At present, the clinical diagnosis of malignant hematologic diseases depends on MICM, which stands for Morphology, Immunology, Cytogenetics and Molecular for comprehensive classification and prognosis stratification of patients. With the development of modern molecular biology technology and the maturity and application of sequencing technology, gene mutation analysis has become a new tool for clinical diagnosis & treatment. Gene mutations have been introduced into the risk prognosis stratification and detailed classification of malignant hematologic tumors in the key clinical guidelines such as NCCN, ELN, WHO, and domestic expert consensus, and the clinically relevant genes have occupied an annually increasing proportion in the updated guidelines.
Annoroad's Gene Testing For Hematonosis - “AnnoBright” can provide a basis for the diagnose of blood diseases by physicians, clarify the gene mutation carrying status of patients with blood disease, and provide a reference for the prognosis stratification of patients with blood disease;make an auxiliary diagnosis of myeloproliferative neoplasm and part of medication guidance, evaluate the efficacy based on gene mutations, and select targeted drugs; monitor the recurrence and abnormal drug resistance.
- 214 Comprehensive Gene Detections
By using NGS technology, a comprehensive gene mutation detection targeting AML can provide clinical basis for prognosis stratification of preliminarily diagnosed patients, indication of the factors leading to prognosis difference, information on targeted medication-related sites, and tools for subsequent auxiliary monitoring of patients’ disease progression.AML has great heterogeneity. In order to ensure precise and accurate diagnosis and treatment, Molecular biology is added on the basis of conventional MIC classification criteria, thus forming an initial complete MICM classification system.However, as the accumulation of clinical data, clinicians increasingly realize that, even under the existing MICM evaluation and stratification system, there are still large prognosis differences between patients: some patients experience a remission without recurrence after treatment, while some patients become recurrent/intractable after treatment, leading to a treatment failure.With the rapid development of NGS, people gradually realize that AML heterogeneity is multi-hierarchical, especially the molecular level provides rich information on AML differentiation.It allows precise and accurate classification and comprehensively distinguished heterogeneity of acute leukemia, and provides advanced and reliable technical methods for clinical practice, so as to better guide the personalized treatment of patients.
1:There are currently 8 types (2008 WHO, accounting for 52%)
t(15;17),t(8;21),inv(16)/t(16;16),t(6;9),inv(3)/t(3;3),MLL fusion genes,CEBPA and NPM1 (temporary).
2:Chromosome-spliceosome complex (18%)
RNA splicing regulators (SRSF2, SF3B1, U2AF1, and ZRSR2), chromatin gene sequences (ASXL1, STAG2, BCOR, MLL-PTD, EZH2, and PHF6), transcriptional regulator genes (RUNX1);
3:TP53 gene mutation, chromosomal aneuploidy, or both (13%)
4:Contains IDH2 gene mutation (1%)
Specifically for IDH2,this mutation is greatly different from IDH2R,and appears in combination with NPM1.
5:Other
Cross-classified (56 bits,4%),unclassified (166 bits,11%)
In a clinical study sponsored by the Wellcome trust Foundation, 111 genes are sequenced in 1,540 AML patients, of which 52% cases could be classified using conventional methods, while the remaining 48% cases are further classified through a gene mutation analysis performed by the investigators. finally, combined with conventional methods such as karyotyping analysis, fusion genes, using this new marker - gene mutation, the investigators classify AML patients into 11 subtypes in total.The investigators state that a larger-scale verification will be carried out subsequently, and they will cooperate with the WHO to explore issues such as genotyping in the updated guidelines.This study result has been published on NEJM in 2016.
In recent years, with the deepening studies of molecular omics, some mutations have been included in the AML stratification system:CEBPA, NPM1, KIT, FLT3-ITD, TP53, RUNX1, ASXL1, etc.At the same time, some studies have demonstrated that: multi-gene mutations often occur concurrently. In such case, a single mutation is no longer sufficient to guide clinical prognosis. A combined analysis on multiple genes on the basis of cytogenetics has gradually become a clinical hot spot. In 2022, the screening of “other mutations” is added to the initial evaluation in the NCCN Guideline for AML, thus contributing to the instructions of the prognosis stratification of AML.
