Antigen expression on residual blast cells in acute megakaryoblastic leukemia (AMKL, classified as AML-M7 by FAB criteria) may change after treatment, potentially affecting both immunophenotypic characterization and minimal/measurable residual disease (MRD) monitoring. This study aimed to characterize post-therapy immunophenotypic alterations in AMKL and to determine whether specific patterns of antigenic change exist between samples obtained at initial presentation (IP group) and those obtained at MRD-positive status after therapy (MRD group). This retrospective descriptive study included 110 patients diagnosed with AMKL at Hebei Yanda Lu Daopei Hospital between January 1, 2009 and December 31, 2024 (male:female = 57:53; 103 pediatric and 7 adult cases). Immunophenotypes at initial diagnosis and after treatment were analyzed by flow cytometry. The chi-square test was used to compare antigen expression between the IP and MRD groups. Flow cytometric immunophenotypes differed by at least three antigens (including CD33, CD61, and CD42b) between initial presentation and post-therapy samples. Compared with the IP group, the MRD group showed a significantly higher frequency of loss of megakaryocytic markers, including CD61 (11/109, 10.1% vs. 30/109, 27.5%; p < 0.05) and CD42b (6/106, 5.7% vs. 22/101, 21.8%; p < 0.05). Partial loss of CD13 expression was also more frequent in the MRD group (18/99, 18.2% vs. 2/83, 2.4%; p < 0.05). No significant differences were observed in the expression of progenitor-associated markers (CD34, CD117), myeloid markers (CD33, CD11b), or other antigens (HLA-DR, CD7, CD56, CD42a) between the two groups (p > 0.05). Lineage-specific markers MPO and CD22, the monocytic marker CD14, and lymphoid markers CD10 and CD5 were negative in both groups. In contrast, aberrant expression of cCD3 (2/89, 2.2%) and CD19 (3/85, 3.5%) was observed in a small subset of IP cases. Overall, 100 of 110 patients (90.9%) showed changes in at least one antigen after therapy. By lineage category, alterations were most frequent in megakaryocytic markers (CD61, CD42b, CD41a, CD42a; 64/110, 58.2%), followed by myeloid antigens (HLA-DR, CD33, CD13, CD11b; 54/108, 50.0%), progenitor-associated antigens (CD34, CD117; 53/110, 48.2%), and lymphoid antigens (CD7, CD56; 24/107, 22.4%). In addition, CD110 was consistently expressed in all 26 AMKL cases tested, whereas only 18% (9/50) of non-AMKL AML cases were CD110-positive (p < 0.05). Significant immunophenotypic differences, particularly involving CD61, CD42b, and CD13, exist between IP and MRD samples in AMKL. Antigenic shifts affecting megakaryocytic, myeloid, progenitor-associated, and lymphoid markers are common after chemotherapy. For MRD assessment, the use of more specific megakaryocytic markers such as CD110, together with comprehensive multiparameter flow cytometry panels, may improve detection accuracy.

Abstract Objective: To find a strong and applicable panel for T-ALL MRD detection by flow cytometry and find the relation with genetics. Methods: From February 28, 2024 to May 29, 2025, 1025 T-ALL patients were tested for MRD in our Hospital using full-spectrum flow cytometry. A total of 1570 tests were completed. All patients were Chinese, without disabilities, with a male-to-female ratio of 768:257, and a median age of 16 years (1-68). There was 1 Uyghur, 2 Tibetans, 5 Hui people, and 1017 Han people. The detectionpanel was CD99 FITC/cCD3 PE/CD3 BV785/CD48 PECy7/CD4 APC Cy7/CD5 APC R700/CD2 BV605/CD7 APC/CD16 efluor 450/CD56 BV711/TdT BV421/CD45 V500/CD34 PerCP Cy5.5/CD94 BV650/CD8a BV570/TCRγδ BV480. A total of 612 people had detectable genes. Ten cases of complete remission and ten cases of MRD positive were selected for correlation testing between the traditional panel and the full-spectrum panel. Data analysis was performed using Kaluza 2.3.0. Statistical analysis was conducted using SPSS 17.0. 68 cases were implemented in Python. Results: (1) The correlation between the full-spectrum flow cytometry and the traditional flow cytometry was good. (2) Among the 1570 tests, 155 tests from 103 individuals were positive. Among the positive patients, the ratio of male to female was 79:24, with a median age of 21 years (ranging from 4 to 68 years). There was 1 Tibetan, 1 Hui, and 101 Han. The median tumor burden was 1.66% (ranging from 0.002% to 94.15%). (3) 68 MRD positive cases had genetics results, 33 had WT1, 2 had EVI, 13 were positive for SIL::TAL1, 1 for PCM1::JAK2, 4 for SET::CAN, 6 for MLL::AF6, 1 for MBNL1::TAL1, 1 for KMT2A::PAP1GDS1, 2 for DIAPH1::PDGFB, and 3 for CALM::AF10. One case of EVI and 6 cases of WT1 had values below the reference range. (4) Among the 52 cases with CD7 negative after CD7 CAR-T, 43 were MRD negative and 9 were MRD positive. Among the MRD positive patients, 2 were cCD3 negative. Among 52 cases with CD7 negative,cases negative for cCD3, CD2, and CD5 was 0. (5) Among the 6 cases with lost cCD3, 2 cases were negative for both CD2 and CD5. (6) The relationship between genetics and immunophenotype: the probability of co-expression of CD7 and CD99 was the highest in the MLL::AF6, CALM::AF10, and EVI1 genetic abnormality groups, which was 100% (P < 0.001). The genotypes that were prone to cCD3 weakening or partial loss were PCM1::JAK2 (100%, 2/2), SET::CAN (75%, 3/4), MLL::AF6 (67%, 2/3), DIAPH1::PDGFB (50%, 1/2), EVI1 (50%, 1/2), WT1 (40%, 14/35), and CALM::AF10 (33%, 1/3). On the MRD recognition markers, strong expression of CD99 and weakened CD48 were common abnormalities. PCM1::JAK2, SET::CAN, and WT1 were prone to losing the characteristic of strong expression of CD99. The probability of weakened or negative CD48 was 100% for MLL::AF6 (6/6), SET::CAN (4/4), CALM::AF10 (3/3), MBNL1::TAL1 (1/1),and KMT2A::PAP1GDS1 (1/1). It was 64% for SIL::TAL1, and 40% for WT1. The positive rate of CD34 in all T-ALL cases was only 19%, while CALM::AF10 and EVI1 was 100% positive, MLL::AF6 was 50%, WT1 was 23%, SET::CAN was 25%. CD45dim/CD7bri is the most commonly used gating method, but should exclude NK cell interference. The probability of CD56 positivity was 67% for CALM::AF10 and 20% for WT1. The positive rate of CD3 was 79% (11/14) for SIL::TAL1, 50% for DIAPH1::PDGFB (1/2) and EVI1 (1/2), 46% (16/35) for WT1, and 25% (1/4) for SET::CAN. MLL::AF6 has a 100% positive rate for CD7briCD99briCD48-CD56-CD34+, theoretically providing the highest degree of identification. Next is EVI1, which is 100% positive for CD7briCD99briCD48+CD56-CD34+ because CD48 could not provide contribution. CALM::AF10 has a 100% positive rate for CD7briCD99briCD48-CD34+, but the CD56 positive rate is 67% (2/3), and it also needs to be differentiated from NK cells, making it an easily identifiable subtype. PCM1::JAK2 and some WT1 subtypes may be relatively more difficult to identify. Conclusion: Full-spectrum flow cytometry can simultaneously perform more than four pan-system markers, theoretically meeting the gating requirements for MRD detection after CAR-T therapy at 98.51%. However, to increase detection coverage, pan-T markers need to be added. There is a certain correlation between genetics and immunophenotype. Different genotypes benefit from different markers, and full-spectrum flow cytometry can simultaneously perform more marker combinations to maximize coverage of common abnormalities in various genetic subtypes.

