Next-generation sequencing analysis of endometrial screening liquid-based cytology specimens: a comparative study to tissue specimens

Akahane, Toshiaki; Kitazono, Ikumi; Yanazume, Shintaro; Kitajima, Masaki; Togami, Shinichi; Sakamoto, Ippei; Nohara, Sachio; Yokoyama, Seiya; Hiraki, Takao; Suzuki, Shinsuke; Ueno, S.; Tanimoto, Akihide

doi:10.1186/s12920-020-00753-6

Cited by 22 publications

(31 citation statements)

References 57 publications

(75 reference statements)

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“…A cancer panel was redesigned by making a minor modification of the previous panel [ 11 ] to include 56 cancer-related genes and 17 microsatellite foci selected from the QIAseq Targeted DNA Custom Panel (Qiagen, Reston, VA, United States), with 2,640 primers for the regions of interest (194,131 bp) and an average exon coverage of 99.87% (Online Resource 2). Whole blood DNA was extracted using QIAamp DNA Blood Mini Kits (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

“…The concentrations of extracted DNA were measured using Qubit 3.0 dsDNA BR assay kits (Life Technologies, Grand Island, NY, United States), and the DNA quality was monitored using the QIAseq DNA quantiMIZE kit (Qiagen). DNA with a quality check score <0.04 was considered high-quality DNA [ 11 ]. NGS was performed using a MiSeq sequencer (Illumina, San Diego, CA, United States) as previously described [ 11 ].…”

Section: Methodsmentioning

confidence: 99%

“…DNA with a quality check score <0.04 was considered high-quality DNA [ 11 ]. NGS was performed using a MiSeq sequencer (Illumina, San Diego, CA, United States) as previously described [ 11 ].…”

Section: Methodsmentioning

confidence: 99%

“…Sequence data were annotated as previously described using the Qiagen Web Portal service ( https://www.qiagen.com./us/shop/genes-and-pathways/data-analysis-center-overview-page/ ) and Mitsubishi Space Software (Amagasaki, Hyogo, Japan) [ 11 , 15 ]. The COSMIC database and human genome reference GRCh37 (hg19) ( https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.13/ ) were used as references.…”

Section: Methodsmentioning

confidence: 99%

“…We previously established a cancer gene panel comprising 60 genes and 17 microsatellite foci. This customized panel was used to analyze genetic profiles using FFPE tissues of endometrial cancers in terms of gene mutations, tumor mutation burden (TMB), and microsatellite instability (MSI) [ 11 ]. In this study, the above panel was further modified to detect POLE for evaluating gene alterations that can categorize DC, CS, SC, and EC with solid proliferation into MMR-deficient (MMR-d), p53 mutation (p53mut)-type, POLE mutation ( POLE mut)-type, and cases with no specific molecular profile (NSMP) according to The Cancer Genome Atlas (TCGA) classification.…”

Section: Introductionmentioning

confidence: 99%

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Molecular Evaluation of Endometrial Dedifferentiated Carcinoma, Endometrioid Carcinoma, Carcinosarcoma, and Serous Carcinoma Using a Custom-Made Small Cancer Panel

Kobayashi¹,

Kitazono

Akahane

et al. 2021

Pathol. Oncol. Res.

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It is often difficult to histologically differentiate among endometrial dedifferentiated carcinoma (DC), endometrioid carcinoma (EC), serous carcinoma (SC), and carcinosarcoma (CS) due to the presence of solid components. In this study, we aimed to categorize these carcinomas according to The Cancer Genome Atlas (TCGA) classification using a small custom-made cancer genome panel (56 genes and 17 microsatellite regions) for integrated molecular diagnosis. A total of 36 endometrial cancer cases with solid components were assessed using IHC, next-generation sequencing (NGS), and the custom-made panel. Among 19 EC cases, six were categorized as MMR-deficient (MMR-d) and eight were classified as having a nonspecific molecular profile. Three EC cases were classified as POLE mutation (POLEmut)-type, which had a very high tumor mutation burden (TMB) and low microsatellite instability (MSI). Increased TMB and MSI were observed in all three DC cases, classified as MMR-d with mutations in MLH1 and POLD1. Except for one case classified as MMR-d, all SC cases exhibited TP53 mutations and were classified as p53 mutation-type. SC cases also exhibited amplification of CCND1, CCNE1, and MYC. CS cases were classified as three TCGA types other than the POLEmut-type. The IHC results for p53 and ARID1A were almost consistent with their mutation status. NGS analysis using a small panel enables categorization of endometrial cancers with solid proliferation according to TCGA classification. As TCGA molecular classification does not consider histological findings, an integrated analytical procedure including IHC and NGS may be a practical diagnostic tool for endometrial cancers.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular Evaluation of Endometrial Dedifferentiated Carcinoma, Endometrioid Carcinoma, Carcinosarcoma, and Serous Carcinoma Using a Custom-Made Small Cancer Panel

Kobayashi¹,

Kitazono

Akahane

et al. 2021

Pathol. Oncol. Res.

