Analysis by Next-Generation Sequencing Panel Covered 60 Genes and 17 Microsatellite Foci in Endometrial Screening Liquid-Based Cytology Specimens A Comparative Study to Tissue Specimens

Liquid-based cytology (LBC) is now a widely used method for cytologic screening and diagnosis of cancers. Since the cells are fixed with alcohol-based fixatives and the specimens are stored in a liquid condition, LBC specimens are also conducive for genetic analysis. Here, we established a small cancer gene panel including 60 genes and 17 microsatellite markers for next-generation sequencing and applied to residual LBC specimens obtained by endometrial cancer screening to compare with corresponding formalin-fixed paraffin-embedded (FFPE) tissues. Results A total of 53 FFPE and LBC specimens (n=24) were analyzed, revealing characteristic mutations for endometrial cancer including those of PTEN, CTNNB1, PIK3CA, and PIK3R1 . Eight cases had higher scores for both tumor mutation burden (TMB) and microsatellite instability (MSI), which agree with defective mismatch repair (MMR) protein expression. Paired endometrial LBC, and biopsied and/or resected FFPE tissues from 7 cases, demonstrated almost the same mutations, TMB, and MSI profiles in all cases. These findings demonstrate that the small cancer gene panel proved to be able to detect therapeutically actionable gene mutations in endometrial LBC and FFPE specimens and that LBC specimens obtained by endometrial cancer screening is an alternative and useful source of molecular testing.


Immunohistochemistry for MMR proteins
To evaluate the expression of MMR proteins, FFPE tissue sections were applied to immunohistochemistry (IHC) using antibodies against MLH1 (clone ES05 M3640), MSH2 (clone FE11 M3639), MSH6 (clone EP49 M3646) and PMS2 (clone EP51 M3647) purchased from DAKO (Tokyo, Japan) [28,29]. Staining was performed on representative 5-μm-thick FFPE sections with Envision FLEX High pH K8000 system (DAKO) according to the manufacturer's protocol. Positive nuclear staining of lymphocytes in the tissue sections was monitored as a positive control. MMR deficiency was defined as complete loss of nuclear staining for both MLH1 and PMS2, both MSH2 and MSH6, MSH6 only, or PMS2 only. When partial loss of each protein was observed, the case was defined as showing heterogeneous expression.

Statistical analyses
All values are expressed as the mean ± standard deviation. Significant differences were analyzed using Welch's t-test. Values of p < 0.05 were considered statistically significant. The cut-off values for the evaluation of TMB-high and MSI-high conditions were determined by receiver operator characteristic (ROC) curves.

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). Ascites LBC contained substantially more inflammatory cells than the endometrial LBC specimens. 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 are summarized in supplemental Table S1. 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 mutations in PTEN, CTNNB1, PIK3CA, and PIK3R1.
The cases of mixed EC/SC, CC, and SC had additional TP53 mutations. Case no. 6 was analyzed from two different FFPE sections, revealing G1 and G2 EC, and NGS analysis exhibited common mutations in PTEN, CTNNB1 and ARID1A, whereas different mutations in PIK3CA and PIK3R1 were also detected in the two parts, suggesting that at least two cancer clones might exist. The three cases of DC harbored mutations in PTEN, CTNNB1, PIK3CA, or PIK3R1, along with multiple mutations in receptortype tyrosine kinase genes such as FGFR, ERBB, RET, and FLT. The mutation profiles of the EC and DC parts were not completely comparable but exhibited similarity with respect to PTEN mutation.

Mutations detected in LBC specimens
Endometrial LBC specimens contained abundant atypical cells, but the number of background inflammatory cells was substantially reduced compared to that in the ascites LBC specimens, 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.
In contrast, LBC samples obtained from ascites frequently contained numerous inflammatory cells, mesothelial cells, and a relatively lower number of atypical cells. When these cells were more abundant than tumor cells, the NGS panel detected no mutation with sufficient VAF (case nos. 4 and 17). In case no. 11, mutations with sufficient VAF scores were not detected even in samples containing more than 10,000 tumor cells and fewer inflammatory cells with (tumor cell ratio: 71%).
Conversely, even many non-tumor cells were found in the specimen, mutations were detected in case no. 8.

Relation 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, 28 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).

