BiLiGeCT Project

The project beneficiary of a grant from the Italian ministry (MUIR) from a call for the presentation of Industrial Research and Experimental Development Projects in the 12 areas of specialization identified by the PNR 2015 - 2020" - Director's Decree July 13, 2017 No. 1735 project code no. ARS01_00492, titled "Liquid Biopsies for Clinical Management of Tumors" (BiLiGeCT), (www.ponricerca.gov.it) 01.07.2019 -31.12.2022, provided 5 lines of research for the development of circulating markers, by liquid biopsy, for the prevention, diagnosis and treatment of both hereditary and sporadic cancers:

1. Our primary objective is to develop cost-effective and highly sensitive diagnostic assays that can detect diseases at an early stage in individuals with BRCA mutations who are at a genetic risk for breast and ovarian cancer. The focus is on early cancer detection using non-invasive and precise methods, specifically targeting a population of healthy subjects. To accomplish this ambitious goal, we have chosen to study a cohort of individuals at genetic risk, particularly those carrying BRCA1 or BRCA2 mutations. These individuals have two experimental advantages compared to the general population: they are at a higher risk of developing cancer and can provide insights into the development of breast and ovarian carcinomas.
   To facilitate our research, we have established a biobank of plasma samples collected from healthy family members who have the same BRCA mutation and are actively participating in surveillance programs. This collection currently includes 20 plasma samples from 16 individuals, and we plan to expand it further. The value of this biobank lies in its ability to conduct retrospective molecular analyses once a subject develops cancer, allowing us to assess the effectiveness of liquid biopsy in detecting the disease at an early stage.
   In addition to technological advancements in liquid biopsy research, we will utilize methodologies derived from the development of the "holistic protocol" to achieve our research goal. Since no subjects in our study have developed tumors yet, the methodologies employed will align with the research described in the subsequent line of research.
   Alongside the specimen collection, we have developed protocols to maximize the yield of circulating DNA (cfDNA) in venous blood samples and prepare plasma from them. We have also created a dedicated database to manage the collected cases, record their clinical characteristics, and document relevant molecular analyses. These efforts have laid the foundation for the next phase of our project.
2. Our objective is to achieve early detection of disease recurrence in individuals with a history of breast or ovarian cancer and BRCA mutations who are under surveillance. We aim to monitor the disease using circulating markers and ensure appropriate therapy is administered.
   To accomplish this goal, we initiated a study to compare the molecular profile of tumors, as determined through whole-exome "next-generation sequencing" (NGS/WES), with the corresponding circulating tumor DNA (ctDNA) present in the bloodstream. Initially, we performed NGS/WES sequencing on over 50 tumors and their respective normal tissues. This comprehensive analysis covered various types of breast and ovarian tumors with different mutations in BRCA1 or BRCA2. It also provided valuable insights into the mutational profile of these tumors, which enabled us to design a proprietary gene panel. This panel, used in conjunction with NGS sequencing, can effectively detect the majority of tumors of this nature. It is a crucial component of our "holistic" protocol, developed within the project, which combines genetic and epigenetic analyses while leveraging the chemical and physical properties of DNA.
   An important aspect of our research involved exploring the feasibility of analyzing the methylome and fragmentome for epigenetic insights. This required the development of specific bioinformatics tools that were validated using data available in public databases. Promising early sensitivity data were obtained using the "holistic panel," indicating its potential as an analytical tool for ctDNA obtained from liquid biopsy. Currently, we are in the process of formalizing the results obtained from analyzing this significant series of BRCA mutated cancers for scientific publication.
3. In the Project, we focused on the early diagnosis of lung and prostate cancers by developing new circulating exosomal markers. These markers offer a more effective and less invasive approach to diagnosing these diseases.
   We evaluated several molecular markers capable of detecting and monitoring prostate, lung, and colon cancers, as well as distinguishing aggressive forms of these cancers and guiding targeted therapies. Our analysis involved studying the molecular content, including proteins, DNA, and RNA fragments, of nano-vesicles called exosomes that are released by tumors. These exosomes carry information about the tumor's tissue of origin and its specific aberrations. We processed and analyzed over 150 exosome samples from patients with prostate, lung, and colon cancer.
   Additionally, we employed a novel technique called Stochastic Optical Reconstruction Microscopy (STORM) to examine individual extracellular vesicles using super-resolution microscopy. This allowed us to evaluate the expression of vesicular proteins at a resolution capable of detecting single molecules. Preliminary data revealed that certain markers present on the surface and within the lumen of vesicles were significantly more expressed in vesicles isolated from the blood of patients with prostate cancer compared to those isolated from patients with benign prostatic hyperplasia. These differences suggest that these markers could enable more accurate and sensitive early diagnosis compared to conventional biomarkers like PSA. Similarly, in patients with locally advanced colon cancer, we identified vesicular proteins that could be associated with prognosis, particularly in patients undergoing conventional therapy or immunotherapy.
   The study also demonstrated the technical applicability of our approach to lung cancers. Currently, early detection of lung cancer primarily relies on expensive and limited-access methods like low-dose CT. Our results indicate that characterizing vesicular proteins through simple and noninvasive methods could serve as a viable alternative for screening these types of cancers in the future.
   
