Patent Application
20230324397
This patent application claims the benefit and priority of Chinese Patent Application No. 202210159447.8, entitled “ANTIBODY COMPOSITION FOR IMMUNOTYPING OF MYELOID TUMOR AND USE THEREOF” filed on Feb. 22, 2022, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.
The present disclosure relates to antibody medicament field, in particular to an antibody composition for immunotyping of a myeloid tumor and use thereof.
Acute and chronic myeloid neoplasms account for a large part of haematological/lymphatic neoplasms. According to the 2017 World Health Organization (WHO) Classification of Tumors of Haematopoietic and Lymphoid Tissues, acute myeloid neoplasms are classified into acute myeloid leukaemia (AML) and related myeloid precursor neoplasms, which are divided into seven main categories as shown in Table 1. As can be seen from the WHO classification, the diagnosis of haematological/lymphatic neoplasms highlights the importance of morphological/pathological, immunotyping, cytogenetic and molecular (MICI) testing.
In the classification of AML, the first category, AML with recurrent genetic abnormalities, is based on cytogenetics and genes as the final diagnostic basis. However, some of these subtypes have characteristic immunophenotypes that allow a general inference as to which genetically abnormal leukaemia they belong to. The second category, AML with myelodysplasia (MDS)-related changes, requires morphology or pathology to determine MDS haematopoietic dysplasia features; the fifth category, myeloid sarcoma, is a mass that forms outside the bone marrow, consists of mature or immature myeloid cells and requires pathological examination of tissue sections. The fourth category, therapy-related AML, and the sixth category, myeloid proliferations associated with Down syndrome are diagnosed by firstly identifying AML and then combining the identification with the medical history. These categories of AML all require immunotyping to help determine the myeloid origin and acute stage of differentiation, and the final MICM diagnosis is then determined in conjunction with the characteristics described above on the basis of AML diagnosis. The determination of AML is not possible without immunotyping. In addition to these categories, immunotyping plays a key role in the fourth-category, AML not otherwise specified (NOS) and the seventh-category blastic plasmacytoid dendritic cell neoplasm (BPDCN), based on which diagnosis is made.
In 2017 WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues, chronic myeloid neoplasms are classified into five categories, as shown in Table 2. Generally, immunotyping is not the primary basis for the diagnosis of CMN and is not required for the diagnosis of most chronic myeloid neoplasms. However, it serves to identify the diagnosis.
Several objectives can be achieved by immunotyping of leukaemia/lymphoma using flow cytometry (FCM).
Objective 1: Determination of the presence and type of a tumor. Objective 2: Determination of the subtype. Objective 3: Identification of markers for the next step of minimal residual disease (MRD) assays to search for leukaemia-associated immunophenotypes (LAIP). Objective 4: Detection of expression of markers associated with therapeutic targets. Objective 5: Prediction of genetic abnormalities by detecting markers for certain phenotypes that are highly correlated with specific genetic abnormalities. Examples include AML with t (8; 21) (q22; q22), RUNX1-RUNX1T1, and APL with t(15; 17)(q22; q12), PML-RARα. It is important to immunotype APL, because such patients have a high mortality rate in the early stage of the disease and need to be given retinoic acid or arsenic-based chemotherapy as soon as possible to save their lives. The long-term efficacy is excellent after the patient has survived the early risk phase. Early diagnosis is therefore crucial. Immunotyping and morphological determination are the fastest methods of leukaemia diagnosis available compared with gene detection and genetic testing, which take at least 2-3 weeks, a time longer than it takes for immunotyping. Therefore, w % ben both immunotyping and morphological testing suggest APL with t(15; 17)(q22; q12). PML-RARα, immediate clinical initiation of targeted chemotherapy is needed to reduce early mortality.
At present, the clinical assays described above require dozens of antibodies. The four-color antibody staining panel for immunotyping of acute leukaemia was described in the Chinese Journal of Haematology in 2015, where for AML typing a total of 10 tubes of 38 antibodies are required for the assay. The consensus on the four-color antibody staining panel is only for acute leukaemia but not for chronic myeloid neoplasms, particularly, e.g. MDS. 2012 Euroflow Europe published an 8-color antibody staining panel for immunotyping of haematological neoplasms. The antibody panel used for further analysis of AML/MDS after the initial detection with screening tubes is shown in Table 3, where 7-tube-8-color antibody panel with 4 common antibodies per tube was used. A total of 56 antibodies were tested and 28 repetitive antibodies were used, leaving 32 valid antibodies.
