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Technical brief

Background

Treatment options for blood diseases

    Blood diseases originate from the hemopoietic system. Of all blood diseases, such hematologic tumors as leukemia, myelodysplastic syndrome and myeloproliterative neoplasm pose tremendous threatening to human life and health. The most common treatments of hematologic tumors are combined chemotherapy and bone marrow transplantation, both of which apply to patients at different levels of severity. Therefore, in clinical practices, it is necessary to evaluate risk stratification and prognosis before choosing a treatment method. Special blood test is an essential part of blood disease treatment.

    Every blood cell has its unique physiological function. For example, red blood cells transport oxygen, and white blood cells are responsible for inflammatory response. Therefore, when the quantity or quality of hemocytes becomes abnormal, the abnormality is manifested as hematal dysfunction.

Multi-gene joint detection helping evaluate blood disease prognosis in a hierarchical manner

    Take acute myelocytic leukemia (AML) as an example. According to the existing prognosis stratification system, patient’s prognosis is divided into three types: high risk, medium risk and low risk. Patients at high risk are advised to receive bone marrow transplantation, and those at low risk usually take chemotherapy. However, 60% patients are evaluated as at medium risk in terms of prognosis, and show no specific treatment indications. For these patients, doctors’ experiences are excessively relied on for choice of treatment protocols. According to long-term statistical results, patients at medium risk of prognosis present the greatest heterogeneity in treatment. For them, more accurate means of prognosis stratification is required.

    An article about precise medical treatment of leukemia published on New England Journal of Medicine in 2012 proposed a brand-new method of prognosis stratification based on somatic mutation. The article concluded as follows: Multi-gene joint detection can be applied to precise prognosis stratification for leukemia.


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Fig 1: Multiple gene mutations in leukemia cells


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Fig 2: Stratification of acute myelocytic leukemia prognosis and its survival period


Multi-gene Testing For Hematonosis

    In Annoroad’s Multi-gene Testing For Hematonosis, bone marrow or peripheral blood sample is used; and 111 genes related to blood diseases are tested on a high-throughput gene sequencing platform. Medical databases and the latest scientific literatures are consulted for interpretation and understanding of biological functions and clinical significance. The test provides important foundation for auxiliary clinical diagnosis and prognosis stratification; and makes a completely new tool of evaluating blood disease prognosis for clinical doctors. Multi-gene joint mutation spectrum detection is also considered as the development trend of precise medical treatment.  

List of Multi-gene Testing For Hematonosis products

    1. Acute myelocytic leukemia (AML) gene test——58 target genes

    2. Acute lymphoblastic leukemia (ALL) gene test——35 target genes

    3. Myelodysplastic syndrome (MDS) gene test——25 target genes

    4. Myeloproliterative neoplasm (MPN) gene test——17 target genes

    5. Comprehensive test of hematologic tumor——111 target genes

Medical application of blood disease gene test

    1.Stratification of acute myelocytic leukemia prognosis

    2. Stratification of myelodysplastic syndrome prognosis

    3. Auxiliary diagnosis and prognosis evaluation of myeloproliterative neoplasm

    4. Detection of Glivec-resistant locus

    5. Pathogenesis exploration for intractable recurrent diseases

Common hematologic tumor diseases

    1. Acute myelocytic leukemia (AML)

    2. Acute lymphoblastic leukemia (ALL)

    3. Myelodysplastic syndrome (MDS)

    4. Myeloproliterative neoplasm (MPN)

    5. Primary myelofibrosis (PMF)

    6. Essential thrombocythemia (ET)

    7. Polycythemia vera (PV)

Note: Aplastic anemia is not a type of hematologic tumor


FAB classification and MICM classification for leukemia

    Hematologists fromFrance,AmericaandBritainheld a discussion in 1976 and set a standard of classifying and diagnosing acute leukemia based on blood cell morphology. The standard is called “FAB” classification for short. According to the standard, acute myelocytic leukemia is divided into seven subtypes from M1 to M7 (the 8 subtypes M0-M7 is currently adopted inChina); and acute lymphoblastic leukemia is classified into L1, L2 and L3. This classification has been broadly used by countries around the world. However, with the advancement of medical science and detection techniques, single cell morphological analysis no longer meets modern clinical detection needs. Even so, bone marrow cell morphology remains one of the standards for leukemia diagnosis.

