Genomic profile of pseudomyxoma peritonei analyzed using next‐generation sequencing and immunohistochemistry

Abstract

Pseudomyxoma peritonei (PMP) is a relatively rare clinical syndrome characterized by neoplastic epithelial cells growing in the peritoneal cavity and secreting mucinous ascites. Our aim was to explore the molecular events behind this fatal but under-investigated disease. We extracted DNA from 19 appendix-derived PMP tumors and nine corresponding normal tissues, and analyzed the mutational hotspot areas of 48 cancer-related genes by amplicon-based next-generation sequencing (NGS). Further, we analyzed the protein expression of V600E mutated BRAF, MLH1, MSH2, MSH6 and p53 from a larger set of PMP tumors (n 5 74) using immunohistochemistry. With NGS, we detected activating somatic KRAS mutations in all of the tumors studied. GNAS was mutated in 63% of the tumors with no marked difference between low-grade and high-grade tumors. Only one (5.3%) tumor showed oncogenic PIK3CA mutation, one showed oncogenic AKT1 mutation, three (15.8%) showed SMAD4 mutations and none showed an APC mutation. P53 protein was aberrantly expressed in higher proportion of high-grade tumors as compared with low-grade ones (31.3 vs. 7.1%, respectively; p 5 0.012) and aberrant expression was an independent factor for reduced overall survival (p 5 0.002). BRAF V600E mutation was only found in one (1.4%) high-grade tumor by immunohistochemistry (n 5 74). All the studied tumors expressed mismatch repair proteins MLH1, MSH2 and MSH6. Our results indicate that KRAS mutations are evident in all and GNAS mutations in most of the PMPs, but BRAF V600E, PIK3CA and APC mutations are rare. Aberrantly expressed p53 is associated with high-grade histology and reduced survival.Pseudomyxoma peritonei (PMP) is a relatively rare subtype of mucinous adenocarcinoma with estimated incidence of one to two persons per million per year. 1 PMP arises most often from mucinous neoplasms of the appendix, which leak tumor cells into the peritoneal cavity, colonize peritoneal cavity and produce abundant mucinous ascites. The classical low-grade (LG) form of PMP has been considered to be a rather benign disease, as it progresses slowly and does not by the definition show invasive characteristics. However, progressive obstruction makes even the LG form of PMP a fatal disease. In contrast, high-grade (HG) PMP possess invasive and metastasizing capacity with the 5-year survival being as low as 23%, while for the LG disease it is 63%. 2 At the moment, the optimal treatment for PMP is aggressive cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC). 3 In addition to PMP-related mortality, these complex treatments are, however, associated with considerable morbidity and mortality. 4 Furthermore, not all patients are amenable to these treatment options. 5 Thus, targeted therapies, even ones that would reduce mucin production, would be important additions to the treatment arsenal for PMP patients. To this end, biomarkers and prognostic factors would be helpful to estimate the aggressiveness of the disease and direct personalized treatment options...

Multiple components of PKA and TGF-β pathways are mutated in pseudomyxoma peritonei

“…Macrodissection was used when applicable to increase the yield of tumor DNA. Two to fifteen tissue flakes were deparaffinized and genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Venlo, the Netherlands) as previously reported [9]. DNA quality was inspected with NanoDrop spectrophotometer (Thermo Fischer Scientific, Waltham, MA) and agarose gel electrophoresis (with Midori Green Advanced DNA Stain; Nippon Genetics EUROPE GmbH, Dueren, Germany), and DNA concentration was determined using Qubit dsDNA HS Assay Kit and Qubit 2.0 Fluorometer (Molecular Probes/Life Technologies, Paisley, UK).…”

“…The pileup files for VarScan somatic were created using SAMTools mpileup (v0.1.19) [16] with default parameters except for -q 1 -Q 10. We estimated tumor cell percentage for each sample both visually from the HE stained slides and from the variant allele frequencies (VAF) of KRAS and GNAS variants obtained from our previous targeted deep sequencing of the same samples [9] (Table 1). The average read coverage of the targeted sequencing was 3,580X, making the VAFs and, thus, tumor cell percentages, more reliable than using VAFs from exome sequencing (average coverage 120X).…”

“…[9]**Tumor cell percentage calculated by multiplying the heterozygote VAF (lower value of either KRAS or GNAS) by factor of two.…”

“…In our previous study covering 212 somatic mutation hotspots in 48 cancer related genes in 19 patients [9], we found that all sequenced PMP tumors had an activating KRAS mutation and 12/19 (63%) of the patients harbored a mutation in the GNAS gene. Here, we have sequenced the whole coding genome of nine of these PMP tumors and paired normal tissues in order to identify common mutated genes and pathways outside the scope of the targeted hotspot panels.…”

Genomics of peritoneal surface malignancies

“…20 Prevalence of KRAS & GNAS mutations in LAMN and PMP tumor samples has been reported in various studies with high variability ranging from 53-100% & 40-63% respectively (Table 1). [21][22][23][24][25][26] Differences in cell enrichment methods, genomic sequencing techniques, regional variation and small number patients in most of these studies may have contributed to this variation. Alakus et al reported KRAS and GNAS mutations in 10/10 and 9/10 tumor samples respectively, which is quite higher than previous studies.…”