Currently available antibody panels are costly due to the repeated use of various gated antibodies, resulting in the use of more total antibodies. More tubes are required and it is difficult to accurately analyze the relationship between the antibodies detected in different tubes, which affects the determination of cell lineage, stage of differentiation and discrimination between benign and malignant cells. There is therefore an urgent need to design an antibody composition that can simultaneously perform comprehensive immunotyping, subtyping, MRD marker and therapeutic target screening, and predictive genotyping of acute and chronic myeloid neoplasms. There are many issues to be addressed by those skilled in the art to design the antibody composition described above, including the selection and combination of antibodies and fluoresceins, and the degree of expertise as well as clinical experience required for those skilled in the art.
In addition, the diagnosis of monocytic leukaemia (AML-M4/5) by immunotyping is often very difficult mainly due to the difficulties in identifying immature granulocytes, abnormal monocytes and promonocytes in the specimen. The differentiation between promonocytes and immature granulocytes determines whether the patient is diagnosed with AML-M2 or AML-M4/5. Secondly, in patients with mononucleosis, whether the monocytes are mature or immature determines whether the patient has CMML or AML-M4. The identification and diagnosis are important as the treatment varies between diseases. Furthermore, in some specimens of MDS, due to granulocyte degranulation, mature granulocytes are difficult to distinguish from monocytes in the CD45/SSC (side scatter) plot, where the SSC intensity is reduced and located in the place of monocytes, seriously affecting their identification. Therefore, there is also an urgent need for a method for identifying promonocytes to solve the above technical problems.
To solve the above problems, provided in the present disclosure is an antibody composition for immunotyping of acute myeloid leukaemia and related precursor neoplasms (together referred to as AML) as well as chronic myeloid neoplasms, specifically including panels of 22 or 24 antibodies. The composition is primarily used for subtyping acute and chronic myeloid neoplasms, screening for MRD markers and therapeutic targets and predicting genotypes, so as to achieve comprehensive immunotyping of myeloid neoplasms.
A panel of 19 antibodies in one tube as described in Application No. CN2021110670743 is used for primary screening of haematological neoplasms (first step of the screen). The haematological neoplasms are classified into 9 categories: AML, T-lineage acute lymphoblastic leukaemia (ALL-T), B-lineage acute lymphoblastic leukaemia (ALL-B), mixed phenotype acute leukaemia (MPAL), B-cell non-hodgkin lymphomas (NHL-B), T-cell non-hodgkin lymphomas (NHL-T), NK-cell non-hodgkin lymphomas (NHL-NK), plasma cell neoplasm (PCN) and chronic myeloid neoplasm, and 7 major categories of neoplasm, AML, ALL-T, ALL-B, MPAL, NHL-B, NHL-T and PCN are clearly diagnosed. Combined with the 22-24 antibodies in one tube for myeloid neoplasm diagnosis (second step of the test) in this application, a total of 2 tubes of antibodies allows for comprehensive immunotyping, subtyping, MRD marker and therapeutic target screening, and predictive genotyping of haematological neoplasms.
To achieve the above objectives, the present disclosure provides the following technical solutions.
The first aspect of the present disclosure provides three panels of antibody composition: a first panel of antibodies including 24 antibodies with 23 colors for immunotyping of myeloid neoplasms, including AML and chronic myeloid neoplasms; a second panel of antibodies including 22 antibodies with 21 colors for immunotyping of AML; and a third panel of antibodies including 22 antibodies with 22 colors for immunotyping of chronic myeloid neoplasms.
In some embodiments, antibody species and fluoresceins in combination therewith of the three panels of antibodies are shown in Table 4.
In some embodiments, the antibodies are monoclonal antibodies.
The second aspect of the present disclosure provides a kit for immunotyping of a myeloid neoplasm, including any panel of antibodies described above.
In some embodiments, the kit further includes an erythrocyte lysing solution and a buffer.
The third aspect of the present disclosure provides a system for detecting an immunophenotype of a myeloid neoplasm, including a test moiety and an analyzing moiety, where
the test moiety is used to attain test results of a sample using a tube of agent for testing the sample with flow cytometry, where the agent includes any panel of antibodies described above;
the analyzing moiety is used to analyze the test results from the test moiety to subtype myeloid neoplasms, identify LAIP markers for MRD testing, screen for therapeutic targets, and predict genotypes.
