Long-term survival of PAC remains stubbornly low and there is an unmet need for early detection and efficient systemic treatment. Although treatment outcomes for many types of cancer has improved, PAC survival has lagged significantly behind in the last decades. One major limitation comes from very few treatment options for PAC. A better understanding of PAC may lead to new treatment options and improved clinical outcomes for this lethal disease . In current study, we hypothesized that by using NGS, genetic alterations guiding the selection of targeted therapies could be identified. By predefining a set of relevant somatic alternations, targeted panels identified variants which could be linked to potentially therapeutic strategies [19–20]. Two commercialized panels, namely the OCP and CHP, were adopted to fulfill the purpose of NGS experiments and novel therapeutics identification. The OCP was designed for compatibility with routine FFPE tissues, with co-isolation of DNA and RNA. The OCP consisted of multiplexed PCR compatible with 20 ng of DNA and 15 ng of RNA input, further augmenting its clinical applicability. The CHP provided a more cost-effectiveness and scalable solution for routine practice as 10 ng of input tumor genomic DNA from FFPE tissues is adequate for targeted library construction.
Initially, 6 Taiwanese PAC FFPE samples were evaluated with metastatic lesion DNA extracted, and 5 with adequate nucleic acid were sequence by the OCP. Up to150 and 200 alternations were found in each sample while most were probable bystanders with no relevant therapy. In part I of the study, there was at least one actionable mutation found in each PAC patient, making personalized therapy achievable. These actionable mutations corresponded to potentially matched treatments, which could be the targets of novel therapeutics.
The presence of KRAS mutation may be a predictive biomarker against the use of anti-EGFR antibody. In lung cancer treatment, KRAS is downstream in the EGFR pathway; therefore, tyrosine kinase-based treatment with gefitinib and erlotinib is ineffective when KRAS is constitutively activated [21–22]. However, many clinical trials which combine other tyrosine kinase inhibitors (TKIs) and chemotherapy are ongoing for these KRAS mutant PACs . In addition, knockdown of mutant KRAS with RNA interference may be a potentially therapeutic strategy in near future . Recently, a subset of KRAS wild-type younger PACs has been identified for whom the recognition of alternative oncogenic drivers which are also targetable is in eagerly need . There was one FGFR2 mutant case (FJU01) and the NGS result showed the possibility of FGFR TKI treatment in the future as there are several FGFR TKI trials [26–27]. Although RNA sequencing showed no fusion gene in current study, there are more and more evidences suggesting that fusion oncogenes present not only in sarcoma, but also in carcinoma . This kind of alternation could also be a potential biomarker for targeted therapy, such as crizotinib for treating lung cancer with EML4-ALK translocation . Many CNVs were also found in this study while their meaning needs further investigation. Further studies to clarify whether the gain of oncogenes or loss of suppressors represent prognostic or predictive biomarkers for PAC are warranted.
The CHP was adopted for part II of the study with more PAC samples assayed. The choice of the CHP rather than OCP was based on economic consideration. The CHP v2 surveyed hotspot regions of 50 oncogenes and tumor suppressor genes, with wide coverage of KRAS, BRAF and EGFR genes, which were evidenced as being PAC-relevant from part I of the study. It deserves notice that it’s also the CHP platform which was adopted in NCI’s MATCH trial . An example of COSMIC 518 KRAS mutation involved in MAPK, EGFR1, IL2, IL3, IL5, and ErbB pathways with a high pathogenic score. Depending on the impacted subject/variant count, the most common actionable mutations came from KRAS (23 samples/variants), HRAS (14 samples/variants), TP53 (11 samples/29 variants), and PDGFRA (10 samples/variants). Supplementary Table 1 showed that up to 5 variants could be detected within the same subject. Although COSMIC database identified KRAS, PFGFRA and KIT as being pathogenic (Supplementary Table 2), only KRAS was predictive in terms of matched alternation-drug combination. A mutated KRAS gene is a biomarker for many cancer types, as this gene has control over cell cycle division and cancer cell growth.
It’s convenient to conduct summarized cohort analysis and subgroup analysis by the method of variant impact heat maps, as moderate sample size of part II made whole cohort and subgroup clustering analyses possible. Variant impact heat map of PACs with mucinous and pancreatic intraepithelial neoplasm (PIN) precursor showed roughly comparable distributions of genetic alternations in TP53, APC, SMAD4, PTEN, PIK3CA and CDKN2A (Supplementary Fig. 4). Although Ryan et al. highlighted that more than 90% of PAC were associated with activating KRAS mutation, the frequency was much higher for intraepithelial neoplasm (> 90%) then mucinous neoplasm (40–65%). Other suppressors such as CDKN2A, TP53 and SMAD4, the aberrant rate increased with higher nuclear grade, albeit alterations in tumor suppressor genes rarely led to targeted therpay. The purposed more GNAS oncogenic mutation was not observed in PAC with mucinous precursor in current study, either .
Singhi et al. conducted by far the largest study on around 3,600 PACs and found that the most frequently mutated genes, i.e. KRAS, TP53, CDKN21, and SMAD4, can’t be targeted with readily available existing drugs . In 2020, FDA approved olaparib for maintenance treatment of germline BRCA-mutated metastatic PAC whose disease has not progressed on at least 16 weeks of a first-line platinum-based chemotherapy, based on phase III POLO trial .The most common pathogenic germline mutations are in BRCA1, BRCA2, and ATM, and more rarely, in PALB2, MLH1, MSH2, MSH6, PMS2, CDKN2A, and TP53, among others, for an aggregate frequency of 3.8–9.7% [33–34]. Although somatic alternations were interrogated in current study, several of aberrant DNA damage repair genes were reported from Taiwanese PAC patients and reflux germline testing should be indicated once alternations in tumor-only sequencing were evidenced.
There were some limitations of the study. First, the modest sample size might hamper externalization of sequencing results. The 50 targeted genes of the CHP had been covered by the OCP, so there was no concern of comparability. In addition to the limited sample size, it should be noted that only 15% of PACs were resectable at time of diagnosis, which means the majority of PAC genetic analyses were conducted on early stage disease [35–36]. In current study, part I samples were derived from metastatic lesions while for part II, all specimens came from primary pancreatic neoplastic tissue in an effort to broaden clinical scenarios of PAC . Second, although PAC is characterized by diverse, largescale chromosomal changes with forms of amplifications, deletions, and rearrangement, only the five OCP assays interrogated CNVs and structural aberrations of the 38 CHP assays were left undetermined. Third, as PAC is hard-to-diagnose due to the difficulty in obtaining tumor samples from patients, the feasibility and prognostic value of circulating tumor DNA (ctDNA) in PAC is being tested rigorously . The knowledge learned from current study and other genetic studies may pave the way for future circulating biomarkers to screen, guide treatment, and monitor disease progress of PAC.