Prognostic Molecular Subtypes of Low-Grade Cancer of the Appendix

Abstract

Background: Appendiceal cancer (AC) patients treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) often demonstrate an unpredictable variability in their survival outcomes. Biomarkers predictive of CRS/HIPEC efficacy could better guide treatment decisions. We hypothesized that variation in the transcriptional programming of AC tumors might distinguish molecular subtypes with differential outcomes after CRS/HIPEC.

Study design: Gene expression profiles of 2 AC cohorts were analyzed using Affymetrix whole-genome expression microarrays. Hierarchical clustering methods, Kaplan-Meier analysis, and Cox regression models were used to discover and validate prognostic molecular subtypes of AC. Gene set enrichment analysis was used to infer pathologic attributes of the molecular subtypes.

Results: Unsupervised hierarchical clustering analysis of tumor expression profiles revealed a 139-gene cassette that distinguished 2 molecular subtypes (based on low vs high expression of the gene cassette) with statistically significant survival differences (disease-specific survival, p = 0.0075; progression-free survival, p = 0.0072). In a second AC cohort, the 139-gene cassette reproducibly partitioned tumors into subtypes with significant survival differences. Tumors showing high relative expression of the genes comprising the cassette associated with poor survival outcomes (disease-specific survival, p = 0.047; progression-free survival, p = 0.0079), and exhibited gene expression patterns enriched for oncogenic processes and pathways. The prognostic value of the molecular subtypes was specific for low-grade appendiceal tumors (disease-specific survival, p = 0.028; progression-free survival, p = 0.0016), and remained significant in the presence of conventional prognostic markers, including grade, surgical resection score, Eastern Cooperative Oncology Group status, and age.

Conclusions: The 139-gene cassette can have actionable clinical utility for identifying low-grade appendiceal tumor molecular subtypes predictive of therapeutic efficacy of CRS/HIPEC.

Figure 1

Histology of peritoneal mucinous tumors. (A) Low-grade tumor showing simple bland appearing epithelium and abundant mucin. (B) High-grade tumor with signet-cell morphology and tissue invasion. Hematoxylin and eosin, 20× (A) and 40× (B) magnification.

Figure 2

Unsupervised hierarchical clustering discerns prognostic tumor subtypes. (A) Heatmap of clustered gene expression patterns (rows) and tumor expression profiles (columns) is shown. Red indicates above-mean expression; green indicates below-mean expression. Degree of color saturation reflects the magnitude of gene expression. (B, C) Kaplan-Meier survival curves corresponding by color to the tumor branches designated in (A) are shown for (B) disease-specific survival and (C) progression-free survival.

Figure 3

Prognostic tumor clusters based on the 139-gene cassette. (A) Genes comprising the 139-gene cassette cluster the tumors into 2 primary branches (subtypes) based on relative high and low gene expression. (B, C) Kaplan-Meier survival curves corresponding by color to the tumor branches designated in (A) are shown for (B) disease-specific survival and (C) progression-free survival.

Figure 4

The prognostic subtypes are reproducible entities. (A) The 139-gene cassette was used to cluster an independent appendiceal tumor cohort. The resulting tumor subtypes were analyzed for survival associations. (B, C) Kaplan-Meier plots for (B) disease-specific survival and (C) progression-free survival are shown. (D, E) Kaplan-Meier plots for (D) disease-specific survival and (E) progression-free survival are shown for patients with low-grade disease only.

Figure 5

Biologic processes and pathways delineate the prognostic subtypes. Gene set enrichment analysis was performed on the genes differentially expression between subtypes. Gene set enrichment analysis gene set titles are listed above each panel. Enrichment scores (green line) and their corresponding false discovery rate-adjusted significance (q-values) are shown. Black vertical bars indicate the position of genes belonging to the gene set along the rank-ordered dataset. Red indicates overexpression (greater mean log fold change [LFC]) in the favorable subtype; blue indicates overexpression in the poor prognosis subtype. KEGG, Kyoto Encyclopedia of Genes and Genomes.