Real-world application of next-generation sequencing-based test for surgically resectable colorectal cancer in clinical practice (本文)

概要

Research Article

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Real-world application of next-generation
sequencing-based test for surgically
resectable colorectal cancer in clinical
practice
Masayo Ogiri1 , Ryo Seishima*,1 , Kohei Nakamura2 , Eriko Aimono2 , Shimpei Matsui1

, Kohei

Shigeta1 , Tatsuyuki Chiyoda3 , Shigeki Tanishima4 , Koji Okabayashi1 , Hiroshi Nishihara2
& Yuko Kitagawa1
1

Department of Surgery, Keio University School of Medicine, Tokyo, Japan
Genomics Unit, Keio Cancer Center, Keio University School of Medicine, Tokyo, Japan
3
Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan
4
Department of Biomedical Informatics, Kansai Division, Mitsubishi Space Software Co., Ltd., Tokyo, Japan
*Author for correspondence: Tel.: +81 3 3353 1211; r.seishima@keio.jp
2

Aim: To evaluate the significance of next-generation sequencing-based gene panel testing in surgically
resectable colorectal cancer by analyzing real-world data. Materials & methods: A total of 107 colorectal
cancer patients who underwent curative surgery were included, and correlations between nextgeneration sequencing data and clinicopathological findings were evaluated. Results: More combination
patterns in gene alteration were identified in advanced-stage tumors than in early-stage tumors. The
copy number alteration count was significantly lower in right-sided colon tumors and early-stage
tumors. Homologous recombination deficiency was more often identified in advanced-stage tumors, and
high homologous recombination deficiency status was useful for identifying high-risk stage II tumors.
Conclusion: Homologous recombination deficiency was identified as a useful result of gene panel testing
with novel utility in clinical practice.
First draft submitted: 5 February 2022; Accepted for publication: 13 June 2022; Published online:
12 July 2022
Keywords: colorectal cancer • copy number alterations • homologous recombination deficiency • next-generation
sequencing • tumor mutational burden

Malignant tumors are traditionally diagnosed and classified based on the organ of origin and histological type,
and treatments are selected according to such classification. However, it has become clear in recent years that
malignant tumors are caused by the accumulation of various genetic mutations. Hence, treatment strategies against
malignant tumors place more emphasis on targeting such genetic mutations. The concept of ‘precision medicine’,
in which genetic mutations in individual malignant tumors are analyzed and individualized treatment targeting
those mutations is employed, has also been gaining ground in recent years [1]. In the case of colorectal cancer
(CRC), it has been reported that tumors are caused by carcinogenic pathways involving various genetic mutations,
such as mutations caused by the adenoma–carcinoma sequence [2]. Accordingly, comprehensive next-generation
sequencing (NGS) studies, such as those of The Cancer Genome Atlas, have been performed and have revealed
that CRC can be classified into some molecular subtypes based on genomic events [3].
NGS-based genomic testing is currently used in clinical settings for the practice of precision medicine. Gene
R
panel tests such as MSK-IMPACT™ (Memorial Sloan Kettering Cancer Center, NY, USA) and Foundation One
CDx (Foundation Medicine, Inc., MA, USA) have been approved by the US FDA, and their use is spreading to
countries worldwide, including Japan. However, the indications for these gene panel tests are limited to locally
advanced or metastatic solid tumors for which standard treatment has been completed or advanced solid tumors for
which no standard treatment is available. Therefore, currently, very few patients can benefit from testing. In fact,
although actionable gene mutations are identified in 37–86% of solid cancer patients, only 11–13% are actually

C 2022 Future Medicine Ltd
10.2217/fon-2022-0122 

Future Oncol. (Epub ahead of print)

ISSN 1479-6694

Research Article

Ogiri, Seishima, Nakamura et al.

identified as targetable mutations [4–6]. Extending the indications of gene panel testing to early-stage tumors may
reveal its utility, but no such studies have been conducted thus far.
In the authors’ institute, in-house NGS-based gene panel testing, which analyzes 160 oncogenes, was performed
for all resectable solid cancer patients in a clinical trial setting [7]. The novelty of this trial is that gene panel testing
was performed immediately after the primary curative surgery – timing that is thus far not available for other
types of insurance-covered tests. In addition, the authors’ in-house gene panel testing is cost-effective, making it
cheaper than other tests. This trial was expected to explore any advantage of genetic information in the decision of
treatment after curative surgery.
Here the authors report the real-world data collected prospectively from patients with CRC who underwent
primary curative surgery at our hospital, including patients with early-stage cancers. The aim of this study was to
investigate any additional information on genetic changes during CRC progression and to explore the significance
of gene panel testing at primary surgery, which will lead to further expansion of testing.
Materials & methods
Patients

