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.

Evaluation of 3 molecular-based assays for microsatellite instability detection in formalin-fixed tissues of patients with endometrial and colorectal cancers

“…In this study, we chose a moderate throughput approach, but broader approaches are described in the literature. Some NGS panels provide, in addition to the MSI status and the genomic profile, the determination of the tumour mutation burden (TMB) score, a complementary predictive tumour biomarker of immunotherapy efficacy 36,49 . Notably, The Food and Drug Administration (FDA) recently approved a NGS-based broad companion diagnostic (FoundationOne CDx (F1CDx) from Foundation Medicine) for the simultaneous analysis of MSI status, genomic aberrations and TMB in all solid tumors.…”

Performance of a 50‐gene next generation sequencing panel with post‐centrifuge supernatant cytology fluid in non‐small‐cell lung cancer

“…These results are in agreement with similar published studies, which have consistently shown concordance between results of molecular testing on FFPE specimens and on liquid cytology specimens, including supernatant with cytopreservative 3,8 and without cytopreservative 7,8 and resuspended cell pellets derived from centrifuged cytology fluid. [9][10][11] Only base pair substitutions and one small deletion were detected in this study. The NGS assay utilized is not designed to detect large insertions or deletions, copy number variations, or gene rearrangements, potentially limiting its utility in nonsmall-cell carcinoma of the lung, where the latter two in particular are recurring genetic alterations.…”

“…The results of the current study lend further support to already published evidence for use of LBC specimens and their routine utilization in molecular diagnostics. Not only have these findings been reproduced across multiple fixative types, they are seen across multiple assay types on differing platforms, including next generation sequencing, 3,[7][8][9] Sanger sequencing, 4,5,14 capillary electrophoresis, 4,5 RT-PCR, 10 and varying genetic alterations, from substitutions and insertions/deletions 3,7,8 to microsatellite instability and tumor mutational burden, 9 loss of heterozygosity 4,5 and copy number variations. 12 Furthermore, the current study adds to the growing data that DNA is stable in LBC media over an extended time, allowing delayed DNA isolation and molecular analysis on an as-needed basis.…”

“…Published data suggest variant allele fraction, as an indirect measure of tumor cellularity in a sample, is not always well correlated between LBC samples and matched (though not necessarily paired) non-LBC samples. [7][8][9] This is not surprising, as tumor cellularity is not known in LBC samples and at best can only be guessed based on cellularity of a paired cell block, smear, and/or monolayer. However, it is worth noting that successful sequencing from LBC samples has been performed when the concurrent FFPE sample was considered not sufficient for molecular analysis, 3 as was also seen in one case in this study.…”

Human Papilloma Virus 18-Positive Submucosal Small Cell Neuroendocrine Carcinoma of the Vagina: An Immunohistochemical and Genomic Study

“…We profiled the patients for their HPV infection status using a polymerase chain reaction (PCR)-based examination, whereas the genomic alterations were profiled using next-generation sequencing (NGS). 11 The 2 cases of vaginal NEC shared a common molecular profile; both were HPV18-positive with a low tumor mutation burden (TMB), low microsatellite instability (MSI) score, and no TP53 and RB gene mutations. This is the first report of an HPV18-infected primary vaginal NEC analyzed using NGS analysis.…”