Expert consensus on standardized management of tumor gene sequencing for cancer patients

2.1 Patient management before gene testing

Before gene testing, clinicians and technicians should comprehensively evaluate the samples of tumor patients to be tested and the selected test items.

For patients who need gene detection, the use of tumor tissue sample is the first choice to obtain tumor-related gene information. The proportion of tumor cells and non-tumor cells in the samples should be evaluated. The number of tumor cells should be greater than 20% of the total number of nucleated cells. Considering that the initial amount of deoxyribonucleic acid (DNA) required for the most basic tumor gene detection at present is about 10−250 ng, while the DNA content of a single nucleated cell is about 6pg, which is equivalent to 6000−25000 living cells for the sample provided. A single Formalin-fixed paraffin-embedding (FFPE) of 60−100 mm² tumor tissue can provide about 2000 cells. For the tissue containing significant mucus and necrotic components, the cell content will be lower.² Therefore, it is recommended to provide 10−15 FFPE tissue slides for tumor gene detection. Whether the storage method is fresh freezing or FFPE treatment has little impact on the test results,³ so there is no requirement for the storage method. In addition, it is also necessary to obtain peripheral blood samples of patients as the germline control.

For tumor patients who do not have tissue samples for various reasons, such as advanced tumor patients who have lost the chance of surgery, patients whose tumor anatomical position is not suitable for surgery, patients whose tumor tissue has been exhausted in other diagnosis or who have received neoadjuvant chemotherapy, it is recommended to immediately conduct puncture or liquid biopsy for detection. The types of samples that can be collected by liquid biopsy include blood (peripheral blood), saliva, urine, cerebrospinal fluid, pleural effusion, etc. Collecting peripheral blood is the most common type of liquid biopsy. Blood samples can overcome the limitations of tissue heterogeneity. For multiple metastasis, since circulating tumor DNA (ctDNA) can come from multiple tumor sites, blood samples are more suitable than biopsy tissue from a single site. Recent research have found the overall consistency was over 80% in lung cancer^{4, 5}; however, the consistency between blood samples and tissue samples is still worth exploring. When the number of tumor cells is more than 10⁶, the ctDNA released into the blood can be detected. Especially in the early stage of tumor, the concentration of ctDNA is extremely low, resulting in relatively low detectable mutation abundance (generally < 1%). Therefore, the detection sensitivity is crucial, and the sequencing depth is also required to be high (generally greater than 10000x). In addition, because the length of ctDNA segment is short, it is difficult to detect the fusion gene.⁶ Therefore, although it is clear that both tissue samples and blood samples can be used for the detection, tissue samples are still preferred. However, puncture samples often have a certain detection failure rate because they are too small or easily contaminated by benign stromal cells. Therefore, if the economic conditions permit, it is recommended to use puncture samples for tumor gene detection as well as ctDNA detection when patients are eager to wait for treatment plans.⁷

Because tumor is a disease caused by multi-gene abnormality, if only a small number of mutation sites are detected, other key sites may not be detected in time, which may delay the disease or miss the best targeted treatment of the patients. Therefore, multi-gene mutation detection, even whole-exome or whole-genome gene sequencing, can better reflect the overall picture of the tumor. There are more than 20 000 genes in the human genome, but the number of genes that may be associated with cancer is not more than 1000. Targeted tumor gene detection panel is to select the genes related to specific tumor treatment, establish the list of specific tumor core genes, and repeat the deep sequencing of the same site. The detection accuracy is often high. Whole exome sequencing (WES) has the obvious advantage of detecting most protein-coding genomes at one time, and can cover all tumor-related genes in the existing targeted tumor detection panel combinations. Whole genome sequencing (WGS) can better detect non-coding genes and some structural changes. In theory, it can detect any genomic changes. However, due to the huge amount of data produced by WES and WGS, and most of the detected mutation sites have no clinical interpretation at present, the application of WES and WGS in routine diagnosis and treatment needs further verification. Moreover, DNA quality (library quality, tumor content, etc.) and sequencing quality (read coverage, bioinformatics analysis) will be the factors influencing the reproducibility of WES and WGS in tumor mutation detection.⁸ In addition, transcriptome sequencing is also a genome-wide sequencing technology. In future clinical practice, integrating DNA sequencing and ribonucleic acid (RNA) sequencing can better detect the expression of alleles, evaluate known or unknown gene fusion, confirm splicing bodies, and analyze signal pathways, etc. Compared with adults, it will be more suitable for the detection of pediatric tumors⁹ because of the less change of recurrence point mutations. However, RNA sequencing also needs to be further verified in routine diagnosis and treatment due to its huge amount of data and complex analysis.