With the deepening of clinical experience of gene mutation detection, the influence of gene mutation burden on clinical significance has been gradually recognized.RUNX1, ASXL1 and TP53 are included in the 2017 ELN Guideline. In addition to newly added genes, the differences in allele frequency of FLT3-ITD (low allelic ratio vs high allelic ratio) combined with the mutation of NPM1 gene have different impacts on the prognosis of AML patients. A definite classification has been made in prognosis stratification:
Moreover, a comprehensive study regarding TP53 published in Leukemia in 2017: difference of TP53 mutation burden in the prognosis of four hematologic diseases (AML, MDS, ALL, and CLL).
| Clinical hot spot 1 | Clinical hot spot 2 |
| CEBPA、 NPM1、 FLT3-ITD、 FLT3、 TP53、 KIT、 DNMT3A、 IDH1、 IDH2、 MLL、 TET2、 RUNX1、 KRAS、 NRAS、 ASXL1、 WT1、 GATA1、 PHF6 | ABCB1、 AKT3、 AMER1、 APC、 ATRX、 BCOR、 BCORL1、 BRAF、 CACNA1E、 CBL、 CBLB、 CDKN2A、 CREBBP、 CRLF2、 CSF1R、 CSF3R、 CUX1、 DIS3、 DNAH9、 EGFR、 ERG、 ETV6、 EZH2、 FAM46C、 FBXW7、 GATA2、 GNAS、 GSTP1、 HRAS、 IKZF1、 JAK1、 JAK2、 KDM6A、 KMT2C、 KMT3A、 MAP2K4、 MLH1、 MPL、 NF2、 NQO1、 NT5C2、 NTRK1、 NTRK2、 PTEN、 PTPN11、 RAD21、 RB1、 SETBP1、 SF3B1、 SH2B3、 SMAD4、 SMC1A、 SMC3、 SRSF2、 STAG2、 STAT5B、 TPMT、 U2AF1、 ZRSR2 |
By using NGS technology, a comprehensive gene mutation detection targeting MDS related genes can provide a justification for clinical prognosis evaluation of patients and monitor the disease progression of patients.
The occurrence and development of MDS is a complex and multifactorial process with great heterogeneity.With the development of NGS sequencing technology, studies have found that mutations are closely related to the occurrence and development of MDS, and increasing evidences support that gene mutations can serve as a molecular biomarker to evaluate prognosis.With the in-depth research in recent years, newly demonstrated epigenetics-related mutations and splicing factor mutations have been proved to play an important role in the occurrence and development of MDS.In addition, an increasing number of studies have demonstrated that these common mutations affect the MDS stratification and prognostic survival (please refer to the 2022 NCCN Guideline for MDS).
Studies have confirmed that MDS undergoes a clonal evolution over time, and subclonal mutations in the MDS stage can often be transformed into more aggressive AML by acquiring driver mutations, and become the main clone of the transformed AML. At present, FLT3, NRAS and SETBP1 mutations are demonstrated to be events at the advanced stage in the MDS disease progression, that is, subclones, and these mutations in patients always predict the transformation to AML and a poor prognosis.
In the 2022 NCCN Clinical Practice Guideline in Myelodysplastic Syndrome, TP53 is considered a factor of poor prognosis. The common mutations of TP53 gene include frameshift mutation, nonsense mutation, splicing mutation and all non-synonymous mutations except P47S and P72R, accounting for 8%-12% of MDS cases. In recent years, relevant studies have found that MDS patients with TP53 mutations have a higher response rate to decitabine.In a study investigating the effect of decitabine in 116 patients with AML or MDS performed in 2016, after decitabine is given in patients for 10 consecutive days per month in a monthly cycle, patients with TP53 mutations achieve a higher response rate to decitabine than those without such mutation [21/21 (100%) vs. 32/78 (41%), p＜0.001].