In some embodiments, when the system is used for detecting immunophenotypes of AML and/or CMN, steps of detection include:
preparing a flow cytometry onboard sample after processing a sample to be tested using the panel of antibodies; performing a flow cytometry onboard assay; where
gating in the flow cytometry onboard assay is as follows:
gating live cells as R1, removing debris and dead cells, and gating lymphocytes, granulocytes, monocytes, immature cells, and erythroblasts within R1 gate using CD45/SSC;
analyzing antigen expression within different cell gates, including:
analyzing AML, which includes immunotyping of myeloid immature cells, granulocytes and monocytes; and
analyzing chronic myeloid neoplasms, which includes immunotyping of immature myeloid cells, granulocytes, monocytes and erythroblasts.
In some embodiments, the immunotyping of granulocytes and monocytes includes distinguishing promonocytes from mature monocytes and/or distinguishing promonocytes from immature granulocytes using two-dimensional CXCR4/CD36 plot. In some embodiments, the expression of CXCR4 in promonocytes is higher than that in mature monocytes and granulocytes, and CD36 is distributed from negative to positive.
Specifically, maturity of monocytes is analyzed using monocyte-related markers included in the antibody composition of the present disclosure, e.g. CXCR4, CD33, CD64, CD14, CD300e, CD36, HLA-DR. CD15. CD11c, CD4, CD45 and SSC.
The fourth aspect of the present disclosure provides use of the antibody composition, the kit or the system in the preparation of a product for myeloid neoplasm diagnosis, therapeutic target screening, and screening of monitoring marker for MRD.
The myeloid neoplasms in the present disclosure include: AML and related precursor neoplasms, and chronic myeloid neoplasms.
In further embodiments, the AML and related precursor neoplasms mainly include 7 categories: 1. AML with recurrent genetic abnormalities; 2. AML with MDS-related changes: 3, therapy-related myeloid neoplasms; 4. AML, not otherwise specified (NOS); 5, myeloid sarcoma; 6, myeloid proliferations associated with Down syndrome, and 7. blastic plasmacytoid dendritic cell neoplasm (BPDCN).
The antibody compositions described herein are used for screening of MRD markers in these 7 disease categories. The first and fourth categories of AML subtypes are immunotyped, and genotypes of part of AML with recurrent genetic abnormalities are predicted.
In further embodiments, the chronic myeloid neoplasms include: myeloproliferative neoplasms (MPN), mastocytosis; myeloid/lymphoid neoplasms with eosinophilia and gene rearrangement, myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and myelodysplastic syndromes (MDS).
After initial screening in the first step, patients with haematological neoplasms are excluded from having AML and identified to have chronic myeloid neoplasms, which are then subtyped (Table 2). In patients with clinically suspected MPN and MDS, three conclusions can be made, i.e., supportive diagnosis, suspicious diagnosis and unsupportive diagnosis. For CMML and JMML, the presence of mononucleosis and predominant presence of mature or promonocytes can aid the diagnosis. For the screening of MRD makers, immunotyping is not used for most chronic myeloid neoplasms, but for MDS, as in AML. Screening for therapeutic targets and genetic phenotypes is mainly conducted on patients positive for CML BCR-ABL, where tyrosine kinase inhibitors can be used. Although immunotyping is not a definitive diagnosis basis for this disease, some diseases have typical immunophenotypic features that are suggestive of a diagnosis.
The fifth aspect of the present disclosure provides a method for identifying promonocytes from mature monocytes and/or promonocytes from immatue granulocytes, including detecting the CXCR4/CD36 expression of cell membrane in a sample to be tested for analysis of promonocytes.
In some embodiments, the method further includes detecting one or more of CD33, CD64, CD14, CD300e, HLA-DR, CD15, CD10, CD11b, CD11c, CD4, CD16, CD45 and SSC intensity as other monocyte markers.
In a specific embodiment, the expression of CXCR4 is higher in promonocytes than in mature monocytes and granulocytes, while CD36 is distributed from negative to positive. In combination with other monocyte-related markers in the antibody compositions of the present disclosure, e.g. CD64+CD14−, CD11c+/−CD4+, CD33st+CD15− and CD14−CD300e−, as well as the location below monocyte R5 in a CD45/SSC plot, the cells are identified as promonocytes, so that CXCR4+CD36dim+/− promonocytes. CXCR4−CD36− promyelocytes and CXCR4+(weaker than promonocytes) CD36st+ mature monocytes are distinguished.