Correlation of the genetic diagnosis from LBC and FFPE specimens
In 10 cases (case nos. 9, 12, 14, 16,18,19,[21][22][23][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. NGS of one ascites LBC sample (case no. 8) detected a mutation profile and a TMB/MSI status that were almost identical to those from FFPE of primary and metastatic sites (supplemental Tables S1 and S2).
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).

Discussion
We showed that the DNA extracted from all the LBC specimens exhibited high enough quality and quantity for genome sequencing and analysis. However, 3 of 4 ascites specimens failed to obtain sufficient VAF to confirm the presence of somatic mutations, even these 4 ascites samples contained more than 10,000 cells with variable tumor cell ratio (around 9.1% to 71%: average 32%). One report analyzing effusion fluid LBC specimens from patients with high-grade serous ovarian carcinoma demonstrated that at least 1,000 neoplastic cells and 50% tumor fraction with 50ng input DNA can yield successful NGS analysis [30]. The differences between the prior report might be attributed to different fixatives (Cytolyt vs. CytoRich Red), extraction methods (scraping from slides vs. directly from residual liquid specimens), and gene panels (capture sequence vs. amplicon sequence), however, one most possible factor for the failed analysis, in the present study, would be a less amount of input DNA (<40ng). Consequently, the application of ascites LBC samples is necessary to be reevaluated for an appropriate preanalytic step in our NGS protocol.
In contrast, endometrial LBC samples contained much higher tumor cell ratio, allowing for NGS analysis to be efficiently performed. The endometrial LBC specimen containing at least 1,450 tumor cells and 10ng of input DNA was successfully analyzed in the present protocol. One report suggested that 5,000 viable cells are necessary for successful NGS using a 50-cancer gene hotspot panel from conventional cytology samples [31]. Therefore, appropriate cytology specimens containing more than several thousand cells with higher tumor ratio could be an alternative source for genetic analysis to FFPE and conventional cytology specimens. However, the use of LBC specimens for NGS analysis faces a few disadvantages [32,33]. Unlike NGS in scraped cells from smears, in which the same cells observed by cytopathologists are subjected to molecular testing, NGS analysis from LBC specimens is started with pooled residual samples. Therefore, it is possible that false negative materials are applied to NGS, although we checked the presence of tumor cells in the residual LBC specimens and then applied to DNA extraction. A rapid on-site evaluation to ensure the sufficient preservation of tumor cells are unavailable in LBC specimens, either. Another disadvantage is, to the best of our knowledge, that an efficient method to enrich tumor cell fraction in LBC specimens is not developed yet. In this regard, the LBC specimens, such as inflammatory cell rich ascites in the present study, may not be always suitable to NGS. Conventional smears and cell blocks are superior to LBC for increasing tumor cell fraction by trimming techniques [33,34]. Conversely, one of advantages of LBC specimens is well preserved DNA quality and yield even after long-term storage [20]. No loss of diagnostic materials by cell scraping and less complicated procedures would be also other advantages in LBC specimens.
Since the loss of MMR protein expression can occur by methylation and mutations in the promoter region, in addition to loss-of-function mutations in the coding region, a validation of the exon sequence would not be sufficient to evaluate the MMR expression and function [41][42][43]. Actually, in the present study, only 3 of 9 MMR-D cases exhibited pathogenic mutations in MLH1, MSH6, or PMS2.
Although IHC is considered a practical tool for speculation of TMB and MSI conditions as a first screening step [44,45], the results of MMR protein IHC were not always comparable to those of MMR gene mutation identification. Thus, evaluation of TMB and MSI using NGS-based genome analysis along with IHC would be a practical strategy for clinical testing [46][47][48]. NGS analysis with endometrial LBC specimens, which were not usually used for immunocytochemistry of MMR proteins, consistently detected TMB-H and MSI-H. Therefore, our custom-made panel would be possibly beneficial to determine TMB-H and MSI-H status. However, the sample size was small and the NGS panel is also too small to precisely detect TMB and MSI relative to whole-exome sequencing [26,27,49,50].

Conclusion
The NGS-based panel with coverage of 60 cancer genes and 17 microsatellite foci demonstrated highly recurrent somatic mutations, TMB, and MSI in FFPE and endometrial LBC specimens. In addition, the endometrial cytology is able to efficiently collect cells from the entire part of endometrial cavity to predict histological subtypes [16] and also is available to detect asymptomatic endometrial cancer by the less invasive cytologic screening [14]. Therefore, the small size cancer gene panel and endometrial LBC specimens would be an alternative tool for a genetic testing as a diagnostic or therapeutic strategy.