2.2 Patient management during gene testing

All patients receiving gene testing should sign a written informed consent form for gene testing. The content of the informed consent should cover the content of the required test items, the significance, advantages, defects, limitations, alternative methods and potential risks of the test. Before signing, the clinician or genetic consultant should explain the relevant contents of the informed consent form in detail. In addition, the patient has the right to decide whether to accept the relevant provisions in the informed consent form, including whether to agree to donate the remaining samples after the test, whether to agree to donate the genetic information produced after the test and other relevant data. The patient has the right to choose to join or exit. For the genetic information that may be detected, the patient has the right to choose whether to be informed all the detection results, including the discovery that is not within the detection scope or the discovery of the status of the carrier of genetic disease. Clinicians or genetic counselors should ensure the best interests of patients, reduce the risk-benefit ratio of patients and ensure the reasonable expectations of patients. If the patient is pregnant or has a history of blood transfusion, organ transplantation, bone marrow transplantation or denaturalization, the patient must inform the detection department when signing the informed consent, otherwise the sample may be considered as contaminated sample in the test.⁷

2.3 Patient management after gene testing

After the gene test, the patient should obtain a clear gene test report with specific conclusions. The report shall include the basic information and diagnosis of the patient, brief description of the test, summary of the test results and main findings, etc. In addition, quality control data should also be listed in the report, including important quality control information such as tumor cell content, sequencing depth, target area coverage, etc. After the patient obtains the report, the technician shall briefly explain the detection information and data quality of the report. The genetic consultant shall explain the mutation genes found in the test and their inheritance pattern. Clinicians should judge whether the results can guide the treatment, and observe the dynamic development of the patient's condition during the treatment, so as to adjust the medication in time.

Since many mutations detected are very rare, we cannot show the importance of each detected mutation. Therefore, if the patient wishes, he can apply to join the clinical trial after obtaining the gene test report to help determine the importance of these mutations. Clinicians can recommend clinical trials according to the actual situation of patients, and patients can also choose whether to participate according to their own conditions.

3.1 Tumor screening

Early detection, early diagnosis and early intervention are the main directions for the development of accurate diagnosis and treatment of cancer in the future. Since most tumors are asymptomatic at the early stage, when most patients are diagnosed, they are already in advanced stage. Therefore, if the high-risk patients can be screened early, and the tumor can be predicted early, the mortality of cancer patients can be significantly reduced.

Early screening of tumors can be applied to asymptomatic populations, which can be divided into single cancer screening and pan-cancer screening. Single cancer screening is applicable to people with obvious family history of cancer, and currently it is mostly applied to liver cancer and colorectal cancer. For example, Lynch syndrome is caused by the germline variation of DNA mismatch repair related genes. The risk of colorectal cancer and endometrial cancer of them is high. Since it has obvious familial aggregation, it is recommended that patients and their families should carry out gene test in time and colonoscopy every 1−2 years.¹⁰ Pan-cancer screening is applicable to the population without clear cancer risk factors.

The early screening of tumors focuses on the assessment of genetic risk and the identification of high-risk populations, while the early diagnosis of tumors focuses on the auxiliary diagnosis of suspicious populations. Liquid biopsy can be used for early diagnosis of tumors. Compared with other liquid biopsy techniques, ctDNA has the advantages of high signal abundance and high signal intensity in detecting tumor-driven gene mutations. In addition, ctDNA can detect abnormal methylation signals, which can indicate ultra-early tumors, and is one of the main development directions of early tumor screening and diagnosis in the future.¹¹

3.2 Molecular classification and companion diagnostics

In 2017, the United States Food and Drug Administration (FDA) approved commercial companies and academic institutions to carry out medical NGS services. After that, a number of pan-cancer NGS products have been approved by FDA. Since 2018, the National Medical Products Administration has also approved several tumor companion diagnostic reagents on the NGS platform, indicating that NGS has officially entered the clinical application stage in China.¹¹

Molecular classification of tumors is to systematically describe the molecular spectrum of tumors and accurately classify tumors according to the characteristics of molecular spectrum. NGS is applicable especially for new characterization of tumors. For example, a distinctive retinoblastoma (Rb) proficient subset of small cell lung cancer was characterized through NGS.¹² However, molecular classification by gene testing is applicable to patients who have failed to make clear classification through previous molecular testing or whose previous molecular testing is incomplete. The accuracy of tumor molecular classification determines the accuracy of tumor treatment. It is also one of the needs of precision medicine to carry out companion diagnosis for patients through molecular characteristic spectrum, and to develop personalized diagnosis and treatment.

