NEJM: The traditional cytogenetics alternative technology has been clinically verified, and the diagnosis rate and accuracy are better

As an important part of the diagnosis of various tumors, genetic testing can be used to predict clinical prognosis and response to targeted therapy.

Gene mutations, including chromosomal rearrangements, gene amplification and deletion, and even single nucleotide mutations, may have clinical significance.
Therefore, the use of multi-platform technology to obtain complete genetic information is essential for the selection of clinical treatment strategies.

The whole genome sequencing (WGS) can basically cover the detection of all types of mutations, and discover unknown or hidden mutations, which can greatly improve the efficiency of gene detection in cancer patients and may replace other detection methods currently used.

 A study (N Engl J Med 2021;384:924-35) from Washington University (St.
Louis) School of Medicine published in the New England Journal of Medicine on March 11, 2021, compared WGS The application of traditional cytogenetic diagnosis in the diagnosis and risk stratification of myeloid hematological tumors shows the feasibility, accuracy and effectiveness of WGS in the diagnosis of acute myeloid leukemia and myelodysplastic syndromes.

It is worth noting that the WGS in this study was completed in a routine clinical sequencing laboratory, rather than a specialized research institution, which is closer to the real clinical scene.

 "NEJM Medical Frontier" invited Professor Yan Hua from the Department of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, to write a review and interpret this paper.

We will release the full text translation of Genome Sequencing as an Alternative to Cytogenetic Analysis in Myeloid Cancers on March 12th.
Please visit NEJM Medical Frontiers official website, APP or click on the WeChat applet picture.Yan Hua Gene abnormalities in the Department of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, are particularly important for the diagnostic classification and prognostic risk assessment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

The current risk assessment system based on chromosomal abnormalities and specific genetic mutations of clinical significance is the basis of the WHO AML risk stratification system[1,2], and the revised International Prognostic Scoring System (IPSS-R) is applicable to MDS patients[3] .

In recent years, as genetic testing technology has made great progress, traditional cytogenetic analysis technology has shown many shortcomings, including the need for more cells, low sensitivity, and low positive rate.

However, the newer FISH and DNA targeted sequencing analysis are unable to obtain complete genetic information.

Whole genome sequencing (WGS) can find a large number of mutations related to clinical diagnosis and prognosis, so it is becoming a standard detection method.

Although WGS currently limits its clinical application due to its cost and complexity, it will become more economical and faster as technology advances.

In this context, researchers at the Washington University, St.
Louise School of Medicine obtained bone marrow and peripheral blood samples from 263 patients with myeloid tumors (AML and MDS), using WGS, karyotyping, and fluorescence isotope Methods such as hybridization (FISH) were used to stratify the risk of patients, and the results were published in the New England Journal of Medicine on March 11, 2021 [4].

Research methods These samples came from 263 patients treated in medical institutions affiliated to the medical school, including 146 retrospective research samples and 117 prospective research samples.

Traditional cytogenetic testing is used to carry out the European Leukemia Network (European Leukemia Network) or IPSS-R risk classification.

For samples that cannot be detected by traditional cytogenetic methods, researchers use FISH, chromosome microarray analysis, and RNA sequencing to verify the samples.

All samples were subjected to WGS analysis with a target coverage depth of 60× (Figure 1), which can detect and automatically analyze 40 gene mutations in myeloid tumors, copy number changes greater than 5 Mbp in the genome, and 612 frequent structural changes.

Researchers used Kaplan-Meier analysis or Cox proportional hazard regression analysis to determine survival differences.

Figure 1.
The WGS process used in this article [4].

Comparison of traditional cytogenetics and WGS diagnosis and risk grading This study conducted a head-to-head comparison between traditional cytogenetics and WGS.

WGS not only detected all 40 recurrent translocations and 91 copy number changes confirmed by traditional cytogenetic analysis, but also found traditional translocations in 17% of patients (40 out of 235 patients).
Clinically significant genetic mutations not detected by cytogenetic methods.

From the beginning of sample preparation to the WGS analysis results, the median time for genome-wide analysis of the 117 patients enrolled in the prospective study was 5 days.

Among them, WGS provided 29 patients (24.
8%) with new genetic information and changed the risk level of 19 patients (16.
2%).

Compared with cytogenetic methods, the standard AML risk categories determined by WGS are closely related to clinical results.

The researchers also successfully used WGS to stratify samples of patients whose cytogenetic analysis could not reach a definite conclusion, and the clinical treatment results of these patients at different risk levels were significantly different.

The detection rate of WGS for frequent translocations found by traditional cytogenetic analysis is 100%, which is highly sensitive.

More importantly, WGS also found more complicated and hidden chromosomal translocations in 13 patients, including the inv(16)(p13.
1q22) fusion gene CBFB-MYH11 in 2 patients, and one patient t(7 ; 21) (p22; q22) fusion gene USP42-RUNX1, and KMT2A gene rearrangement in 10 patients. In terms of gene mutations, the study found that compared with 102 patients with traditional high-coverage targeted sequencing, WGS had a sensitivity rate of 84.
6% for single-nucleotide mutations and 91.
5% for indel mutations.

For AML risk stratification, the overall detection rate of single nucleotide variants and indels in genes such as ASXL1, CEBPA, FLT3, NPM1, RUNX1, and TP53 has reached 87.
5% (Figure 2).

Figure 2.
Comparison of WGS with traditional cytogenetics and targeted sequencing analysis [4].