Based on the above technical solutions, it is obvious that the embodiments of the present disclosure have the following beneficial effects.
Panels of 22-24 antibodies are utilized for immunotyping of myeloid neoplasms. When a primary screening tube for haematological neoplasms is used in conjunction, only 2 tubes of antibody combination are required for the comprehensive immunotyping of haematological neoplasms. Currently, it is common to perform conventional flow cytometric immunotyping of AML, CML and MDS using 8-10 colors staining antibody panels, each disease requiring 4-5 tubes of antibody panels (36-40 antibodies), whereas only 1 tube of antibody composition (22-24 antibodies) is used in the present disclosure to subtype acute or chronic myeloid neoplasms, which therefore reduces the requirement for specimen volume and operation procedures, reduces labor intensity and saves operation time. The present disclosure reduces the repeated application of gated antibodies, and increases the number of effective antibodies used. Meanwhile, it allows for observation of the simultaneous expression of 22-24 antibodies and analysis of the relationship between phenotypes and antibody panels, increasing the accuracy, specificity and sensitivity of the diagnosis of myeloid neoplasms. Also disclosed in the present disclosure are methods of analysis for identifying promonocytes from mature monocytes and immature granulocytes using a two-dimensional CXCR4/CD36 plot; the above methods are used to identify AMLM4/5 from other AML and from CMML. In MDS, granulocytes are often difficult to be distinguished from monocytes when granularity decreases, and the method of the present disclosure can also be used to distinguish monocytes from abnormal granulocytes.
Abbreviations in the figures are as follows: Mo-monocytes, MM-mature monocytes, PMC-promonocytes, Ly-lymphocytes, EC-erythrocytes, GC-granulocytes, IMC-immature myeloid cells, Eo-eosinophils, Bo-basophils.
The following examples are intended to illustrate the disclosure, not to limit the scope of the disclosure. Unless specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art. The experimental methods used in the following examples are conventional if not otherwise specified. All materials, reagents etc. used in the following examples are commercially available, if not otherwise specified.
In the present disclosure, flow cytometry is used to immunotype specimens of bone marrow fluid, thoraco-abdominal fluid and peripheral blood from clinical patients, and specimens identified as possible AML and chronic myeloid neoplasms by primary screening is subjected to a second step of comprehensive immunotyping. The panel of 19 antibodies used in the primary screening is described in CN patent application No. 2021110670743.
Three panels of antibodies were used for the diseases.
The first panel of 24 antibodies with 23 colors of fluoresceins was prepared according to the antibody combination for myeloid neoplasms in Table 4. The antibodies and the fluoresceins were mixed accordingly, put into a container, and used for immunotyping of all specimens identified as myeloid neoplasms (AML and chronic myeloid neoplasms).
The second panel of 22 antibodies with 21 colors of fluoresceins was prepared according to the antibody combination for AML in Table 4. Components were the same as those in the myeloid group except that anti-CD105-SB436 and anti-CD10-BV711 were not added. The antibodies and the fluoresceins were mixed accordingly, put into a container, and used for immunotyping of specimens identified as AML.
The third panel of 22 antibodies with 22 colors of fluoresceins was prepared according to the antibody combination for chronic myeloid neoplasms in Table 4. Components were the same as those in the myeloid group except that anti-CD41-FITC and anti-CD61-FITC were not added and anti-CD9-PerCP-eF710 was replaced with anti-CD38-PerCP-eF710. The antibodies and the fluoresceins were mixed accordingly, put into a container, and used for immunotyping of specimens identified as chronic myeloid neoplasms.
The antibodies above are commercially available, and the antibodies used in the examples were purchased from BD Biosciences, Biolegend, Beckman Coulter, and Thermo Fisher Scientific.
The above three panels of antibodies were prepared into a kit for detecting myeloid disease, AML and chronic myeloid disease. The kit further included an erythrocyte lysing buffer and phosphate buffer saline (PBS). The erythrocyte lysing buffer was either self-made or purchased from a company (BD Biosciences, for example).
1. Experimental Materials and Instrument
1. Materials: 10×PBS, and a BD fluorescence activating cell sorter (FACS) Lysing Solution dedicated for flow cytometer.
2. Instruments: CytekNL-3000 full spectrum flow cytometer, provided with 3 lasers (405 nanometers (nm), 488 nm and 635 nm) and 38 fluorescence detectors; a desktop low-speed centrifuge, and a vortex mixer.