This study included patients with colorectal cancer (CRC) who underwent curative surgery from July 2018 to
February 2020 at Keio University Hospital. The study protocol was approved by the ethics committee of the Keio
University School of Medicine (approval number: 20180015). All study participants provided informed consent.
This study was performed following all relevant guidelines and regulations. The Union for International Cancer
Control tumor, node, metastasis classification was used for stage classification, and European Society for Medical
Oncology clinical practice guidelines were used for high-risk stage II classification – specifically, lymph nodes
<12, poorly differentiated tumor, presence of vascular/lymphatic or perineural invasion, pT4 stage and clinical
presentation with intestinal occlusion or perforation [8].
Next-generation sequencing

Tumor tissue was collected from surgical specimens of CRC patients who provided consent to undergo comprehensive genomic testing. Details of the panel have been previously reported [7,9,10]. Briefly, genomic DNA was extracted
from 10-μm thick formalin-fixed, paraffin-embedded tissue sections of tumor specimens using the Maxwell RSC
FFPE Plus DNA Kit (AS1720; Promega Corporation, WI, USA) according to the manufacturer’s instructions.
DNA quality was checked by calculating the DNA integrity number (DIN) using a 4200 TapeStation (Agilent
Technologies, Waldbronn, Germany); all analytes had DIN ≥2.0. Libraries were generated from 80 ng (DIN
≤2.5) or 160 ng (DIN >2.5) of DNA per sample using the Human Comprehensive Cancer Panel, GeneRead
DNAseq Panel PCR Kit V2, GeneRead DNA Library I Core Kit and GeneRead DNA Library I Amp Kit (Qiagen,
Hilden, Germany), and the library quality was assessed using a D1000 ScreenTape (Agilent Technologies). Targeted
amplicon exome sequencing was performed using a 160 cancer-related gene panel as previously described. The
targeted regions of all 160 genes were specifically enriched using oligonucleotide probes. The enriched libraries
were sequenced with a paired-end (150 bp × 2) sequencing method using the NextSeq sequencing platform
(Illumina, CA, USA), resulting in a mean depth of 500. The sequencing data were analyzed using the GenomeJack
bioinformatics pipeline (Mitsubishi Space Software Co., Ltd., Tokyo, Japan; http://genomejack.net/) as previously
described [11]. The proportion of tumor cells ranged from 5 to 80% (median: 45%). Tumor mutational burden
(TMB) was defined as the number of nonsynonymous and synonymous mutations in the target. The estimated
copy number (CN) of the tumor cells was calculated using the following formula: measured CN - 2/proportion of
tumor cells + 2 = estimated CN.
Homologous recombination deficiency (HRD) was evaluated by determining the ‘HRD score’. The score was
R
CDx (Myriad
calculated using an algorithm similar to the loss of heterozygosity (LOH) score in myChoice
Genetics, Inc., UT, USA). Although the LOH score is calculated by the sum of LOH, telomeric allelic imbalance
and large-scale state transitions, the latter two factors cannot be calculated in targeted gene panel sequences because
of the limited number of genes. Thus, a unique method of counting CN alterations (CNAs) has been used to
ensure measurement sensitivity. In detail, the score is defined as the percentage of detected breakpoints in the
whole genome and differences in the CNA status of adjacent probe genes. CNA status includes three categories:
loss, neutral and amplification. LOH regions spanning ≥90% of a whole chromosome or chromosome arm are
considered to be due to non-HRD mechanisms [12]. Thus, chromosomes with fewer than two probe genes (no.
8, no. 18, no. 21 and X in this test) were excluded from the calculation of the HRD score. Chromosomes with

10.2217/fon-2022-0122

Future Oncol. (Epub ahead of print)

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Research Article

Real-world application of NGS for surgically resectable CRC

Table 1. Summary of patient clinicopathological characteristics in each histological type. ...

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