For drugs with clear targets, the principle of target detection must be followed before use. It is not allowed to use drugs blindly without relevant examination. Because of the high specificity of NGS detection, the patient should have the opportunity to carry out targeted treatment immediately when the detection locus has passed the positive verification and there are matching targeted drugs.⁷ However, if the detected gene mutation cannot match the drug recommended in the clinical guidelines, or the drug recommended by the gene mutation is not applicable to the cancer, it should be carefully applied to clinical practice. There were data showed that about 9% patients could be made molecular diagnosis. The positive yield was 14.8% for colon/stomach cancer, followed by 13.4% for ovarian cancer and 9.7% for breast cancer.¹³

In addition, some new markers have gradually entered clinical application. For example, homologous recombination repair is the preferred repair method for DNA double strand break. The detection method of the beneficiaries of Poly adenosine diphosphate (ADP)-ribose polymerase (PARP) inhibitors has expanded from the detection of breast cancer susceptibility gene (BRCA) mutations to the detection of homologous recombination deficiency (HRD). Tumor patients with positive HRD are more sensitive to PARP inhibitors.¹⁴

The results of gene testing can also provide information for immunotherapy. Immunogenomics is a relatively new research field. It obtains the genome map of immune cells and tumor cells through NGS which can provide support for tumor immunotherapy, thus providing patients with new alternative treatment.¹⁵ The detection of immune checkpoint inhibitors of NGS includes microsatellite instability (MSI), tumor mutation burden (TMB), etc. Microsatellite is a simple and repetitive nucleotide sequence in DNA. When tumor cells invade, it will lead to the instability of the sequence. When the test results show the MSI-H (microsatellite instability-high, MSI-H) phenotype, it suggests that the tumor can benefit more from the treatment of immune checkpoint inhibitors.¹⁶ TMB represents the number of mutations per megabase in the tumor cell genome. The increase of TMB suggests that the body can produce more new antigens and attract more T cell attacks, thus improving the ability of the immune system to recognize and clear tumors, and making tumors more benefit from the treatment of immune checkpoint inhibitors.¹⁷ Although MSI and TMB have been proved to have certain predictive value in clinical practice, their scope of application and clinical standards need to be further explored.

3.3 Recurrence monitoring

Traditionally, tumor relapse is monitored through radiographic imaging; however, their utility is controversial due to suboptimal specificity and radiation exposure.¹⁸ Minimal residual disease (MRD) refers to a small number of tumor cells that remain in the patient's body due to incomplete removal of tumor cells or tolerable treatment. It is one of the main reasons for tumor recurrence. CtDNA is a small DNA fragment in the peripheral blood that carries tumor tissue information from the apoptosis or necrosis of tumor cells in the primary and metastatic foci of tumor.¹⁹ It can be used for tumor recurrence monitoring due to its high specificity.²⁰ Since the half-life of ctDNA in circulation is between 16 min to 2.5 h,²¹ the change of ctDNA can reflect the real-time dynamic information occurring in the body. Because the sample acquisition is convenient and non-invasive, it can truly achieve real-time dynamic monitoring and can also detect tumor recurrence earlier than imaging. The elevated level of ctDNA is closely related to the clinical staging, tumor metastasis and prognosis. The detected tumor mutations can be used to monitor disease progress and detect residual lesions after treatment. If tumor progression is detected, targeted intervention can be carried out against the rising clones according to the detected mutation information. Rapid tumor progression can be avoided through timely drug adjustment.²²

In terms of prognosis, patients with ctDNA clearance after surgery have a good long-term prognosis and do not need adjuvant treatment, while patients with residual ctDNA in the blood suggest a high risk of recurrence and need timely systemic treatment to reduce the risk of disease recurrence. Successful systemic treatment will be accompanied by a rapid decrease in ctDNA concentration. If the ctDNA concentration remains unchanged, it indicates that the treatment effect is poor.

At present, MRD mainly has two assays: tumor-uninformed assay and tumor-informed assay. The former does not rely on the information provided by the primary tumor tissue, which is simple and universal. The latter needs to rely on the mutation information detected in the primary tumor tissue to establish an individualized information base and algorithm, and carry out targeted tracking and monitoring, which is more personalized. No matter which assay is selected, patients will undergo periodic tumor monitoring. If the condition is stable, they will maintain treatment. If the condition progresses, they will adjust the treatment plan in time, so that the tumor will gradually become a “chronic disease” management mode.