II. Methods
1. Sample Collection
1-3 mL of human bone marrow fluid was sampled and immediately placed into a heparin anticoagulation tube, which was then quickly inverted several times to prevent coagulation of the sample. Chest and abdominal fluid, lavage fluid and other cells were sent to the laboratory immediately after collection, and stored in a refrigerator at 4° C. Flow cytometry (FCM) assay was completed within 48 hours, as per instructions.
2. Sample Preparation.
(1) Cell counting: 10 μl of bone marrow fluid was taken, 150 μl of PBS was added and mixed well, the number of cells per microliter (μl) was counted using Myriad FCM, the cell concentration was adjusted to 5-10×106/100 μl according to the results, and 50 μl-100 μl of cells were added to a flow tube.
(2) Antigen Staining.
a) The corresponding fluorescein-labeled monoclonal antibody premix according to Table 4 and bone marrow samples were added to each tube separately, mixed thoroughly, incubated for 15 min at room temperature and protected from light.
b) Haemolysis: 2 ml of 1×FACS lysing solution was added, vortexed at low speed to mix well with the samples, allowed to stand for 8-10 min at room temperature and protected from light. Centrifugation at 300 g for 5 min and washing were carried out and a resulting supernatant was discarded.
c) Washing: 1 ml of PBS containing 0.1% NaN3 and 1-2% BSA were added for washing by centrifugation at 300 g for 5 min, and a resulting supernatant was discarded. 200 μl of PBS was added to re-suspend the cells for further onboard assay.
(3) Onboard Assay.
a) Determination of the optimum voltage and compensation: the voltage was set according to the routine operation of the spectral flow cytometer and single-stained samples were prepared with reference to the fluorescent color combination of the kit for instrument setting.
b) Instrument set-up, calibration and quality control (QC): CytekNL-3000 was switched on to warm up the machine for more than 20 min and rinsed with deionized water, and the internal QC was tested to ensure that the values were within the normal range. AL-PANAL© was called for sample loading to collect data.
c) On-board assay: 50-100,000 cells per tube were obtained according to the instrument parameters. When the assay could not be run on time, 0.5 ml of 1% paraformaldehyde was added, mixed well with the cells, and stored in a refrigerator at 4° C., and the assay was conducted within 24 hours.
III. Data Analysis Using Kluzaa© Software.
(i). Analysis on AML
1. Debris, adherent cells and dead cells were removed using forward scatter (FSC)/SSC, and single live cells were gated as R1.
2. R1 gate cells were shown, and a CD45/SSC plot was constructed. According to the distribution of CD45 and SSC, lymphocytes, monocytes, granulocytes, erythroblasts and immature cells were gated and various colors were assigned. Lymphocytes (R1) had the highest CD45 expression and the lowest SSC intensity; monocytes (R5) had lower CD45 expression than lymphocytes, higher SSC intensity than lymphocytes but lower SSC intensity than granulocytes; granulocytes (R4) had lower CD45 expression than monocytes and the highest SSC intensity: erythroblasts (R6) showed negative CD45 expression and the same SSC intensity as lymphocytes; immature cells (R3) had lower CD45 expression than lymphocytes and lower or equal SSC intensity than/to lymphocytes. In normal bone marrows, the normal proportions of cell populations are as follows: lymphocytes 20% to 40%, monoyctes 2% to 8%, erythroblasts 2% to 15%, and immature cells less than 5%. It was observed whether the proportion of each population was normal and whether the proportion of immature cells increased.
A series of two-dimensional dot plots with 2 antibodies was constructed, mainly including CD34/CD117, CD33/CD117, HLA-DR/CD123, CD13/CD11B, CD13/CD16, CD15, HLA-DR/CD11B, CD14/CD16, CD14/CD16 CD14/CD300E, CD36/CXCR4, CD64/CD15, CD64/CD14, CD33/CD15, CD11C/CD4, etc. First, R1 gate cells were observed for the expression of antigens in these plots. Given that different cell populations were marked with different colors, the antigen expression in different cell populations could be analyzed. Among the specimens of some myeloid neoplasms, the boundary of immature cells in the CD45/SSC plot was unclear. Herein, CD34+CD117+ or CD34−CD117+ cells were gated using CD34/CD117 plots so that the immature myeloid cells could be gated and analyzed better.
