Results
First stage (May 2018- December 2018)
One of the priorities in the first stage of PANCNGS project was represented by the design and optimization of the protocols. Seven researchers have joined the team, including 3 Research Assistants at Fundeni Clinical Institute, one Research Assistant at IBPC, 1 PhD Student at the University of Bucharest, one Research Assistant at the University of Medicine and Pharmacy Iasi, and one Research Assistant, who is also a PhD Student, at the University of Medicine and Pharmacy in Craiova.
- According to the research design, we have started including patients in the first stage of the project. The Coordinating Institution, Fundeni Clinical Institute, selected 38 patients with pancreatic cancer. For these patients, we have collected, registered and stored in the biobank biological samples consisting of tumor and non-tumor tissue, and also blood. In the 4th subproject, the team has performed Fine Needle Aspiration (FNA) in order to obtain tumor samples from patients with non-operable cancer. Whole-Exome Sequencing and Whole-Transcriptome Sequencing were performed for 8 samples of tumor and non-tumor tissue, and a total of 250 Gb of data were obtained. Furthermore, using the somatic mutation registry COSMIC, we have selected a panel of 40 genes, relevant for the diagnosis, prognosis and personalized treatment of pancreatic cancer.
- The first partner in the project (P1), the Surgical Clinic of the Regional Oncology Hospital in Iasi contributed to the project by including 10 patients diagnosed with resectable cancer, considered for curative treatment.
- University of Bucharest (P3) performed the RT-PCR analysis for the expression of TRPM8 in different pancreatic cancer cell lines (MIA PaCa-2, Panc-1 and PK-9), but also in the non-cancer line HPDE. Also, in the 5th subproject, the team optimized the transfection methods for the TRP receptors in cells that express the regulatory protein.
- IBPC Institute (P4) optimized the different steps in the laser microdissection protocol, including the fixation, coloring and dehydration of the cryosections.
Second Stage (January 2019- December 2019)
In the second stageof the Complex Project 66PCCDI/2018, which took place between January 2019 and December 2019, whole exome analysis, whole transcriptome analysis and targeted sequencing analysis were performed. For human resources improvement, in this interval, 7 new researchers: 2 research assistants – ICF, 3 PhD students – UB, 1 research assistant, PhD student – UMF Craiova were recruited.
In this study, CO has included 100 patients with pancreatic tumors (prospective, in Pr 1, Pr 2 and Pr 3). The pancreatic ductal adenocarcinoma (PDAC) diagnosis was confirmed for 27 of the 100 patients from which tumor tissue samples, non-tumor tissue samples and blood were collected. In the Surgery Clinic I of the Iași Regional Oncology Institute, 9 patients diagnosed with curative-intent resectable pancreatic cancer were included. In the sub-project P4 the nucleic acid extraction from FNA tissue samples or ccfDNA from pancreatic adenocarcinoma patients protocol was developed and implemented. The prospective clinical database, at the moment containing 86 subjects (55 included by CO ICFundeni and 31 included by Partner P3 UMF Craiova), was updated. The samples were banked and stored in optimal conditions at the Translational Medicine Center, Fundeni Clinical Institute (CEMT), obtaining thus a valuable biological material bank (tumor and non-tumor tissue, blood and plasma) from PDAC patients.
From the sequencing analysis of 20 micro-dissected tumor tissue samples (confirmed as PDAC) by whole exome sequencing (WES), about 200 GB of data were generated and more than 1200 indel-type mutations were found for each analysed sample. From the whole transcriptome sequencing analysis (WTS), about 250 GB of data were obtained and after bioinformatic analysis, more than 600 genes with differential expression between the tumor tissue and non-tumor tissue were found. In sub-project 4 a panel of 1121 somatic mutations comprising hot spots of 40 genes significant for pancreatic adenocarcinoma was constructed. These genes were selected after studying the COSMIC database of somatic mutations, including the genes with the highest significance in the diagnosis, prognosis and personalised therapy of pancreatic adenocarcinoma. Using the Illumina Nextseq500 sequencing platform (CEMT ICFundeni) new generation targeted sequencing was performed in a group of 28 patients with histopathologically confirmed PDAC. As result of this sequencing, 114 and 104 genetic variants were identified in FNA and ccfDNA, respectively, with over 90% agreement between the two DNA matrices. This is the first study of its kind conducted in Romania. In sub-project 5, the function and structure of pancreatic adenocarcinoma (ADK) – expressed TRP receptors were studied. Thus, results on TRP proteins expression in ADK tissues from the Fundeni Clinical Institute patient tissue database were obtained. At UB, the glycosylation status of TRPM8 was assayed by Terahertz spectroscopy and molecular modeling. These results were submitted for publication at the ”Biophysical Biochimica ACTA General Subjects” journal. The expression and function of TRPA1 proteins were studied in ADK cell lines and the results were presented at the International Biophysics Congress in Madrid, 20-24th of July, 2019. To assess the function of TRPM8 receptor in gentamicine resistance, pilot studies on gentamicine-conditioned cell lines were performed.
