Next-Generation Sequencing Analysis of Endometrial Screening Liquid-Based Cytology Specimens: A Comparative Study to Tissue Specimens

Abstract

Background: Liquid-based cytology (LBC) is now a widely used method for cytologic screening and cancer diagnosis. Since the cells are fixed with alcohol-based fixatives, and the specimens are stored in a liquid condition, LBC specimens are suitable for genetic analyses. Methods: 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 49 FFPE and LBC specimens (n=24) were analyzed, revealing characteristic mutations for endometrial cancer, including PTEN, CTNNB1, PIK3CA, and PIK3R1 mutations. 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, presented almost identical mutations, TMB, and MSI profiles in all cases. Conclusion: These findings demonstrate that our ad hoc cancer gene panel enabled the detection of therapeutically actionable gene mutations in endometrial LBC and FFPE specimens. Endometrial cancer LBC specimens offer an alternative and affordable source of molecular testing materials.

Direct next‐generation sequencing analysis using endometrial liquid‐based cytology specimens for rapid cancer genomic profiling

“…Since good quality DNA can be obtained from preserved LBC specimens, they could be successfully applied for NGS. 16,17 However, the most important factor for the successful NGS analysis using cytology specimens is the number of tumor cells and input DNA 7,25 . In this study, when input tumor cells were greater than approximately 10,000 and tumor fraction was greater than 50%, resulting in more than 17 ng of input DNA (17 cases), successful detection of mutations and analysis and calculation of MSI/TMB, which is necessary for WHO molecular classification, were attained 20…”

“…DNA from FFPE tissues was obtained from 3 to 6 sections with of 10 μm thickness, including more than 30% of the cancer area. DNA extraction from FFPE and LBC specimens, as well as DNA quality monitoring were performed as previously reported 7,16 …”

“…NGS library generation and quality checks were performed as previously described 7,16 . The libraries were applied to a MiSeq sequencer (Illumina, San Diego, CA) after dilution with a hybridization buffer to a final concentration of 20 pM.…”

“…The non‐tumor cell count ( N ) was calculated from an average of five HPFs, from which the tumor cell to non‐tumor cell ratio and tumor fraction ( T / T + N ) were calculated. Estimated tumor cell numbers subjected to NGS were calculated as following formula: Average cell count × [SurePath spot area (113.04 mm 2 )/400× HPF area (0.2376 mm 2 )] × [LBC sample volume used for NGS (1 mL)/LBC sample volume used for SurePath preparation (0.25 mL)] × [applied volume of PCR library for NGS (μL)/fixed total volume for NGS reaction (16.8 μL): these volumes are indicated by QIASeq DNA panel system 7,16 ]. For example, the estimated tumor cell number of case #1 (Table S2) was calculated as 43,429 = 127.8 × [113.04/0.2376] × [1 mL/0.25 mL] × [3 μL/16.8 μL], and that of case #18 (Table S2) was 62,419 = 134.4 × [113.04/0.2376] × [1 mL/0.25 mL] × [4.1 μL/16.8 μL].…”

“…Furthermore, the feasibility of LBC specimens for NGS analysis was validated by retrospective studies using residual LBC specimens obtained from various organs, such as the thyroid gland, breast, endometrium, and lymph nodes after long‐term storage 16,17 . The present study sought to expand the above methods by using a modified custom cancer gene panel 16 to verify whether direct NGS from residual LBC specimens could be practically applied as a standard, routine technique in laboratories.…”