    At present, the internationally used method of classifying leukemia is MICM, which stands for Morphology, Immunology, Cytogenetics and Molecular. Comprehensive judgment of several test results becomes the important foundation for modern leukemia classification, based on which leukemia treatment choices are made. Clinically, common test methods include flow cytometry, karyotype analysis and fusion gene.

Introduction of common hematologic tumors

Acute myelocytic leukemia (AML)

    AML is a malignant disease of medullary hematopoietic stem/progenitor cell. Its main feature is dysplasia of primitive and immature myeloid cells in bone marrow and peripheral blood. Clinically, karyotype analysis and fusion gene are the main bases of risk prognosis stratification for AML patients. However, among acute leukemia patients, only 40% show specific chromosome abnormality or fusion gene. For the patient group with the same chromosome abnormality or fusion gene, the effect of treatment is heterogeneous. 

    Gene mutation is an emerging detection index of molecular features, and exists in almost all leukemia cells. NCCN (National Comprehensive Cancer Network) Guidelines on Acute Myelogenous Leukemia has integrated the four gene mutations of c-KIT, FLT3, CEBPA and NPM1 into cytogenetic classifications, thus further refining risk prognosis stratification for patients with AML. Other gene mutation plays a guiding role in the diagnosis, prognosis stratification and treatment and drug choices of AML to varying degrees.

 

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Fig 3: Test process for AML patients


Myeloproliterative neoplasm (MPN)

    The original myeloproliferative disorder (MPD) was changed to the name MPN after revision by WHO in 2008. MPN is a disease that originates from hematopoietic stem cell and features excessive proliferation of one or multiple medullary systems (such as granulocyte system, erythrocyte system, megakaryocyte system and mastocyte system). In addition to adjustment of MPN subtypes, an important change of the revised version is inclusion of the application of gene mutation to disease diagnosis.

    BCR/ABL fusion gene remains the standard of chronic granulocytic leukemia (CML). Moreover, since V617F mutation of JAK2 gene exists in most cases of polycythemia vera (PV), about half of primary myelofibrosis (PMF) cases, and essential thrombocytosis (ET), major changes are made to the diagnosis processes of the three diseases. JAK2 gene, other similar activated mutations, and histological characteristics related to bone marrow biopsy are included as the diagnosis standards. When JAK2, MPL and CALR gene mutations are jointly detected, patients with MPN show a positive rate of 97%. According to clinical study findings, the positive type and negative type of JAK2 gene V617F mutation are not only different in molecular pathogenesis, but also in clinical feature, laboratory examination and disease transformation. This has certain guiding significance to the choice of therapeutic regimens.

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Fig 4: Test process for MPN patients


Myelodysplastic syndrome (MDS)

    MDS is a set of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells, and features myeloid cell differentiation and dysplasia, which are manifested as ineffective hematopoiesis, refractory hypocytosis and hematopoietic failure. A third of patients with MDS can progress to acute myelocytic leukemia. 15% patients with MDS experience secondary attack after chemotherapy or radiotherapy of other primary tumors, and extensive methylation of genes may occur to late-stage patients.

