Discrimination of low- and high-grade appendiceal mucinous neoplasms by targeted sequencing of cancer-related variants

Abstract

DNA was obtained from matching micro-dissected, primary tumor cells, paired metastases, and peripheral blood mononuclear cells (germline) from patients with appendiceal mucinous neoplasms. We compared specimens from patient cohorts comprising low-grade adenomucinous neoplasm versus high-grade mucinous adenocarcinoma using a targeted, amplicon sequencing panel of 409 cancer related genes (Ion Torrent Comprehensive Cancer Panel, Thermo-Fisher, Waltham, MA). Copy number variants, single nucleotide variants and small insertions/deletions were identified using a multiplex algorithm pipeline (GATK, VarScan2, MuTect2, SIFT, SIFT-INDEL, PolyPhen-2, Provean). There were significantly more damaging variants in high-grade versus low-grade tumor cohorts. Both cohorts contained damaging, heterozygous germline variants (catenin β1; notch receptor 1 and 4) in pathways associated with cell-lineage specification (WNT, NOTCH). Damaging, somatic KRAS proto-oncogene, GTPase mutations were present in both cohorts, while somatic GNAS complex locus mutations were confined to low-grade neoplasms. Variants predominantly affected transcription factors, kinases, and stem cell signaling molecules in canonical pathways including epithelial to mesenchymal transition, stem cell pluripotency, p53, PTEN, and NF-қB signaling pathways. High-grade tumors demonstrated MYC proto-oncogene, bHLH transcription factor (MYC) and death domain associated protein (DAXX) amplification and damaging somatic variants in tumor protein p53 (TP53), likely to amplify an aggressive phenotype. Damaging APC, WNT signaling pathway regulator (APC) deletions were identified in metastatic tissue of both cohorts suggesting a role in invasive disease. Our data suggest that germline dysregulation of WNT and/or NOTCH pathways predisposes patients toward a secretory cell phenotype (i.e., goblet-like cells) upon acquisition of somatic KRAS mutations. Additional somatically acquired variants activating oncogenes MYC and DAXX and inhibiting the critical tumor suppressor, tumor protein TP53, were consistent with manifestation of a high-grade phenotype. These additional changes within the epithelial to mesenchymal transition signaling network (WNT, NOTCH, RAS/ERK/PI3K, PTEN, NF-қB), produce aggressive high-grade tumor characteristics by actively driving cells towards dedifferentiation, proliferation, and migration.

Distinct cell states define the developmental trajectories of mucinous appendiceal neoplasms towards pseudomyxoma metastases

“…The 8q and 21q gains were unique to the PMPs. We observed CNV gains in chromosome 1q and 20q that have been previously identified in mucinous neoplasms of the appendix [25,26].…”

Germline and somatic genetic alterations in two first‐degree relatives with appendiceal low‐grade mucinous carcinoma peritonei

“…The high incidence of KRAS mutations in appendiceal cancers distinguishes them from colorectal cancers and may partially explain their limited response to systemic chemotherapies 40 . Likewise, GNAS mutations are reported in 50%‐72% of AMN, but without correlation to clinical outcomes 41‐43 . Both patients had GNAS mutations at the same loci, but different base substitutions (mother: R201H; daughter: R201C).…”

“…40 Likewise, GNAS mutations are reported in 50%-72% of AMN, but without correlation to clinical outcomes. [41][42][43] Both patients had GNAS mutations at the same loci, but different base substitutions (mother: R201H; daughter: R201C). Borazanci et al profiled 588 appendiceal tumors and found that AMN have approximately the same incidence of GNAS mutations as intraductal papillary mucinous neoplasms, suggesting that it may play a role in disease development and mucin production, and have similar molecular profiles to pancreatic cancers.…”

K-RAS, B-RAF and GNAS Gene Status and Immunohistochemistry Analysis of Mucinous Neoplasm of Appendix

“…This relatively low rate of GNAS mutation in LAMNs might be due to the low sensitivity of Sanger sequencing and limited exon coverage. Alakus and other researchers [24][25][26][27] also proposed that coexisting mutations of KRAS and GNAS were characteristic to LAMN and KRAS mutation occurs earlier in the course of tumorigenesis. This pathway was also shared by IPMN.…”