A total of 5 cases of stage IIIa-IIIc HGSC treated with 3–5 cycles of platinum-based NACT were selected (Table 1). This included three cases with platinum-resistant (Case 1–3) and two with platinum-sensitive disease (Case 4–5). The biochemical response in terms of reduction of Ca125 level ranged from 66 to 84% for platinum-resistant cases and 96 to 98% for platinum-sensitive cases. The average time to recurrence was 4.2 months in platinum-resistant, and 20.2 months in platinum-sensitive cases. All women with platinum-resistant HGSC died of their disease within 12.1–21.4 months from diagnosis, while both patients with platinum-sensitive HGSC were alive at the last follow-up 42.2 and 47.9 months after the diagnosis.
Whole exome sequencing was performed in buffy coat and macro-dissected pre-NACT (N = 5 samples) and post-NACT tumor (N = 6 samples). A summary of the somatic mutation counts is shown in Table 2 (exonic) and Additional File 1 (non exonic). When comparing exonic non-synonymous mutations in pre-NACT and post-NACT samples from the same patient, an average of 41% (1–68%) of genes were mutated at both time points (Table 2). There were no visible trends in increase vs. decrease in the mutational burden following exposure to NACT in resistant vs. sensitive cases
The comparison of genes with non-synonymous exonic mutations detected at any time point (pre-NACT, post-NACT, or both) is shown in Fig. 1A. The majority of mutated genes were unique to each case, including 666/730 (91%) in Case 1, 88/112 (79%) in Case 2, 45/66 (68%) in Case 3, 125/151 (83%) in Case 4 and 183/234 (78%) in Case 5. All recurrently mutated genes (> 1 case) are listed in Additional File 2. Of note, all cases had detected mutations in MUC2, while mutations in DDX11, TP53 and TUBA3D were observed in 4 of 5 cases. Genes that were exclusively mutated in > 1 resistant or sensitive cases are highlighted in Table 3; 16 genes were exclusively mutated in > 1 resistant case and 5 genes were exclusively mutated in both sensitive cases.
The comparison of genes with non-synonymous exonic mutations detected within a given time point is shown in Fig. 1B (pre-NACT) and 1C (post-NACT). The majority of mutations were case-specific, including 85–96% among pre-NACT samples and 68–96% among post-NACT samples. However, a few genes were commonly mutated in pre-NACT samples in exclusively resistant (CSPG4, SLC35G5, TUBA3D) or sensitive (CYP2D6, NUTM1, DNAH5) cases. Among post-NACT samples, 10 genes were exclusively mutated in > 1 resistant case (ADGRV1, AOC1, CACNA1S, MTMR11, MUC17, MUC20, OR52N5, PAK2, PCDHB11, TMEM14B); no genes were exclusively mutated in both sensitive cases at this time point. As shown in Table 2a small proportion of the same mutations were present at both time points (pre- and post-NACT) within the same case; the comparison of these stably mutated genes is shown in Fig. 1D. Of note, only 2 genes were shared between the resistant cases (SLC35G5, TUBA3D), while there were no shared genes between the sensitive cases. To compare the potential impact of NACT in our cases, we next looked at the genes that were mutated in post-NACT, but not pre-NACT samples, from the same case; this included 683/691 (99%) of post-treatment genes from Case 1, 32/65 (49%) from Case 2, 26/53 (49%) from Case 3, 56/98 (57%) from Case 4 and 25/60 (42%) from Case 5. The comparison of these emergent mutations is shown in Fig. 1E; of note, 6 genes were shared between the resistant cases (highlighted in Fig. 1F). While two of these genes showed a different pattern of mutation in sensitive cases (CACNA1S in pre-NACT only and KIR2DL3 at a low level in both pre-NACT and post-NACT), the remaining 4 genes were exclusively mutated in platinum-resistant cases (ADGRV1, MUC17, MUC20, PAK2). There were no genes with treatment-emergent mutations shared between the two sensitive cases.
The inclusion of post-NACT samples from two different tumor sites (right ovary and omentum) for platinum-resistant Case 3 allowed us to compare the impact of both sites and exposure to NACT on mutational status within the same patient (Fig. 2A; see Additional File 3 for more detailed gene list). Of note, 8 genes were commonly mutated among all samples (highlighted in Fig. 2B; CDK12, FOXJ1, HMCN1, MMRN1, MTMR11, PCDHA6, RBM12, REV3L). The same mutation was detected in all samples, with no significant changes in allele fraction following NACT. Both omental samples had detected mutations in 6 genes irrespective of treatment status, while 2 genes had the same emergent mutations in both post-NACT samples irrespective of tissue site (OR10G9, ZNF28). There were also site-specific differences in post-treatment samples, including 10 genes with emergent mutations only at the ovarian site and 14 genes with emergent mutations only at the omental site (highlighted in Fig. 2C). While five of the genes with emergent mutations showed a different pattern of mutations in sensitive cases (FRG1, KIR2DL3, MUC2, SETD8, ZNF28), the majority were exclusive to platinum-resistant cases (ARHGAP5, ARMC4, CFAP47, CLCNKA, GOLGA6L2, GPR101, KRTAP4-11, MUC17, MUC20, NUP50, OR10G9, PAK2, PCDHB11, PCDHGB6, PGAM1, PLEC, SGSM2, SPATA31D1, TBC1D3B/F, TRIM49, UGT2B11).
The results of targeted deep sequencing of 75 candidate genes selected from our exome data are summarized in Fig. 3 (see Additional File 4 for full panel gene list and selection criteria). Mutations were validated in several genes in platinum-resistant cases, while mutation of DNAH5 was confirmed in both pre-NACT and post-NACT samples from Case 4, the one platinum-sensitive case with sufficient material for targeted sequencing (Fig. 3A). Of note, emergent mutations in post-treatment samples were confirmed in seven genes in platinum-resistant Case 2 (ANKRD12, HSD17B4, KIAA1217, NBEA, SH3RF2, SH3TC2, VTI1A) and one gene in platinum-resistant Case 3 (SPATA31D1). Copy number changes were also observed in several genes prior to and/or following NACT (Fig. 3B). Of note, copy numbers of ≥ 7 were observed in 2 genes in Case 1 (TMED8, PTCD3), six genes in Case 2 (WSCD1, OR52N5, PRB1, KANK4, KLHL38, ATP1A2), and six genes in Case 3 (MMRN1, CDAN1, HSPB7, LRP1, MTMR11, RBM12). Furthermore, copy number loss was observed in SPATA31D1 following NACT in Case 4 only.