4. The expression of the antigens described in the present disclosure was analyzed mainly in immature cells, granulocytes and monocyte gates. The function of the antigens and their expression on normal blood cells are shown in Table 5. The criteria for subtyping AML using the 22-24 antibodies in the present disclosure are shown in Table 6.
CXCR4/CD36 was used to differentiate promonocytes from immature granulocytes and mature monocytes.
In some AML or MDS specimens, conventional gating methods such as CD45/SSC and CD33/CD15 or CD64/CD14 do not effectively gate monocytes and granulocytes, and promonocytes are often included in the granulocyte gate. Therefore, the present disclosure adopted CXCR4/CD36 to analyze promonocytes, and CXCR4 expression was stronger in promonocytes than in mature monocytes and granulocytes, while CD36 was distributed from negative to positive. In combination with other monocyte-associated markers in the panels, such as CD64+CD14−, CD11c+/−CD4+, CD33st+CD15−, CD14−CD300e−, and the location below monocyte R5 in the CD45/SSC plot, promonocytes were identified. Promonocytes were identified from immature granulocytes (
Promyelocytes had the phenotype of CD64st+CD33st+CD14−CD300e−CD11c−CD4−, but did not express CXCR4. They were located at the lower edge of the R4 gate in the CD45/SSC plot and had higher SSC intensity than mature granulocytes. In some CMML specimens, granulocytes and monocytes were overlapped with each other and neither CD45/SSC nor CD64/CD14 could effectively separate monocytes from promyelocytes, so a CD36/CXCR4 plot was used in the present disclosure to identify CXCR4−CD36− promyelocytes from CXCR4+CD36dim+/− promonocytes. Thus, AML-M2 versus AML-M4, and AML-M5 versus CMML were distinguished.
5. Determination of LAIP Markers.
Antigens that are aberrantly expressed on leukemic cells are called LAIP. LAIP is one of the important characteristics that distinguishes leukemic cells from normal haematopoietic cells and is the basis for FCM detection of MRD. The antibody panels of the present disclosure were used to screen for abnormal immunophenotypes in patients with AML and chronic myeloid neoplasms for MRD monitoring.
The LAIP described primarily includes:
(1) cross-series antigen expression or cross-lineage antigen expression, e.g. in the case where AML cells express lymphoblastic antigens: CD19, CDT CD56, CD5, etc. (2) Synchronous expression of early and late antigens. Under normal circumstances, the expression of antigens in different series of cells at different stages of differentiation is strictly controlled by genes, and CD34, CD15, or CD11b are not expressed at the same time. The antigens are expressed in sequence. However, leukaemia cells do not express in the normal order. Therefore, CD34+CD15+ or CD1b+ is LAIP. (3) Abnormal antigen expression. Normally, cellular antigen expression intensity of each series at each stage is consistent. AML cells often show enhanced or reduced expression, or even no expression of antigens such as CD34, CD117, CD33, CD13, CD38, and HLA-DR.
Each of the 25 antigens included in this disclosure was analyzed individually to determine if they were LAIP markers.
6. Screening of markers related to therapeutic targets. There are more than 10 targeted drugs for AML, among which the only one targeting the cell surface antigen phenotype is Gituzumab Ozogamicin, which consists of a chemotherapeutic drug coupled to a monoclonal antibody (artificial immune protein) targeting the CD33 protein. The disclosure therefore provided information on CD33 expression abnormalities, thereby providing guidance for clinical administration.
7. Prediction of genotypes. Certain phenotypes are highly correlated with specific genetic abnormalities, and the detection of these markers can predict whether genetic abnormalities are present or not. Several types of AML with recurrent genetic abnormalities with typical phenotypic features are listed in Table 6, where AML with t(8:21)(q22; q22), RUNX1-RUNX1T1 tends to be manifested morphologically and immunophenotypically as AML-M2, and immunophenotypically immature myeloid cells tend to express CD34, CD117. CD38 and HLA-DR. CD33 is typically weakly expressed and about 60% abnormally express the B-lineage-associated marker CD19 and the NK-associated marker CD56. If there are typical immunophenotypic features, there is more than 90% correlation with AML with t(8; 21)(q22; q22), RUNX1-RUNX1T1 genotype leukaemia. For APL with t(15; 17)(q22; q12), PML-RARα genotype leukaemia, the typical patient's bone marrow morphology tends to show a marked increase in promyelocytes and more granules in cells; the immunotyping shows a marked increase in the proportion of CD117+ immature cells, and high side scatter (SSC) intensity of cells, with the cells characterized by the absence of CD34 and HLA-DR expression, strong expression of CD33, and simultaneous expression of CD9, CD123 and CD64 (weak). This phenotype is often indicative of APL with t(15:17)(q22; q12), PML-RARα genotype leukaemia. There's high similarity between AML with mutated NPM1 and APL, while they were distinguished by the low SSC intensity and weak MP0 expression.