| Clinical hot spot 1 | Clinical hot spot 2 |
| TET2、 DNMT3A、 ASXL1、 EZH2、 SF3B1、 SRSF2、 U2AF1、 ZRSR2、 TP53、 STAG2、 NRAS、 CBL、 JAK2、 RUNX1、 ETV6、 IDH1、 IDH2、 SETBP1、 PHF6、 BCOR、 PTPN11、 KRAS、 JAK3、 GATA2、 CEBPA、 BRAF、 CSF3R、 CALR、 MPL、 TERC、 NPM1、 PIGA、 DKC1、 PRPF8、 NF1、 STAT3、 TERT、 ETNK1、 DDX41、 SRP72、 ELA2、 HAX1、 GFI1、 BLM | ATRX、 BCORL1、 CDKN2A、 CREBBP、 CUX1、 DNAH9、 FLT3、 GNAS、 KDM6A、 KIT、 NT5C2、 PTEN、 RAD21、 SMC3、 SH2B3、 WT1 |
Acute lymphocytic leukemia (ALL) is a malignant neoplastic disease originating from abnormal hyperplasia of B or T lymphocytes in bone marrows. Abnormally proliferating primitive cells can aggregate in the bone marrow and inhibit normal hematopoiesis, and simultaneously invade tissues outside the bone marrow. They have diverse biological characteristics and great clinical heterogeneity.The incidence of ALL may peak at 2-4 years of age, and then decreases with the increasing age. After the age of 60 years, the incidence reaches the second small peak in the elderly. Therefore, ALL is prevalent in the elderly and the infants.
Although multi-drug combination chemotherapy can achieve complete remission in more than 80% of treatment-naive adult patients with ALL, over 50% of patients eventually relapse, and the 5-year disease-free survival rate is only 30%-40%.The overall curative effect of children with ALL is relatively satisfactory, about 80%-90% of ALL children achieve long-term disease-free survival, but there are still 15%-20% of children experiencing a relapse after remission, for which it is difficult to improve the curative effect even after the intensity of chemotherapy is further increased. Serious toxic side effects may occur in some patients.
Therefore, according to the specific cellular/molecular genetic variations of ALL, measures such as dynamic adjustment of risk grouping, rational selection of treatment regimens, and combined application of targeted drugs will contribute to a precise and accurate treatment of ALL.
Figure. Abnormalities of genes related to lymphoid differentiation and development in Ph-like ALL
——Commonly observed IKZF1, PAX5, ETV6, ERG, etc.
MPN is a set of heterogeneous diseases originating from pluripotent lymphoid-myeloid stem cells or myeloid progenitor cells, with a proliferative phenotype predominately, and occasional pathological hematopoiesis.In 2008, the WHO has changed myeloproliferative disorder (MPD) to myeloproliferative neoplasm (MPN). In 2016, the WHO revision has supplemented the weight of molecular indicators in the definition of disease (type) and in the diagnosis.Currently, JAK2, MPL, and CALR mutations are associated with the differential diagnosis of polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Compared with patients carrying JAK2 V617F mutation, ET and PMF patients carrying CALR mutations are characterized by a lower average age, lower risk of embolism, and prolonged overall survival time; as a newly added gene, CSF3R mutation is highly specific to chronic neutrophilic leukemia (CNL).With the deepening of molecular research, the prognosis-related genes such as ASXL1 and RUNX1 have gradually attracted academic attention. Multi-gene mutation research is particularly important in triple-negative MPN patients (negative for all three genes of JAK2/CALR/MPL). Gene mutations such as ASXL1, EZH2, TET2, IDH1, IDH2, SRSF2, and SF3B1 are recommended in guidelines for the diagnosis and prognosis evaluation of ET and prePMF, providing molecular basis for the exploration of survival differences.
Oh ST et al. Therap Adv Hem. 2011;11-19.
Delhommeau F et al. Int J Hematol. 2010;91:165-173.