4. Liquid Biopsy in colorectal cancer for choice of second-line therapies and beyond;
   Within the project, the team responsible for this objective recruited colorectal cancer patients and collected serial blood samples using venous sampling. Both tumor tissue DNA and circulating free DNA (cfDNA) from the blood samples were extracted for biomolecular analyses. The tumor DNA and cfDNA underwent sequencing using Next-generation Sequencing (NGS) technique, and subsequent bioinformatics analysis identified "trunk" mutations (mutations present in all regions of the tumor) or "driver" mutations (mutations responsible for tumor phenotype and potential resistance to targeted therapies) in specific genes of the tumor tissue.
   To detect the same trunk/driver mutations, droplet digital PCR (ddPCR), a highly sensitive DNA quantification technique, was performed on the cfDNA of the same patients. The quantification of cfDNA through liquid biopsy, as a noninvasive, highly sensitive, and repeatable diagnostic test, enables monitoring disease progression and response to therapies. In fact, for some patients included in these analyses, clinical biopsy helped predict clinical disease progression.
   The data obtained from this project validate the clinical application of liquid biopsy in defining the prognosis and treatment strategies for colorectal cancer. It highlights the potential of liquid biopsy as a valuable tool for guiding the selection of second-line therapies and beyond for patients with colorectal cancer.
5. Development of innovative cell-based assays for screening new drugs or repositioning known drugs for mutations detected by liquid biopsy.
   We aimed to develop new cell-based assays for screening potential drugs or repurposing existing drugs to target mutations detected through liquid biopsy. Our primary goal is twofold: firstly, to develop a methodology for assessing the functional significance of gene variants associated with cancer predisposition, specifically variants of unknown significance (VUS); secondly, to address therapeutic challenges, particularly those involving PARP inhibitors. These inhibitors rely on the presence of pathogenic variants in BRCA genes, making them useful for identifying VUS that respond or do not respond to this treatment. The issue of VUS is critical in genetic counseling because it does not allow for a definitive diagnosis. To tackle this, our project involves two partners. One partner focuses on creating engineered cellular models through genome editing (CRISPR-Cas9) and has developed a highly efficient transfection protocol for characterizing VUS in BRCA genes. Using this methodology, we constructed five co-isogenic cell lines. The first line, called the Parental Line (MCF7 BRCAnull/BRCAwt or HAPLO), was created by deleting one wild-type allele of BRCA1. Subsequently, five mutations were inserted into the remaining BRCA1 locus: three with known pathogenicity, one neutral, and one VUS (of unknown significance). These co-isogenic lines allow us to test various approaches for functionally defining VUS by comparing them to the Parental Line. In parallel, another line of research focused on converting the susceptibility phenotype to treatment resistance in multiple cell lines chronically exposed to the drug. This approach not only identifies genes and pathways related to drug resistance but also reveals molecules with "dual" activity that can reverse resistance to Olaparib. Overall, our findings indicate that targeting PARP in combination with CDK or mTor inhibitors produces antagonistic effects. Conversely, the activity of Gem122, a dual PARP/HDAC antagonist, suggests that combining PARP inhibitors with HDAC inhibitors may have therapeutic relevance that warrants investigation at the clinical level.

