DNA quality and quantity obtained from FFPE and LBC specimens
The results of the pathological diagnosis, DNA quality, and input DNA for NGS of the 24 endometrial cancer cases are summarized in Table 2. The storage period of FFPE and LBC specimens ranged from 2 weeks to 3 years. The tumor fraction of the FFPE sections ranged from 30% to 90%. In endometrial LBC specimens, estimated tumor cell count (T) subjected to NGS varied from 1,500 to 140,000, and the tumor cell ratio varied from 27% to 95% (T/N+T). All of the genomic DNA extracted from the FFPE and LBC samples demonstrated high-quality and sufficient quantity for library construction and successful sequencing.
Mutations detected in FFPE specimens
The detected mutations in endometrial cancers and genomic information of the variants are summarized in supplemental Tables S1 and S2. Among the 24 cases, 18 were finally diagnosed as endometrioid carcinoma (EC) by biopsy or surgical resection, including 12 cases of G1, 5 cases of G2, and 1 case of G3 EC. The other 6 cases consisted of 3 dedifferentiated carcinomas (DC), and 1 case each of mixed EC/serous carcinoma (SC), SC, and clear cell carcinoma (CCC). These endometrial cancers showed common mutation profiles, including PTEN, CTNNB1, PIK3CA, and PIK3R1 mutations.
The cases of mixed EC/SC, CC, and SC had additional TP53 mutations. Two different FFPE sections relative to case no. 6 were analyzed, revealing G1 and G2 EC. While NGS analysis unveiled common mutations in PTEN, CTNNB1, and ARID1A, different PIK3CA and PIK3R1 mutations were also detected, suggesting the existence of at least two cancer clones. The three DC cases harbored PTEN, CTNNB1, PIK3CA, or PIK3R1 mutations along with multiple mutations in receptor-type tyrosine kinase genes, such as FGFR, ERBB, RET, and FLT. The mutation profiles of the EC and DC sections were not completely comparable but exhibited similarity with respect to PTEN mutation.
Mutations detected in LBC specimens
Endometrial LBC specimens contained abundant atypical cells, resulting in a higher frequency of mutation detection in the endometrial LBC specimens (9 out of 10 cases; supplemental Table S1). Mutations in PTEN, CTNNB1, and PIK3CA were identified in 1 case of atypical cell cytology (case no. 14), in which the diagnosis of G1 EC was confirmed by endometrial curettage biopsy.
Relations between MMR protein expression, TMB, and MSI
The overall relationships between MMR protein deficiency (MMR-D), TMB, and MSI status are shown (Fig. 1). In cases of MMR-D (9 cases, 21 samples), the TMB score was significantly higher than in cases of MMR protein proficiency (MMR-P) (15 cases, 27 samples) (p < 0.001; Fig. 2A, left). The MSI score of MMR-D cases was also significantly higher than that in MMR-P cases (p < 0.001; Fig. 2B, left). Estimated cut-off values for the evaluation of TMB-high (TMB-H) and MSI-high (MSI-H) were >31.1 and >4.2, respectively, as determined by the ROC curve (Figs. 2A and 2B).
The IHC results, TMB, and MSI status are summarized in supplemental Table S3. The majority of MMR-D cases (8 of 9 cases), but not MMR-P cases, showed both TMB-H and MSI-H. The exception was the case no. 6, exhibiting heterogeneous loss of expression in MLH1, PMS2, and MSH6, which was only classified as TMB-H. One case with high TMB and MSI scores (case no. 16) presented a pathogenic mutation in MSH6 and the corresponding loss of MSH6 expression. Three cases of DC with MMR-D were found to be both TMB-H and MSI-H, among which 2 cases harbored pathogenic MLH1 mutations and loss of MLH1 and PMS2 protein expression. In 3 cases (case nos. 1, 3 and 5), no loss of MMR protein was detected despite a TMB-H status. In 1 case (case no. 12), despite being MSI-H positive, no MMR protein loss was detected. The photomicrographs of H&E staining and IHC from a representative MMR-D case of G2 EC (case no. 23) with loss of MLH1 and PMS2 expression are shown in Fig. 3. Very similar findings were obtained from cases no. 20 and 22, and both the TMB and MSI scores were over the cut-off values in these cases.
Correlation of the genetic diagnosis from LBC and FFPE specimens
In 10 cases (case nos. 9, 12, 14, 16, 18, 19, 21-24), the FFPE tissues from biopsy and/or resection along with endometrial LBC, were subjected to gene panel analysis. For cases no. 14 and 22, biopsied and endometrial LBC samples were analyzed together, revealing the same mutations. In 8 cases (case nos. 9, 12, 16, 18, 21, 23, and 24), 3 paired endometrial LBC, biopsied, and resection FFPE specimens were available for genetic studies. Seven of these 8 cases (except for case no. 18) exhibited almost identical mutation profiles and a similar TMB/MSI status.
The VAF detected from 7 endometrial LBC specimens (case nos. 9, 12, 14, 16, 21, 23, and 24) showed a remarkably similar VAF to that of the same gene mutations from the corresponding biopsied FFPE (Fig. 4A). In case no. 22, VAF of APC markedly changed from 85.6 % in LBC to 58.9 % in biopsied FFPE, possibly because chemotherapy resulted in tumor clonal selection after LBC sampling and before the biopsy, and, therefore, the data were excluded from the Fig. 4A. All together with the results obtained from these 7 cases, a fine concordance was observed between endometrial LBC, and biopsied and/or resected FFPE tissue specimens in numbers of mutations, TMB and MSI scores, and MMR protein expression (Fig. 4B).