The study found that among 71 patients who had not undergone stem cell transplantation, WGS was 89% consistent with the risk grouping of traditional diagnosis.

About 20% of AML patients cannot obtain cytogenetic results for various reasons.
The authors believe that WGS will benefit these patients the most.

Therefore, the investigators performed WGS assessment on 27 such AML patients (6 failed tests, 13 had uncertain results, and 8 had unknown results).

The authors found that 2 patients had KMT2A and RUNX1-RUNXT1 rearrangements, 2 patients had complex karyotypes, and 23 patients had normal chromosomes or had only 1-2 abnormal chromosomes.

Survival analysis showed that the risk prediction based on WGS was closely related to clinical prognosis.
Among them, the median OS of 21 low- and medium-risk patients reached 20.
5 months, and the median OS of 6 high-risk patients was only 3.
3 months (Figure 3).

Figure 3.
Using WGS to assess the risk stratification of AML patients [4].

Feasibility analysis researchers found that the optimized WGS process provides rapid and accurate genetic analysis methods for patients with AML and MDS.

Compared with traditional cytogenetic analysis, WGS can provide a broader diagnostic field of vision and more effective risk stratification.

The optimization of the process allowed the WGS in this study to get the sequencing report as fast as 3 days.

The analysis of sequencing results can make effective risk prediction based on the existing risk stratification system.

Although more large-scale studies are needed to establish the clinical application of WGS, the existing research results show that this method can be used for risk stratification for more patients with myeloid tumors, especially for patients with uncertain results through cytogenetic analysis.
, Has a good prognostic value, and quickly provides a basis for the choice of clinical strategies.

At the same time, the continuous reduction in the price of WGS also makes the cost of this method close to the cost of other sequencing platform technologies.

WGS can provide a unified, stable, and expandable technology platform for clinical testing, minimize laboratory errors and achieve standardization.

Although the current research is only focused on myeloid hematological tumors, the great advantages of WGS can also be reflected in other tumors.

Based on personal diagnosis and treatment experience, the author believes that WGS can further improve the accuracy of clinical diagnosis.

In our clinical work, we have found a young AML patient, which was detected by traditional sequencing methods to carry NPM1 mutation.

According to the current guidelines and clinical experience, the clinical prognosis of this type of patient is good; however, during the course of treatment, this patient quickly relapsed.

After reanalysis using WGS, we found that this patient also had other genetic mutations with poor prognosis, and adjusted the treatment strategy.

In the same way, MDS often has clonal evolution.
Although patients only have one or two mutations at the beginning, as the disease progresses, the possible mutations gradually increase, and the probability of turning into AML is also getting higher and higher.

Therefore, monitoring multi-gene mutations in patients with MDS can prompt the patient's disease progression, and adjust the clinical treatment plan at an appropriate stage.

At present, WGS also has certain limitations.

First of all, in the process of data analysis and result interpretation, the number of detected genes to be tested that meet the quality control requirements is too small, the number of detected variants significantly deviates from expectations, and the sample label sequence is contaminated, and other factors may cause the results to deviate.

Secondly, for the description of mutations, especially for mutations that have not yet been reported in the literature or are uncertain whether there are clinically relevant mutations, laboratories need to formulate corresponding standards to make statements and have follow-up treatment strategies.

The clinical application of WGS technology also involves a series of ethical issues and requires the improvement of relevant laws and regulations to ensure its healthy and orderly development.

This is a question that scientists, clinicians, laboratory technicians, and even medical administrative departments who use this technology must think about.

The author introduces Yan Hua, chief physician and doctoral supervisor of the Department of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine.

Dedicated to the basic and clinical research work of blood diseases for a long time, focusing on multiple myeloma.

In 2008, he went to Massachusetts General Hospital to specialize in myeloma bone disease.

Published more than 30 SCI articles.

Responsible for the key research projects of Shanghai Municipal Science and Technology Commission of Biomedicine and many projects of the National Natural Science Foundation of China, deputy leader of the 863 Program.

Academic part-time jobs include executive director of the Chinese Geriatrics Association Council, member of the 8th Laboratory Diagnostics Society of the Chinese Medical Association Hematology Branch, member of the Plasma Cytology Group of the Hematology Branch of the Chinese Medical Association, and Executive Committee of the Chinese Anti-Cancer Association Clinical Oncology Cooperative Professional Committee Committee member, member of the Myeloma Special Committee of the Hematologists Branch of the Chinese Medical Doctor Association, member of the Hematological Oncology Committee of the Chinese Anti-Cancer Association, etc.

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National Comprehensive Cancer Network.
NCCN guidelines for patients: acute myeloid leukemia (https:// 3.
Greenberg PL, Tuechler H, Schanz J, et al.
Revised international prognostic scoring system for myelodysplastic syndromes.
Blood 2012;120:2454-65.
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Duncavage EJ, Schroeder MC, O'Laughlin M, et al.
Genome sequencing as an alternative to cytogenetic analysis in myeloid cancers.
N Engl J Med 2021;384:924-35.
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Wetterstrand KA.
DNA sequencing costs: data from the NHGRI Genome Sequencing Program (GSP).
National Human Genome Research Institute, 2020 ( 6.
Cressman S, Karsan A, Hogge DE,et al.
Economic impact of genomic diagnostics for intermediate-risk acute myeloid leukaemia.
Br J Haematol 2016;174:526-35.
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