Third Stage (January 2020- December 2020)
In the 3rd stage of the Complex Project 66PCCDI/2018, which took place between January 2020 and December 2020, whole exome analysis, whole transcriptome analysis and targeted sequencing analysis were performed. In this study, CO has included (prospective, in Pr 1, Pr 2 and Pr 3) 101 patients with PDAC from which tumor tissue samples, non-tumor tissue samples and blood were collected. Among these patients, 19 were included in this stage (CO and P1). Moreover, the samples were banked and stored in optimal conditions at the Translational Medicine Center, Fundeni Clinical Institute (CEMT), obtaining thus a valuable biological material bank (tumor and non-tumor tissue, blood and plasma) from PDAC patients. In sub-project 4, 10 patients with unresecable pancreatic tumors (CO) and 31 patients from P2 (22 with confirmed PDAC diagnosis) were included in the study. Also in sub-project 4, the group for prospective validation of NGS sequencing-generated mutational profile data was generated. In this group 68 new patients diagnosed with unresecable pancreatic cancer were included. From the sequencing analysis of 20 micro-dissected tumor tissue samples (confirmed as PDAC) by whole exome sequencing (WES), about 200 GB of data were generated and more than 1200 indel-type mutations were found for each analysed sample. From the whole transcriptome sequencing analysis (WTS), about 250 GB of data were obtained and after bioinformatic analysis, more than 600 genes with differential expression between the tumor tissue and non-tumor tissue were found. During activity 3.22, CO has investigated the mutational profile in unresecable pancreatic cancer – diagnosed patients from activity 2.4.2, using new generation “targeted sequencing” technique, using a 1% filter for genetic variants, raising thus the accuracy of the assay and the chance of detecting somatic mutations specific for PDAC. In activity A.3.23, Co and P1 researched multiple methods for the validation of sequencing data such as: sequencing using TAQMAN, Sanger sequencing, CAST-PCR, pyrosequencing, digital droplet PCR etc. One of the most useful methods for clinical use, also investigated in Pr4, is genotyping using TAQMAN probes, due to its reduced cost and capacity to investigate the mutational profile in individual cases. The research showed a good agreement between results obtained by TAQMAN genotyping using gDNA extracted from FNA and cfDNA, respectively. In activity A.3.24, CO and P2 used TAQMAN, DD-PCR and SNV Nanostring sequencing techniques for the validation of the results obtained in the previous activity. Mutational profiles were systemized in groups significant for personalised medicine and a protocol for the genetic assay of patients with unresecable locoregional advanced pancreatic cancer was proposed, having clinical applicability in view of personalised medicine. After clinical validation, this protocol could adress about 40% of the total number of patients with unresecable ADK at diagnosis, recommending personalised therapies available in the present for the treatment (A3.25). In sub-project 5 (Pr5), TRP function was analysed during tumorigenesis. Thus, we obtained results on TRPA1 protein expression in cell cultures and their role in cell cycle and cell migration. The P3 partner performed experiments to assay the expression and function of TRPA1 proteins in ADK cell lines, whose results were sent for publication and are now reviewed by the Scientific Reports journal. We also performed pilot studies on gentamicine-conditioned cell lines to assess the effect of gentamicine resistance on the TRPM8 function. The results on TRPM8 protein glycolisation status obtained by Terahertz spectroscopy and molecular modelling were published in the ”Biophysical Biochimica ACTA General Subjects” journal. We continued training a number of four PhD students in our research team. CO performed experiments to determine the gene expression of TRP family members using RT-PCR on a 95-patients lot (pairs of tumor and normal tissues).
Fourth Stage (January 2020- September 2021)
In the 4th stage of the Complex Project 66PCCDI/2018, which took place between January 2021 and September 2021, the analysis and interpretation of genomic data and validation of mutational profiles in PDAC patients were performed. During this stage, 55 patients with pancreatic tumors were evaluated (prospectively in Pr 1, Pr 2 and Pr 3). In this study has been included 18 patients confirmed with the ductal pancreatic adenocarcinoma (PDAC) and were collected biological samples (tumor, non-tumor tissue and blood). Within Surgery Clinical I of the Iași Regional Institute of Oncology, between January 2021 and September 2021, a patient diagnosed with pancreatic cancer (PDAC) resectable were included. Moreover, in the Fundeni Clinical Institute, CEMT, and the samples were preserved and stored in optimal conditions, thus creating a valuable biobank of biological material (tumor and non-tumor tissue, blood and plasma) derived from PDAC patients. In subproject 4, 22 patients with non-resectable pancreatic tumors were included in the study. Following the sequencing of the whole exome (WES) for 30 tumor and non-tumor tissue samples (confirmed PDAC), approximately 225 GB of genomic data was generated (FASTQ + BAM files). The results indicated that, according to the PolyPhen score, a mutational profile was obtained for the group of tumor samples derived from microdissected tissue divided as follows: 76% are considered benign mutations (without known implications in tumorigenesis), 14% mutations with a potential risk in tumorigenesis processes and 10% mutations with an increased risk in tumorigenesis processes in patients with PDAC, being similar to those obtained in bulk tumor samples. The genes with the best concordance and with mutational frequency > 90% in both groups of samples are: TP53, SYNE1, ERBB2, CFTR. In subproject 4, a customized panel was set up comprising 40 genes of significance for pancreatic adenocarcinoma. After reviewing the COSMIC somatic mutation database, these genes were selected as being the most important for diagnosis, prognosis, and personalized therapy. Subproject 5 aimed to establish the culture conditions of the cell line for the PDAC study, also optimized the transfection methods of TRP receptors in cells that express the regulatory protein.