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Evaluation and diagnostic value of next‐generation sequencing analysis of residual liquid‐based cytology specimens of pancreatic masses

Sekita-Hatakeyama

Fujii

Nishikawa

et al. 2021

Cancer Cytopathology

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Background Liquid‐based cytology (LBC) is a widely used method for processing specimens obtained by endoscopic biopsy. This study evaluated next‐generation sequencing (NGS) analysis of LBC specimens to improve the diagnostic accuracy of pancreatic lesions. Methods Upon the diagnosis of a suspected pancreatic mass, LBC residues were used retrospectively. The quantity and quality of DNA extracted from residual LBC samples were evaluated, and an NGS analysis targeting 6 genes (KRAS, GNAS, TP53, CDKN2A, SMAD4, and PIK3CA) was performed. Results The library was prepared from LBC specimens taken from 52 cases: 44 were successful, and 8 preparations failed. An analysis of DNA quantity and quality suggested that the success or failure of NGS implementation depended on both properties. The final diagnosis was achieved by a combination of the pathological analysis of the surgical excision or biopsy material with clinical information. Among the 33 cases of pancreatic ductal adenocarcinoma (PDAC), KRAS, TP53, CDKN2A, and SMAD4 mutations were identified in 31 (94%), 16 (48%), 3 (9%), and 2 (6%), respectively. Among the 11 benign cases, only a KRAS mutation was identified in 1 case. On the basis of NGS results, 18 of 33 PDACs (55%) were classified as highly dysplastic or more, and 10 of 11 benign lesions were evaluated as nonmalignant, which was consistent with the final diagnosis. Conclusions NGS analysis using LBC specimens from which DNA of appropriate quantity and quality has been extracted could contribute to improving the assessment of pancreatic tumor malignancies and the application of molecular‐targeted drugs.

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

Akahane

Kitazono

Kobayashi

et al. 2021

Diagnostic Cytopathology

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Background Genomic examination of cytology specimens is often performed on cell blocks or conventional smears rather than on liquid‐based cytology (LBC) specimens. Since LBC specimens preserve high‐quality DNA, cancer genome profiling using next‐generation sequencing (NGS) is also attainable from residual LBC specimens. One of the advantages of using LBC specimens for NGS is that it allows direct extraction of DNA from residual specimens, avoiding a sacrifice of smear slides and minimizing genomic profiling processing time. Methods Endometrial LBC specimens were subjected to NGS analysis to validate the practicality of rapid cancer genomic profiling in a pathology laboratory. The extracted DNA was subjected to NGS using a customized cancer gene panel comprising 56 genes and 17 microsatellite regions. The workflow strategy was defined, and the processing time estimated for specimen sampling, cell counting, NGS run, and genome profiling. Results NGS analysis of most LBC specimens revealed somatic mutations, tumor mutation burden, and microsatellite instability, which were almost identical to those obtained from formalin‐fixed paraffin‐embedded tissues. The processing time for direct NGS analysis and cancer genomic profiling of the residual LBC specimens was approximately 5 days. Conclusion The residual LBC specimens collected using endometrial cytology were verified to carry a high tumor fraction for NGS analysis and could serve as an alternate source for rapid molecular classification and diagnosis of endometrial cancers, as a routine process in a pathology laboratory.

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Next-generation sequencing analysis of endometrial screening liquid-based cytology specimens: a comparative study to tissue specimens

Cited by 22 publications

References 57 publications

Molecular Evaluation of Endometrial Dedifferentiated Carcinoma, Endometrioid Carcinoma, Carcinosarcoma, and Serous Carcinoma Using a Custom-Made Small Cancer Panel

Molecular Evaluation of Endometrial Dedifferentiated Carcinoma, Endometrioid Carcinoma, Carcinosarcoma, and Serous Carcinoma Using a Custom-Made Small Cancer Panel

Evaluation and diagnostic value of next‐generation sequencing analysis of residual liquid‐based cytology specimens of pancreatic masses

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

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