BackgroundRecent genetic studies have highlighted that alterations in MEN1, chromatin remodeling genes, and mammalian target of rapamycin (mTOR) pathway genes are the most frequent molecular events identified in pancreas neuroendocrine tumors (pNETs). The prognostic or predictive impact of these biomarkers and other less frequently observed aberrations, i.e. PTEN, TSC2 and PIK3CA are relatively unknown. The aims of this targeted next generation sequencing (NGS) study were to assess tumor cytology genotype diversity, to survey for potential adverse prognostic biomarkers and the prevalence of mTOR pathway variants.MethodsUsing a custom 15 gene gastroenteropancreatic neuroendocrine tumor panel, targeted NGS of archived (2002-2013) primary pNETs (n=90) and pNET liver metastasis (n=32) cytology smears was performed.ResultsThe genetic variant landscape revealed that 21% and 28% of primary and metastatic liver pNETs harbored ≥ 2 variants per tumor, respectively. The most prevalent primary tumor variants were in the MEN1 (42%), DAXX (11%), ATRX (10%), and TSC2 (8%) genes. Patients harboring aberrations in TSC2, KRAS or TP53 were more likely to experience disease progression and reduced overall survival, when compared to individuals who were wild-type. The prevalence of these potential prognostic biomarkers in early disease was observed in 3.3% of the primary tumor cohort. mTOR pathway variants including alterations in PTEN, TSC2 and PIK3CA were identified in 10% and 12.5% of primary tumors and pNET liver metastasis, respectively.ConclusionCytology based tumor genotyping revealed a broad spectrum of genetic variants including possible adverse prognostic biomarkers, reflective of an aggressive phenotype. It also demonstrated the prevalence of potential predictive biomarkers for mTOR pathway inhibitor sensitivity.

In patients with PDAC, EUS-FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with predilection for peritoneal dissemination. Accurate peritoneal staging is imperative for treatment recommendations, as one-third of patients develop peritoneal recurrence after resection. Because >90% of PDAC tumors harbor mutant KRAS (mKRAS), we sought to determine feasibility of mKRAS DNA detection in peritoneal lavage (PL) fluid using droplet-digital polymerase chain reaction (ddPCR) via a prospective trial. STUDY DESIGN: Patients with nonmetastatic PDAC undergoing staging laparoscopy with PL were included. PL fluid was sent for cytologic examination, CA19-9/CEA levels, and mKRAS ddPCR assay. Clinically positive laparoscopy was defined as gross metastases or positive cytology. PL mKRAS status was compared with gross findings, cytology, and CA19-9/CEA levels. RESULTS: There were 136 patients enrolled; 70 of 136 (51%) patients received neoadjuvant therapy before PL, and 32 of 136 (24%) patients had clinically positive laparoscopy. Cytology was positive in 17 of 136 (13%) patients, and 22 of 136 (16%) patients had gross metastases. Of patients with gross metastases, only 8 of 22 (36%) had positive cytology; 97 of 136 (71%) patients had mKRAS in PL. PL mKRAS was present in 27 of 32 (84%) clinically positive laparoscopies, with higher mean copy number in clinically positive patients (643 vs 10, p = 0.02). Peritoneal mKRAS was positive in an additional 70 clinically negative patients. CONCLUSIONS: This prospective study establishes the feasibility of PL mKRAS detection. Clinically positive disease was identified in 1 in 4 staging laparoscopies. Although PL mKRAS was highly associated with clinically positive findings, many clinically negative laparoscopies had detectable PL mKRAS, suggesting that standard staging may be inadequate. Longer follow-up will elucidate utility of this promising molecular assay.

Background: Biomarker testing in lung cancer is often limited by a lack of sufficient formalin fixed, paraffin embedded (FFPE) tissue for comprehensive genomic profiling. To promote personalized therapy for lung cancer, a multiplex FISH assay was developed to simultaneously assess aberrations in ROS1, RET, and MET on a single FFPE specimen slide. Methods: Specimens included primary tumor (N = 39) as well as biopsies from a variety of metastatic sites (N = 16). These included 12 samples with ROS1 rearrangements, 3 samples with RET rearrangements, and 11 samples with MET amplification reported by a previously validated laboratory test method. A probe mix contained 6 differentially labeled fluorescent probes: 3' ROS1, 5' ROS1, 3' RET, 5' RET, MET and CEP7. The probes were formulated in Vysis IntelliFISH Hybridization Buffer to allow for a 2 h hybridization time. BioView imaging platform and Duet software algorithm were used to perform automated slide scanning and digital analysis. Specimens were considered positive for ROS1 or RET rearrangement if >15% evaluated cells contained a break apart (rearranged) signal. Specimens were considered positive for MET amplification if >15% of cells had MET/CEP7 ratio >2 and positive for polysomy if >15% of cells had 5 or more MET signals copies. Results: The 6 color FISH assay was 97% concordant for ROS1 rearrangement and 100% concordant for RET rearrangement. The average background percentage of positive tumor cells in cases without known gene rearrangements was approximately 5%, yielding a negative cutoff threshold of approximately 15%, in accordance with cutoff thresholds reported in literature. The 6 color FISH assay was 91% concordant for MET amplification or polysomy. Results were interpretable for 98% of targets analyzed by the 6 color FISH method. Four samples failed on analysis for one of the targets due to lack of sufficient cells or lack of adequate hybridization signal. Conclusion: A newly developed 6-color FISH assay allows simultaneous detection of three genomic abnormalities using only 1 specimen slide. This feature combined with rapid hybridization in IntelliFISH buffer and automated BioView slide imaging and analysis can significantly increase the yield of molecular testing on limited lung cancer tissue samples. Careful pathologic correlation for tumor cell identification and careful assessment of hybridization quality are necessary to optimize the accuracy of this test method. Citation Format: Irina A. Sokolova, Patrick Bedroske, Tatyana A. Grushko, Amber R. Schneider, Kristine Jacobson, Jesse S. Voss, Yishay Tauber, Yossi Avisror, Vitaliy Shkolnik, Katerina Pestova, Katherine B. Geiersbach. Multiplex fast FISH assay for detecting ROS1, RET and MET aberrations in FFPE specimens using BioView image analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4256.

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