Received Date: May 12th, 2021; Accepted Date: May 17th, 2021
Context Pancreatic cancer is the fourth most common cause of cancer death in Europe characterized by disappointing tumor response rates and high levels of resistance to standard treatments. Understanding genomic variations in pancreatic cancer is crucial, as they are known to contribute to pancreatic carcinogenesis and may provide fundamental knowledge for new and effective treatment strategies. Objective The main purpose of this exploratory study was to try to characterize the genomic profile of nine pancreatic tumors of patients treated at our institution. Methods Primary or metastatic tumor samples from patients with pancreatic cancer were analysed by FoundationOne CDx (F1CDx) Next Generation Sequencing (NGS) diagnostic test and clinical data were retrieved from the patients electronic medical record. Results Nine patients with cytological or histological documentation of pancreatic cancer treated at the Medical Oncology Department of Hospital da Luz Lisboa between October and December of 2017 were included. F1CDx NGS identified genomic variants with clinical significance in all 9 samples in the following genes: KRAS (7/9), TP53 (5/9), SMAD4 (3/9), CTNNB1 (1/9), CDKN2A (1/9), MDM4 (1/9), ARID1A (1/9), ARID2 (1/9), PIK3C2B (1/9) and FANCA (1/9). No tumor sample had microsatellite instability or high mutational burden. Actionable genomic alterations were identified in 7 tumors. However, no patient underwent targeted therapy. Conclusion This exploratory cohort, although small in size, documents the genetic heterogeneity of pancreatic carcinoma and confirms RAS, TP53 and SMAD4 as the most common genetic alteration in this tumor type. However the utility of this test to foster inclusion in clinical trials is also conditioned by their accessibility (at the time no such trials were open in Portugal). Further studies are needed to validate the clinical utility of F1CDx in clinical practice.
Pancreatic; Cancer; Next generation sequence; Genomic profile
Pancreatic cancer is the fourth most common cause of cancer death in Europe, with stable or slightly increasing mortality rates [1, 2]. Despite decades of research and therapeutic development, five-year survival rate remains below 5%. The disappointing response rates and high levels of resistance to standard treatments highlight the urgent need for novel treatments for patients with pancreatic cancer. The study of genomic alterations of pancreatic cancer may provide insight into targets for treatment . Precision medicine clinical trials such as IMPACT , SAFIR 01 , MOSCATO  and SHIVA , suggest that this strategy is challenging but feasible. The recently published POLO clinical trial, brought new hope for target therapy in pancreatic cancer. It documented the benefit of olaparib, a poly adenosine diphosphate ribose polymerase (PARP) inhibitor, as maintenance therapy, in patients with germline BRCA mutation and metastatic pancreatic carcinoma without progression after at least 16 weeks of platinumbased chemotherapy metastatic pancreatic carcinoma with germline BRCA mutation without progression after at least 16 weeks of platinum-based chemotherapy. The median progression-free survival was significantly longer in the olaparib group than in the placebo group (7.4 months vs. 3.8 months; hazard ratio for disease progression or death, 0.53; 95% confidence interval [CI], 0.35 to 0.82; P=0.004) .
Also, patients with MSI-H and NTRK fusion-positive tumours presented meaningful clinical benefit with matched therapies in multi-histology studies [9, 10]. Currently, it is not recommended to perform tumour multigene Next-generation sequencing (NGS) in patients with pancreatic cancer in routine clinical practice unless in the context of molecular screening programmes, to allow access to innovative drugs . The main purpose of this study was to characterize the genomic profile of nine pancreatic tumors of patients treated at our institution.
We applied FoundationOne®CDx (F1CDx) NGS test (provided by Roche Foundation Medicine®) to primary or metastatic lesions obtained from patients with pancreatic cancer. Eligible patients had to be 18 years or older and had to have a histologically/cytologically confirmed pancreatic cancer, American Joint Committee on Cancer (AJCC) stage I-IV, submitted to surgery, chemotherapy or/and radiotherapy and followed up in the Oncology Outpatient Clinic between October and December of 2017. Patients were excluded if they had documented hereditary cancer susceptibility syndromes, other neoplasms in the previous 5 years, except for non-melanoma skin cancer or life expectancy less than 3 months.
F1CDx uses DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens and applies NGS to the detection of point mutations (substitutions, insertions and deletions), copy number alterations (CNAs) and selected gene rearrangements, in 324 genes. F1CDx also enables the evaluation of genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) . The test report describes the clinically relevant alterations found in each patient´s tumor and identifies target therapies and clinical trials are indicated for those genomic events. The cut-off date for the data included in this report was 31 March 2021.
An observational descriptive cross-sectional study of 9 patients with pancreatic cancer with tumor samples analysed by F1CDx NGS was done. Clinical data were retrieved from the electronic medical record (EMR) and NGS results from the F1CDx report. The median age of patients was 65 [range 25; 85]; 6 were females and 3 males. Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 in 6 patients, of 1 in 2 patients, and of 2 in 1 patient (Table 1). The diagnoses were made between October 2013 and December 2017 (median of 9.9 months between cancer diagnosis and test request).
There were seven were ductal adenocarcinomas, 1 a solid pseudopapillary tumor of pancreas (SPTP) and 1 a carcinoma not otherwise specified (NOE). The study samples were obtained from the primary tumor in all but in 2 cases, both with samples obtained from peritoneal tumor metastases (Table 2). Stage at diagnosis was (8th edition of the American Joint Committee on Cancer (AJCC)): stage IIA (n = 1), stage IIB (n = 3) and stage IV (n = 4) (the other patients had an SPTP). Among the patients with advanced disease (n=4), three had received 2 or more lines of systemic treatment. At the time of this analysis, 6 patients have died, and 3 patients are alive and disease free. The overall survival for each patient is described in Table 1.