The BiLiGeCT project, focused on liquid biopsies for the Clinical Management of Tumors, has been awarded a prestigious funding from the MIUR's National Operational Program (PON). Aging, chronic and degenerative diseases, and quality of life are the three key factors identified as priority areas in the "Health" sector of the national strategic agenda. The project, titled "Liquid Biopsies for the Clinical Management of Tumors" (acronym: BiLiGeCT), proposed by Cogentech, a Benefit Company of IFOM, in collaboration with the National Interuniversity Consortium for Innovative Synthesis Methodologies and Processes (CINMPIS, involving the University of Catania and the University of Messina), the Italian National Institute of Health (ISS), the University of Turin (UniTO), the Mediterranean Oncological Institute (IOM), and CaReBios, aims to improve the clinical management of oncological diseases. The ambitious BiLiGeCT project has secured funding from the National Operational Program for Research and Innovation 2014-2020, managed by the Ministry of Education, Universities and Research (MIUR). BiLiGeCT proposes innovative research to develop new and improved technological solutions to address two crucial aspects in the field of oncology: early diagnosis and therapeutic appropriateness.
Regarding the first point, the project focuses on the emerging issue of specific types of tumors, primarily resulting from population aging. Cancer is increasingly considered a disease associated with aging, and the project is based on the concept that early diagnosis almost always leads to better disease outcomes.
Regarding the second point, next-generation oncological therapies aim to transform tumors into chronic diseases. This is evidenced by the fact that continuous administration of the appropriate drug is required to control the disease.
Finally, the quality of life of cancer patients assumes an increasingly important role, considering the concept of chronicization of the disease. Patient associations also emphasize this aspect.
The project aims to address these objectives using an innovative tool, liquid biopsy, which holds promise as a new and more effective approach for the clinical management of tumors. In addition to traditional tissue biopsies, liquid biopsy involves minimally or non-invasive collection of biological fluids such as urine, oral washes, or primarily blood. Sophisticated molecular technologies are then used to detect various biomarkers indicative of the presence of a tumor. Liquid biopsy is well-suited for "longitudinal studies" that involve collecting biological samples at different time points during the clinical and instrumental surveillance program, enabling the monitoring of the dynamic evolution of cancer over time. Therefore, liquid biopsy not only aids in diagnosis but also allows for adapting therapy over time by following the molecular changes that occur during the disease's progression.
Through a liquid biopsy approach, the BiLiGeCT project aims to address specific questions related to early diagnosis in healthy individuals with a high genetic risk of tumors, particularly those with hereditary/familial BRCA (1 and 2) gene mutations. The objective is to develop a more sensitive, effective, and cost-efficient diagnostic tool compared to current instrumental screening methods.
The second line of research, similar to the first, focuses on individuals with a previous history of tumors in the context of hereditary/familial BRCA-related cancers. The aim is to identify tumor recurrence early and characterize it molecularly to monitor the effectiveness and appropriateness of therapy.
The third line of research focuses on early diagnosis using liquid biopsies, following similar approaches as described for hereditary/familial tumors, but specifically targeting lung and prostate cancers. The objective is to make disease diagnoses more effective and less invasive while introducing technological innovations for molecular diagnosis and prognosis of these tumors.
The fourth line of research will address the issue of early diagnosis, especially in the case of metastatic disease recurrence and the choice based on the molecular profile of the evolving disease during second-line treatment and beyond in colorectal tumors.
The fifth line of research aims to develop an innovative cellular test for screening new therapeutically active molecules or the repositioning of already known drugs for mutations in the BRCA genes and those genetic elements that collaborate with them to ensure the integrity of DNA through its repair mechanisms (BRCAness). These drugs, in general, belong to the category of so-called PARP enzyme system inhibitors. These inhibitors have been found to be effective not only for hereditary-familial tumors with the aforementioned germline mutations but also for sporadic tumors of the same histotype with somatic mutations in these genes. With this functional model, the problem posed by BRCA mutations of unknown functional significance (VUS) will also be addressed. These mutations will be tested for their sensitivity to PARP inhibitors active on tumors with clearly pathogenic BRCA mutations.
The ultimate goal of the project is rightly ambitious and is at the forefront of current technology. However, it appears feasible thanks to the infrastructure and previous experience in the use of innovative technologies of the participating institutions and the qualified expertise available within them. Furthermore, an undeniable added value is provided by the availability of a cohort of healthy individuals or those with previous experience of tumor disease belonging to the population at genetic risk of developing breast and/or ovarian cancer because they carry a germline mutation in one of the BRCA genes. This opportunity appears unique for the diagnostic objectives of liquid biopsy, both because the type of tumor that will appear can be predicted "a priori" due to its characteristic of earliness, and because several molecular aspects of the tumors in question are well-known and will allow the development of highly personalized molecular diagnostic tools for these patients.
Additionally, with the liquid biopsy approach, it will be possible to monitor the cost-effectiveness of the introduction of these new diagnostic methodologies.
The successful achievement of the project's final objectives will allow for the introduction of both innovative laboratory techniques and original diagnostic assays in the market.

The operational headquarters of Cogentech Società Benefit SRL for the BiLiGeCT project is at the Science and Technology Park of Sicily, Zona Industriale, Blocco Palma 1, 95121 Catania (CT).

Nina Offenhauser, Project Manager
nina.offenhauser[@]cogentech.it

Marco Pierotti, Scientific Director
marco.pierotti[@]cogentech.it