Cohort 1: Whole-exome sequencing of multi-regions of primary tumor and matched cfDNA identified genomic heterogeneity
The WES was successfully performed in those two patients of 9 samples. The quality of WES data with mapping ratio of >99% and exome enrichment of >99% was acceptable (supplementary Table S1) (Additional files 2). Overall, the most common type of mutation was C>T transition and the major type of mutation was missense mutation (supplementary Figure S2) (Additional files 3), which had been observed in previous study of ESCC . The total mutations (silent and non-silent) for solid tumor ranged from 290 to 483, with a median value 342 (221 for silent ones and 123 for non-silent ones). We found 915 and 863 mutations (376 silent and 340 non-silent ones) for two cfDNA samples respectively. Tumor mutation burden (TMB) for solid tumor samples ranged from 4.92 to 8.19 mutations /M, whereas cfDNA samples had higher TMB from 14.63 to 15.51 mutations /M. A total of 694 non-silent mutations were discovered in 9 samples. In E102, a total of 712 mutations (silent and non-silent mutations) were identified in tumor tissues, 119 of which were ubiquitous in T1, T2, T3 and T4 tumor regions. Meanwhile, 796 mutations (silent and non-silent mutations) were identified in E110, 234 of which were ubiquitous in T1, T2 and T4 tumor regions. To explore intratumoral heterogeneity and the genomic evolution of ESCC, phylogenetic trees were constructed on the basis of somatic mutations (both silent and non-silent mutations) identified in each region. The phylogenetic trees varied extensively in two cases, which showed evidence of spatial intratumoral heterogeneity, with an average of 76.6% (E102 of 83.2% and E110 of 70.6%) of somatic variants having spatial heterogeneity (Figure 1A).
13 genes previously reported in ESCC were confirmed in this cohort, including TP53, MUC16, CTNND2, DNAH9, EP300, NOTCH3, SYNE1, RP1, TTN, ATM, KMT2D, NOTCH1 and USH2A [31, 32]. However, only MUC16, CTNND2 and NOTCH3 were detected in matched cfDNA (Figure 1B). In addition, 12 of 20 (60.0%) tumor-associated mutations identified were found in cfDNA, which were the important driver genes in four oncogenic pathways, including NOTCH (NOTCH3, NCOR2, SPEN), RTK-RAS (ARHGAP35, KSR1, IRS1), WNT (DVL3, FRAT1) and Hippo (TAOK2, LLGL2, TEAD2) signaling pathways (Figure 2). In addition, we found two novel cancer-related genes (MUC4 and MUC17) in both of the two patients which were not previously reported in ESCC. The details of somatic mutations and prevalence in two ESCC patients were showed in supplementary Table S2 and S3 (Additional files 2). The Pearson’s correlation analysis of VAFs between cfDNA and matched solid tumor showed the positive linear relationship (R2 =0.78, P <0.0001) (Figure 3). The VAFs of ubiquitous and heterogeneous somatic mutations in cfDNA and solid tumor were listed in supplementary Table S4 (Additional files 2).
Cohort 2: Targeted captured sequencing revealed correlation of genomic alterations between cfDNA and matched solid tumor
Since ESCC has low tumor fraction in cfDNA, in order to better identify mutations with low VAFs, we performed targeted captured sequencing by a panel of 560 cancer-associated genes with higher sequencing coverage (~1000x) for another cohort (E104, E111 and E121) (supplementary Table S5) (Additional files 2). The most common type of mutation was C>T transition and the majority of variant type was missense mutation, which were the same as Cohort 1 via WES (supplementary Figure S3) (Additional files 3). The total mutations (silent and non-silent) for solid tumor ranged from 107 to 194, with a median value of 142. We found 494, 769 and 262 mutations (245, 407 and 121 non-silent ones respectively) for three cfDNA samples respectively. A total of 333 genes were mutated (non-silent mutations) in 12 samples (supplementary Table S6 and S7) (Additional files 2). The phylogenetic trees were constructed on the basis of somatic mutations (both silent and non-silent mutations) identified in each region of those three cases. The phylogenetic trees varied extensively as well, which showed evidence of spatial intratumoral heterogeneity, with an average of 91.7% (1011/1103; range, 90.5%-92.7%) of heterogeneous somatic mutations (Figure 4A).
32 mutated genes previously reported in ESCC were confirmed in this cohort [31-33]. In patient E104, 323 variants were detected in three sub-regions, 27 out of which were ubiquitous variants. KMT2D, TP53, UBR5 from the identified variants were previously confirmed as frequent mutations in ESCC[31, 32]. In patient E111, 423 mutations were detected totally, 39 out of which were ubiquitous variants in three regions. Among them, CSMD3, PTCH1, TP53, PIK3CA were important and associated with ESCC. In Patient E121, 357 mutations were detected totally, 26 of which were ubiquitous variants. Among them, FBXW7, LRP1B, TP53, MTOR and EP300 were confirmed as frequent mutations in ESCC (Figure 4B). To determine the VAFs of tumor mutations in cfDNA. The VAFmin we setup in algorithm for detection of mutations was 0.1%. The first quantile and third quantile of VAFs in cfDNA were 0.49-0.88%, 0.44-0.77% and 0.41-0.875% for patient E104, E111 and E121 respectively (supplementary Figure S4) (Additional files 3). In total, there were 14 mutations with VAFs of >5% detected in cfDNA. The results showed ESCC had low ctDNA in the blood, which was similar to one previous study on EAC . Moreover, the variants called from cfDNA were much more than the ones called from solid tumor tissues. Some true positive calls were missed in our solid tumor samples due to the intratumor heterogeneity and sampling biases. Some were potential false positive calls possibly due to clonal hematopoiesis (CH). Therefore, we focused on the ESCC recurrent genes to answer the following questions: how many mutated genes in solid tumor were identified in cfDNA? How many important mutations were found in cfDNA only? For patient E104, 10 out of 16 (62.5%) ESCC-associated mutations detected in solid tumor were recovered in paired cfDNA, including CSMD3, KMT2D, LRP1B, SYNE1, ATR, EP300, PRDM1, UBR5, FSTL5, LIFR. In E111, 16 out of 21 (76.2%) ESCC-associated mutations were recovered in cfDNA, including KMT2D, LRP1B, SYNE1, BRCA2, ATR, EP300, ASXL1, MTOR, PKHD1, PTCH1, UBR5, BRIP1, KMT2A, NUP214, NOTCH2, CREBBP. In E121, 35.7% (5/14) ESCC-associated mutations were found in both solid tumor and cfDNA, including KMT2D, LRP1B, EP300, PKHD1, PRDM1. Moreover, KMT2D, SYNE1 and UBR5 were shared by cfDNA and all regions of tumor in E104. PTCH1 was the shared one in E111 and LRP1B was shared by three sub-regional tumors and cfDNA in E121 (Figure 5). Mutations in TP53 were often detected in solid tumor only, whereas mutations in CBL, POLE, PTCH1 and NFE2L2 were only detected in cfDNA. Furthermore, the correlation of VAFs between cfDNA and matched solid tumor was strong (R2 =0.92) with a significant P-value of <0.0001 (Figure 6). The VAFs of somatic mutations in cfDNA and solid tumors were showed in supplementary Table S8 (Additional files 2).