All F1CDx NGS were performed on tumor samples obtained prior to systemic therapy administration. The average time between the date of test request and receipt of the report was 14 days [10; 20], and between the receipt of the report and the date of death was 180 days [15; 440]. Genomic variants with known clinical significance were found in all tumor samples involving the following genes: KRAS (7/9), TP53 (5/9), SMAD4 (3/9), CTNNB1 (1/9), CDKN2A (1/9), MDM4 (1/9), ARID1A (1/9), ARID2 (1/9), PIK3C2B (1/9) and FANCA (1/9) (Table 2). Breakdown of mutations according to the histological subtype are listed in Table 3. Major pathways targeted by genomic alterations are shown in Table 4. Ras-ERK and DNA damage response pathways were the intracellular pathways more commonly involved (8 and 6 samples respectively) (Table 4). Variants of unknown significance (VUS) are listed on Table 5. No tumor sample had microsatellite instability or high mutational burden (but in 2 cases the result was undetermined due to low tumor purity). Drug able genomic alterations were identified in 6 mutated RAS tumors (cobimetinib and trametinib). However, no clinical trials were available in Portugal. No patient received targeted therapy.
Pancreatic cancer treatment is an unmet medical need. Understanding genomic alterations in pancreatic cancer is crucial, as they are known to contribute to pancreatic carcinogenesis and may provide fundamental knowledge for new and effective treatment strategies . In these nine patients evaluated by F1CDx, the most prevalent genomic alteration was in the KRAS gene [G12V (n=3), G12D (n=2), G12R (n=2)]. The proto-oncogene KRAS is known to be mutated in almost 95% of pancreatic ductal adenocarcinoma (PDAC) and unfortunately, there are no therapeutic options that successfully target mutant KRAS . Approximately 90% of patients with PDAC harbour the G12 mutation in KRAS . The presence of a KRAS mutation seems to negatively influence the prognosis, although large-scale studies are certainly required. Targeting of KRAS to treat PDAC has been applied at different stages of the RAS molecular pathway . Proteins downstream of KRAS, such as the RAF/MEK/ ERK pathway have also attracted increasing interest . The clinical trials proposed to the patients with KRAS mutations used MEK inhibitors as therapies: cobimetinib and trametinib.
TP53 is somatically mutated in up to 85% of pancreatic cancers  and it was the second most frequent genomic alteration in our cohort. TP53 has an effect on DNA repair and responds to diverse cellular stresses to regulate target genes that induce cell cycle arrest, thus inducing growth arrest or apoptosis , and is the most commonly inactivated tumour suppressor in PDAC . However, there were no clinical trials targeting TP53 at the time in Portugal. SMAD4 is inactivated in approximately 55% of pancreatic cancers, either by homozygous deletion or by an intragenic mutation in association with loss of the second copy . SMAD4 was the third most frequently identified genomic alteration. SMAD4 mediates the pleiotropic signalling network downstream of the transforming growth factor-β (TGF-β) pathway and exerts paradoxical effects on tumorigenesis. In PDAC, SMAD4 mutations interfere with the trimeric assembly of its C-terminal domain, thus therefore preventing the normal transduction of TGF-β signals . Studies Clinical studies have suggested that SMAD4 inactivation is associated with a poor prognosis [18, 19].
In contrast to ductal adenocarcinomas, the genomic alteration found in SPTP was in the somatic β-catenin coding gene (CTNNB1 S37F) (Table 2). Genomic alterations of KRAS, SMAD4, TP53 and CDKN2A have never been detected in SPTP, differing from the molecular changes seen in adenocarcinoma of the pancreas. Almost all patients with SPTP have mutations of the CTNNB1, and multiple proteins associated with β-catenin have been detected as dysfunctional [20, 21]. Our cohort, although small in size, illustrates the genetic heterogeneity of pancreatic carcinoma. We stress the short time between sample dispatchment and test results, a logistic variable that may be relevant in clinical practice. Furthermore, the report of tests results is easy to interpret and the information on available clinical trials is helpful.
This study has limitations. First, given the small sample size it is not possible to make a correlation between the identified mutations and risk of recurrence or survival. Second, the tumor samples used for analysis were collected at diagnosis, prior to therapy. While this represents the true genetic changes of pancreatic cancer, it will miss treatment-induced changes that may be drug able. According to the European Society for Medical Oncology recommendations, it is acceptable to propose tumor multigene NGS may be offered to patients with advanced PDAC in the context of molecular screening programmers, to screen for access to innovative drugs . However the application of this diagnostic test to foster clinical trials enrolment is also conditioned by their availability.
F1CDx, a NGS diagnostic test allows the rapid characterization of tumor genomic alterations and is a helpful tool to identify potential molecular targets for cancer treatment. This exploratory study of patients with pancreatic cancer studied by F1CDx validates RAS, TP53 and SMAD4 as the most common genomic alteration in this tumor type. The genomic profiling of pancreatic cancer, a disease with limited systemic therapeutic options, may lead to the identification of druggable intracellular pathways targets to be tested in early phase clinical trials. However, further studies are needed to validate its application in clinical practice.
The FoundationOne CDx NGS test was provided by Roche®, for a user experience program, in the launch period of FoundationOne in Portugal.
The author Patrícia Machado has declared associations with Roche Farmacêutica Química as an employee for the last 2 years. The author Catarina Pulido has declared receiving speaker fees from Roche.