Assessment of pancreatic neuroendocrine tumor cytologic genotype diversity to guide personalized medicine using a custom gastroenteropancreatic next-generation sequencing panel

Abstract

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.

Advances in the cytologic diagnosis of gastroenteropancreatic neuroendocrine neoplasms

“…Genotyping these neoplasms can aid in diagnosis, potentially predict behavior, and guide therapy, particularly with regard to agents affecting the mTOR pathway. Cytology is known to be highly accurate for diagnosis and staging of these neoplasms, but it is also a highly effective, nonoperative technique for acquiring concentrated cellular tumor samples with well preserved genetic material that can be used successfully for next‐generation sequencing . Areas of opportunity for further advancement in cytology in the field of gastroenteropancreatic neuroendocrine neoplasms include validation of surrogate markers for NET genotype and investigation into the potential role of cytology in assessing for emerging epigenetic modifications and biomarkers with potential prognostic and/or therapeutic importance.…”

“…Retained nuclear staining for these markers is not helpful. Immunohistochemistry for p53 is another candidate marker for this differential diagnosis, because tumor protein p53 ( TP53 ) mutations are common in NEC and are relatively infrequent in NET; however, currently, there is less evidence indicating the appropriate threshold for a positive stain, and the utility is uncertain …”

“…Next‐generation sequencing of cytology specimens that contain neuroendocrine neoplasms demonstrates genotypes similar to those identified in histologic studies . The most common somatic mutations of panNECs are TP53 , v‐Ki‐Ras2 Kirsten rat sarcoma 2 viral oncogene ( KRAS ), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PIK3) catalytic subunit α/phosphatase and tensin homolog ( PIK3CA / PTEN ), and B‐raf proto‐oncogene serine threonine kinase ( BRAF ) .…”

“…Overall, in panNETs, the main genetic pathways affected are DNA damage repair, chromatin remodeling, telomere alteration, and activation of the PI3K/mammalian target of rapamycin (PI3K/mTOR) signaling pathway. The most common recurrent genetic mutations are multiple endocrine neoplasia type 1 ( MEN1 ) (42%‐45%), DAXX (11%‐25%), and ATRX (10%‐18%) . Mutations in the mTOR pathway genes tuberous sclerosis complex 2( TSC2 ) (8%), PTEN (7%), and PIK3CA (1%) also are observed in panNETs.…”

“…Immunohistochemistry for p53 is another candidate marker for this differential diagnosis, because tumor protein p53 (TP53) mutations are common in NEC and are relatively infrequent in NET; however, currently, there is less evidence indicating the appropriate threshold for a positive stain, and the utility is uncertain. 25,36,37 Clinical history is helpful for supporting the differentiation of neuroendocrine neoplasms. Grade 3 NETs can (but rarely do) present de novo, and most patients have a known history of grade 1 and 2 NET that may have originated up to 10 years prior.…”