Regional and temporal heterogeneity of epithelial ovarian cancer tumor biopsies: implications for therapeutic strategies

Abstract

Stage III/IV epithelial ovarian cancer (EOC) is a systemic disease. The clonal relationship among different tumor lesions at diagnosis (spatial heterogeneity) and how tumor clonal architecture evolves over time (temporal heterogeneity) have not yet been defined. Such knowledge would help to develop new target-based strategies, as biomarkers which can adjudge the success of therapeutic intervention should be independent of spatial and temporal heterogeneity. The work described in this paper addresses spatial and temporal heterogeneity in a cohort of 71 tumor biopsies using targeted NGS technology. These samples were taken from twelve high grade serous (HGS) and seven non HSG-EOC, both at the time of primary surgery when the tumor was naïve to chemotherapy and after chemotherapy. Matched tumor lesions growing in the ovary or at other anatomical sites show very different mutational landscapes with branched tumor evolution. Mutations in ATM, ATR,TGFB3,VCAM1 and COL3A1 genes were shared across all lesions. BRCA1 and BRCA2 genes were frequently mutated in synchronous lesions of non HGS-EOC. Relapsed disease seems to originate from resistant clones originally present at the time of primary surgery rather than from resistance acquired de novo during platinum based therapy. Overall the work suggests that EOC continues to evolve. More detailed mapping of genetic lesions is necessary to improve therapeutic strategies.

Keywords: drug resistance; ovarian cancer; spatial heterogeneity; targeted next generation sequencing; temporal heterogeneity.

Figure 1. Patient cohort enrolled in the study.

Graphical representation of clinico-pathological features of patients (n = 19) and tumor biopsies (n = 71) enrolled in the study. Matched blood samples (light blue circles, n = 19) were used as reference to exclude germline variants. At primary surgery, 21 samples were from the ovary, and 24 from different anatomical sites. After chemotherapy, 24 samples were from second surgery, while two samples from patient 20724, were at third surgery. Green circles, sensitivity to Pt-based treatment (PFS > 6 months from the end of chemotherapy); red circles, resistance against Pt-based treatment (PFS < 6 months from the end of chemotherapy). Black circles, information is missing. Tumors are grouped into high grade serous (n = 12) and non high grade serous (n = 7). Detailed anatomo-pathological features are reported in Supplementary Table 1.

Figure 2. Mutational load.

The diagram describes for each sample the mutational load. Mutations are categorized according to their predicted effect. Blue bars, non synonymous mutations; green bars, synonymous mutations; red bars, indel; light blue bars, VUS. Indel, insertion/deletion. VUS, variant of unknown significance. Complete list of sample names are reported in Supplementary Table 2.

Figure 3A. Unsupervised cluster analysis.

Unsupervised clustering of somatic mutational allelic fractions (AF) depicted for HGS-EOC patients for each gene (row) and for each patient (column), AF is defined as the percentage of reads that carried the mutation versus the total reads. Complete list of sample names are reported in Supplementary Table 2.

Figure 3B. Unsupervised cluster analysis.

Unsupervised clustering of somatic mutational allelic fractions (AF) depicted for non HGS-EOC patients. Color bars in the upper part of Panel B show information at diagnosis as reported in Table 1: grade (red, high grade; green, low grade) and histotype (orange, serous; green, endometrioid; blue, mucinous). For each gene (row) and for each patient (column), AF is defined as the percentage of reads that carried the mutation versus the total reads. Complete list of sample names are reported in Supplementary Table 2.

Figure 4. Concordant somatic mutations.

Heatmap showing the distribution of concordant somatic mutations. The Number of concordant mutations summarized to single gene was reported for each patient in a false color scale. Grey boxes indicate the absence of concordant mutations. Genes are grouped into pathways, depicted by color palette, as described in Supplementary Table 2. Color bars in the upper part show information at diagnosis as reported in Supplementary Table 1: grade (red, high grade; green, low grade) and histotype (orange, serous; green, endometrioid; blue, mucinous).

Figure 5. Phylogenetic tree.

Phylogenetic tree depicting the clonal relationship among multiple biopsies at primary surgery (green leaves, originally pt sensitive) and at relapse (red leaves, platinum resistant or black, unknown) for both HGS and non-HGS EOC. The root of the tree is represented by OVCAR-8 cell lines used as unrelated control (see Supplementary Section Methods 3.1.2.4). The construction of the tree was based on mutant allelic fractions. Ovaries, synchronous diseases and metachronous diseases were considered. Complete list of sample names are reported in Supplementary Table 2.