    The prognosis of MDS depends largely on the percentage of primitive cells in bone marrows, degree of hemocyte drop, and whether there is cytogenetical abnormality. Karyotype detection should be conducted for all patients with suspected MDS. Karyotype abnormality of acute myelocytic leukemia is mostly about chromosome translocation.       However, MDS usually involves partial or complete loss of chromosome, and the most common abnormal karyotypes of chromosome are -5/5q-, -7/7q-, +8, and -20/20q-. The new classification of MDS set by the WHO in 2008 is more based on genetic research. With the development of sequencing techniques, gene mutation and other molecular biological markers, in addition to cell genetics, have gradually been included in MDS diagnosis and treatment guidelines. In 2014 Agreement of Chinese Experts on the Diagnosis and Treatment of Myelodysplastic Syndrome, gene mutation was listed as an optional test item, and some gene mutations were specified and considered to have potential value of application. 1460605068178325.png

Fig 5: Test process for MDS patients


Acute lymphoblastic leukemia (ALL)

    ALL is a malignant neoplastic disease originating from abnormal hyperplasia of B or T lymphocytes in bone marrows. In China, the incidence rate of ALL is around 0.67/100,000. ALL mainly occurs to children from 0 to 9 years old, and can make up over 70% of children leukemia. ALL accounts for about 20% of adult leukemia. Currently, the treatment protocols made according to different biological characteristics of ALL have achieved good curative effect. As a result, about 80% children patients and 30% adult patients enjoy disease-free survival for a long time and the possibility of cure. Since the major group suffering ALL is children, their long-term quality of life is attracting more and more attention. Despite the good effect of existing treatment methods, it is necessary to conduct more clinical studies, explore molecular markers, and therefore provide guidance on personalized treatment.


Technical advantages

The test achieves comprehensive gene mutation detection, and serves as a new diagnosis tool for areas from prognosis stratification to targeted drug use.Wide coverage:

    1. Multiple target genes and all clinical hot spots are covered at the same time, and one-stop detection is achieved;

    2. Strong sensitivity: Mutation with a frequency as low as 5% can be detected;

    3. Precise quantification: Difference of 1% can be identified;

    4. High resolution: The change of single base can be detected;

    5. High scalability: facilitates conjoint analysis for multiple sites and multiple genes;

    6. Professional database for localized exploration;

    7. Precise analysis of FLT3-ITD, MLL-PTD and other special variation types.


Testing process

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Test period: 10 workdays since the laboratory receives the sample.

Sample requirements:

    1. Bone marrow/peripheral blood: 2-3ml; placed in EDTA anticoagulative tube (purple-head tube); stored and transported under a temperature of 4℃.

    Bone marrow is the preferred specimen; and the proportion of primitive cells or juvenile cells is greater than 5%. For samples with more than 5×109/L white blood cells in peripheral blood, over 5% primitive cells or abnormal cells, and less than 5×109/L white blood cells, the sample amount (>5ml) must be increased accordingly.

    2. DNA: The total amount is higher than 1μg; OD 260/280 stands between 1.8~2.0, and concentration is greater than 30ng/μL. The electrophoretic band is larger than 20kb after agarose electrophoresis. The DNA sample shall be put in an Eppendorf tube the size of 1.5ml or 2ml and be kept under a temperature of 4℃.


Note: Individuals with history of hematopoietic stem cell implantation or blood transfusion in the past 3 months are advised against taking the test. To ensure the quality of test, the sample must be sent to the laboratory within 24 hours.

For people

    1. Patients who are preliminarily diagnosed with different types of leukemia;

    2. Patients who are preliminarily diagnosed with myelodysplastic syndrome (MDS);

    3. Patients who are preliminarily diagnosed with myeloproliterative neoplasm (MPN);

    4. Patients who are preliminarily diagnosed with myelodysplastic syndrome/myeloproliterative neoplasm (MDS/MPN);

    5. Patients suspected of hematologic neoplasms whose types are not confirmed.

Patients with intractable recurrent diseases can also benefit from the test afterwards:

    1. Detection of the whole mutation hot spots for the purpose of understanding the causes of diseases;

    2. Monitoring sub-clone low-frequency mutations to prevent recurrence or transformation to disease subtypes;

    3. Detection of mutation site for targeted drug resistance.

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