(ii) MDS Analysis.
MDS belongs to chronic myeloid neoplasms and is listed separately herein because of the complexity of analysis. The gating method is shown in
1. Debris and dead cells were removed, each population of cells was gated, and the proportion of each population was shown, as described in steps 1-2 of the AML analysis above.
2. Gating of CD34+ cells: Sequential gating was adopted for CD34/SSC and CD34/CD45 plots to remove non-specific cells. CD34+ cells were gated and CD13 and HLA-DR expression within CD34+ cell gate were analyzed.
3. Gating of CD117+ cells: Sequential gating was adopted for CD117/SSC and CD117/CD45 plots to gate CD117+ cells and remove CD45st+ and non-specific cells. The distribution of CD33/CD13 and CD13/HLADR within CD117+ cells was analyzed.
4. Analysis of granulocytes: sequential CD45/SSC, CD33 or CD64 and CD15 gating was adopted for granulocytes with weak CD33 and CD64 expression and strong CD15 expression. Granulocytes were broadly classified into li1 to li5 stages based on CD13/CD11b, CD10/CD16, which roughly corresponded to promyelocytes, myelocytes, metamyelocytes, stab granulocytes, and segmented granulocytes, respectively. Normal bone marrows at li1 and li2 stages were less than 10%. Some MDS showed an increased proportion of li1+1i2 cells.
5. Analysis of monocytes: sequential CD45/SSC, CD33 or CD64 and CD15 gating was used to gate monocytes with strong CD33 and CD64 expression and weak CD15 expression. The analysis was focused on the proportion of promonocytes, in the same way as AML.
6. Analysis of the phenotype of erythroblasts: sequential CD45/SSC, CD71 or CD36 gating was carried out on CD71+CD45 weakly expressing or negative erythroblasts. The proportion and expression intensity of CD71, CD36, CD105 and CD117 positive cells were observed. Normal phenotype showed strong CD71 and CD36 expression as shown in
7. Analysis of CD123/HLA-DR plot: the proportion of CD123st+ HLA−DR+(pDC) and CD123st+ HLA−DR-basophils (normally <1%) were analyzed.
The diagnosis was then made in conjunction with the results of the primary screening tube as follows: (1) Supported MDS: more than 1% of increase in proportion of immature myeloid cells, or ogata score more than 2, or abnormal immature myeloid phenotype e.g. increased CD34+CD38−%, or CD34st+/dim+ or CD117st+/dim+ or CD33st+/dim+ and CD13st+/dim+ and HLA-DRdim+. (2) Suspected MDS: abnormal granulocytes or erythrocytes only. (3) Excluding MDS: no significant abnormalities in immature myeloid cells, granulocytes or erythrocytes. See Chinese patent application No. 2021110670743 for the method of diagnostic analysis of the primary screening tube.
(iii) Analysis of Chronic Myeloid Neoplasms
The analysis was performed in the same way as for MDS. Kaluz© software was used to remove debris and perform gating analysis on a CD45/SSC plot. Lymphocytes, monocytes, granulocytes, erythroblasts, eosinophils and immature cells were gated and their proportions in the sample were determined. It was determined whether the proportions and the phenotypes of each cell populations were abnormal. The diagnosis was moved on based on the followings in combination with clinical presentation.
1. Chronic myeloid leukaemia (CML): The manifestations that the proportion of granulocytes is 79-90%, and the percentage of CD34+CD117+ cells is normal or slightly increased, usually below 5%. The proportion of CD11b-granulocytes (li1 and li2 stages, as shown in
2. Chronic neutrophilic leukaemia (CNL): No CML phenotype is present. The disease mainly presents with an increased proportion of granulocytes, of which the granulocytes are mainly CD10+CD16+ mature granulocytes, with a normal proportion of CD34+CD117+ and no significant abnormalities in the rest of the cells.
