latter possibility and to identify possible variants that associate with ALCL development after breast implant, whole exome sequencing (WES) was performed on blood samples from a case series of 82 patients who received a breast implant Of these 82 patients, 41 had developed lymphoma and 41 did not. Unfortunately, the results did not identify any variants that clearly associate with lymphoma development, suggesting that either a larger cohort needs to be analyzed to have sufficient statistical power to reveal an association or that environmental factors/life style play a more important role than anticipated. This Project, commissioned by the Ministry of Health, was carried out in collaboration with the Istituto Superiore di Sanità (ISS), University La Sapienza and the European Institute of Oncology (IEO). The results of this study were presented at the Conference "National breast implant registry: A tool to improve patients' safety," held in Rome on 12/15/2022 at the Ministry of Health (manuscript in preparation.

Identification of germline molecular alterations as possible risk factor for non-small cell lung cancer (NCLC) in non-smoking subjects.

In a retrospective study, carried out in collaboration with the National Cancer Institute of Milan, on a case series of 75 subjects who, despite being non-smokers ("never smokers") developed non-small cell lung cancer (NSCLC), Cogentech performed Whole Exome Sequencing (WES) on frozen samples of genomic DNA extracted from peripheral blood in order to identify germline alterations that might constitute risk factors and predispose to the onset of the disease. Bioinformatics analysis showed that a total of 33 pathogenic or likely pathogenic variants were identified in 31 genes. Of them, 13 were located in cancer predisposing genes (nine were lung cancer drivers), and most of the others in genes that are associated with NSCLC. These findings confirmed and extended data already in the literature that pathogenic variants of genes involved directly or indirectly in tumor development can be identified in never-smokers who developed lung cancer. This indicates that certain pathogenic variants present in the germline may be considered a risk factor for lung cancer, suggesting that certain categories of lung cancer patients should be included in the guidelines for germline molecular testing.The results of this study will be presented at the 2nd National Meeting of the Italian Association Familial and Hereditary Tumors (AIFET) Rome 23-24 November 2023 and a (manuscript in preparation).

Development of isogenic cell models by genome editing for the functional study of specific BRCA1/2 variants and their sensitivity to targeted therapies.

It is known that individuals who carry certain germline mutations in the BRCA1/2 genes involved in the DNA damage repair process have a significantly higher risk than the rest of the population of developing cancer, especially breast and/or ovarian cancer, during their lifetime. Indeed, there are variants capable of generating alterations in the structure and/or function of BRCA1/2 proteins, termed pathogenic. In addition to these, about 15 percent of the variants detected have uncertain clinical significance (Variants of Unknown Significance or VUS) for which there is no data associated with an increased risk of developing the cancer pathology. This uncertainty poses an important problem about the most appropriate prophylaxis and treatment of the disease in these individuals. In fact, the diagnosis of these variants still remains very unsatisfactory because it does not provide a clinically useful response to individuals who carry these variants and their family members. The main goal of the project was to develop an innovative in vitro cell model for functional characterization of VUS and for screening PARP inhibitors, new therapeutically active molecules. To achieve the goal, CRISPR/Cas9 genome editing technology was used to generate cellular models suitable for the functional study of gene variants of BRCA1/2 genes with as yet uncertain clinical significance.

ARETHUSA: patient stratification for clinical trials

Cogentech has been actively participating in the first non-profit, IFOM-sponsored multicenter clinical trial called ARETHUSA (PMID: 35522273) offering an NGS sequencing service to assess the number of mutations present in tumor biopsies of colorectal cancer patients. Colorectal cancers with mutations in Mismatch Repair (MMR) genes are known to be sensitive to immunotherapy because of an increased tumor mutational burden (TMB). The majority of colorectal cancers instead are MMR proficient, display low TMB and hence are little responsive to immunotherapy. ARETHUSA was designed to demonstrate how such tumors can become sensitive to immunotherapy following treatment with specific drugs that increase their TMB. During the timeframe of the project Cogentech, with its cutting-edge genomics service, has performed NGS sequencing of tumor biopsies for TMB analysis after drug treatment for timely stratification of these patients.