Vannucchi AM et al. Haematological. 2008;93:972-976.
| Disease/suspected disease | Gene detection | |
| First-line | PV | JAK2 V617F |
| PMF/ET | JAK2 V617F, MPL W515L, CALR | |
| Second-line | PV | JAK2exon12,LNK |
| PMF | ASXL1, TET2, SRSF2,IDH1/2 | |
| PV (44% of patients, mut>1) | TET2(18%), ASXL1(11%), SH2B3(5%),SF3B1(3%), SETBP1(<2%), IDH1, DNMT3A CEBPA,CSF3R,SUZ12, SRSF2, ZRSR2, TP53, CBL, NRAS,KIT, PTPN11, FLT3 |
| ET(46% of patients, mut>1) | TET2(13%),ASXL1(11%), DNMT3A(6%), SF3B1(5%), CEBPA(4%), P53(<2%) SH2B3, EZH2, CSF3R |
| PMP (83% of patients, mut≥1) | ASXL1(36%), TET2(18%), SRSF2(17%), U2AF1(17%), ZRSR2(11%), SF3B1(10%), DNMT3A(9%), CEBPA(9%), P53(7%), CBL(5%),IDH1/2(5%) |
| Sum (90% of patients, mut>1) | Multi-gene mutation analysis can provide a clonal marker reference for the diagnosis of patients, Mayo Clinic, US |
< J Clin Intern Med, March 2016, Vol.33, No.3>
| Clinical hot spot 1 | Clinical hot spot 2 |
| JAK2、 MPL、 CALR、 CSF3R、 ASXL1、 EZH2、 TET2、 IDH1、 IDH2、 SRSF2、 SF3B1、 CBL、 DNMT3A、 IKZF1、 KIT、 ABL、 SETBP1、 SH2B3、 KRAS、 NRAS、 ETV6、 GATA2、 RUNX1、 NF1、 TP53、 U2AF1 | CBLC、 CSF1R、 EGFR、 FAM46C、 SMC3 |
“AnnoBright” clinically beneficial chemotherapy gene detection set, based on the needs of commonly used clinical chemotherapy regimens, solicits the advice of a number of national hematology experts, summarizes complete pharmacogenomic related database PKGB information created by NIH (National Institutes of Health, US) and dosage recommendations from CPIC (Clinical Pharmacogenetics Implementation Consortium), the competent authorities responsible for issuing international precise and accurate medication guidelines, selects 14 commonly used chemotherapeutic drugs for hematologic tumors, includes polymorphism gene detection of 46 loci, presents different levels of evidence in the report, and provides a rigorous and detailed reference for the toxic and side effects of chemotherapy drug metabolism.
Chemotherapy is the main treatment approach for acute leukemia, and “early stage, sufficient dose, combination and individualization” are the distinctive features of chemotherapy regimens for acute leukemia.Although the reasonable regimen and sufficient dose of chemotherapy often achieve complete remission within one course of treatment, the toxic and side effects of chemotherapeutic drugs cannot be ignored, especially for young or elderly patients or those with poor constitutions.Taking anthracyclines as an example, anthracycline chemotherapeutic drugs, including adriamycin, epirubicin, daunorubicin, and aclacinomycin, are widely used in the treatment of hematologic malignancies, and their toxic and side effects are also of clinical interest.In the China Guidelines for the Prevention and Treatment of Anthracycline Cardiotoxicity, there is no absolute “safe dose” for anthracyclines, which may be due to individual differences in patients, that is, interindividual differences in hereditary drug metabolism, leading to different susceptibilities to anthracyclines, thus causing the difference of toxic and side effects.
In addition, the metabolism and genetics of chemotherapeutic drugs for various hematologic diseases are related, such as asparaginase for remission induction therapy in ALL, 6-mercaptopurine (6-MP) and methotrexate (MTX) for maintenance therapy in ALL, typical drug cytarabine in conventional chemotherapy, etc., which all have genetically related differences in metabolic capacity.
The clinical benefit of chemotherapeutic drugs refers to the gene detection of the metabolism toxic and side effects induced by chemical drugs, that is, the detection of gene polymorphism sites that affect the sensitivity of chemotherapeutic drug metabolism, to scientifically predict the efficacy of drugs, and provide guideline for clinical medication.In this process, more drug-related information, higher reference levels, and more comprehensive interpretation are the keys to serving personalized medication in clinical practice.