3. MPN-thrombocytosis (essential thrombocythaemia, ET): Patients with ET often manifest a marked increase in platelets, accompanied by a mild increase in WBC. The CD45/SSC and CD34/CD117 plots show no abnormalities in the ratio of each cell populations. The proportion of granulocytes CD11b-immature cells is not high (normally <10%) and there may be a slightly weaker expression of CD11b. The remaining cells are not significantly abnormal. Such manifestation is indicative of MPN-ET.
4. Chronic myelomonocytic leukaemia (CMML) in MDS/MPN: For patients with more than 1×109/L monocytes in peripheral blood, the percentage of monocytes in the bone marrow is mainly analyzed and attention is paid to whether they are mature or promonocytes, identified in the same way as in AML. Predominantly mature monocytes are increased, with >8% monocytes. If CD14, CD300e, CD CD11b, and CD36st are expressed, mature monocytes are identified. Other monocyte related markers may be positive, but HLA-DR, CD11b, CD13 and even CD15 can show abnormal intensity of expression. CD56 is also expressed in some patients. Granulocytes can also show an increased proportion of CD11b-cells. When the percentage of CD34+CD117+ cells are normal or slightly increased and promonocytes are less than 20%, combined with the indication of more than 1-109/L monocytes in the peripheral blood, CMML are diagnosed.
5. Other Chronic Myeloid Neoplasms.
In patients with myelofibrosis (MF), there may be an abnormal immature cell phenotype, e.g. CD38dim+, and other cellular abnormalities may not be evident, and an increase in basophils may be seen.
Analysis of mast cells: The cells are CD117st+, and express CD33 and CD9. In mastocytosis, the antibody panels can be used to detect an increased proportion of CD117 st+ mast cells and analyze whether CD33 and CD9 expression is abnormal.
Analysis of basosphils: HLA-DR-CD123st+ is used to gate eosinophils, which have a small SSC intensity, are weak CD45+ cells, also express CD9st, CXCR4st, CD13, CD33, CD11b, and CD11c and do not express CD15, CD10, CD16, CD64, and CD14. The antibody panels can be used to analyze whether the proportions (normally <1%) and phenotypes are abnormal.
Analysis of eosinophils: Eosinophils have the highest SSC intensity, are positive for CD45st, expresse CD33, CD13, and CD11b, weakly express CD15, and does not express CD16 or CD10. Determination of whether the proportion of eosinophils (normally <5%) and their phenotype is abnormal and whether there are immature eosinophils that can help to determine eosinophilic disease.
IV. Results.
A total of 103 bone marrow specimens were tested using the antibody panels of the present disclosure, all of which underwent primary screening tube testing. Of the specimens, 27 were proved to have AML and were tested using the AML antibody panel. The remaining 76 specimens were tested by primary screening tests. They were excluded from having lymphoid neoplasms or suspected myeloid neoplasms, and thus were tested using the antibody panel for chronic myeloid neoplasms.
It was shown that in 27 cases of AML, the phenotypes after the test were: 12 cases of AML-M2, 10 cases of AML-M4/5 and 1 case of AML-basophilic; 4 cases of AML with recurrent genetic abnormalities: including 1 case of AML with Mutated NMP1, 1 case of AML with t(8:21) and 2 cases of APL with t(15:17). 44 cases were diagnosed with chronic myeloid neoplasms, including: 15 cases of supportive MDS, 5 cases of suspected MDS, 5 cases of MPN, 5 cases of MDS/MPN (including 3 cases of CMML): 6 cases of suspected CML; 4 cases of eosinophilia: and 4 cases of hypoplasia. 32 specimens were approximately normal.
All 103 specimens were simultaneously immunotyped with the conventional 8-10 color 4-8 tube antibody panels and the results were consistent between the 2 methods.
6 cases of CML identified by initial determination were confirmed by genetic testing, and the diagnosis of CML was confirmed in 6 cases with positive CML genes. The above results demonstrate the high sensitivity of the antibody panels of the disclosure for the diagnosis of MDS.
In particular.
As shown in
Although, the disclosure has been described in detail above with a general description and specific examples, some modifications or improvements can be made on the basis of the present disclosure, as will be apparent to those skilled in the art. These modifications or improvements, which do not deviate from the spirit of the disclosure, therefore fall within the protection scope claimed by the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210159447.8 | Feb 2022 | CN | national |