Background: Minimal Residual Disease(MRD) detection by multicolor flow cytometry(MFC) is an important parameter for monitoring therapy effect in acute leukaemia. However, MFC panels for MRD detection after CD19-CAR-T treatment should change because the expression of CD19 will lose or partially lose. Aims: To discuss the significance of MRD detection by MFC with cytoplastic CD79a(cCD79a) gating panel in relapsed or refractory B-cell acute lymphoblastic leukemia(r/r B-ALL) and evaluate its value for prognosis in CD19-CAR-T bridging to allogeneic-hematopoietic stem cell transplantation (allo-HSCT). Methods: 37 r/r B-ALL patients who received CD-19-CAR-T infusion bridging to allo-HSCT were enrolled at Hebei Yanda Lu Daopei Hospital from January 2019 to July 2019. Patients had not been treated with CAR-T and/or allo-HSCT before. MFC is used to monitor the patient’s MRD and the dynamic of B cells. The median follow-up time of 37 patients after treatment was 890 days (104-1062). Binary logistic regression was used to retrospectively analyze the data. Results: 8 patients (8/37, 21.62%) died and 29 (29/37, 78.38%) survived. when using death/survival as the end point in the dependent variable, MFC-MRD-positive recurrence (P=0.0006), extramedullary infiltration (P=0.047), and chromosomal karyotype changes (P=0.047) showed significant differences between the two groups, as well as the remission rate of MRD on 15d (P=0.011), the remission rate of the MRD on the 28d (P=0.002), and the time from CD19 positive recovery to CAR-T before allo-HSCT (P=0.020) after CAR-T reinfusion to allo-HSCT. P values of these indicators were all less than 0.05. the rest factors were not significant between the two groups. In the binary Logistic regression model of related factors during treatment and disease outcome, FCM-MRD remission on 28d can be used as a potential indicator to evaluate disease death or survival outcome, while other factors are excluded from the equation. The area under the ROC curve for the analysis of disease prognosis affecting factors was 0.762 (P=0.026), OR=12.667, the sensitivity was 0.625, and the specificity was 0.897. Image:Summary/Conclusion: The cCD79a antibody gated MFC-MRD monitoring can play a certain guiding significance in the prognosis judgment of the disease and the selection of the type of CART.

Full spectrum flow cytometry brings a breakthrough for minimal residual disease (MRD) detection in acute myeloid leukemia (AML). We aimed to explore the role of a new panel in MRD detection. We established a 24-color full-spectrum flow cytometry panel. A tube of 24-color antibodies included CD45, CD117, CD34, HLA-DR, CD15, CD64, CD14, CD11c, CD11b, CD13, CD33, CD371, CD7, CD56, CD19, CD4, CD2, CD123, CD200, CD38, CD96, CD71, CD36, and CD9. We discovered that when a tube meets 26 parameters (24 colors), these markers were not only limited to the observation of MRD in AML, but also could be used for fine clustering of bone marrow cells. Mast cells, basophils, myeloid dendritic cells, and plasmacoid dendritic cells were more clearly observed. In addition, immune checkpoint CD96 had the higher expression in CD117+ myeloid naive cells and CD56dimNK cells, while had the lower expression in CD56briNK cells in AML-MRD samples than in normal bone marrow samples. CD200 expression was remarkably enhanced in CD117+ myeloid naive cells, CD4+ T cells, T cells, activated T cells, CD56dimNK cells, and CD56briNK cells in AML-MRD samples. Our results can be used as important basis for auxiliary diagnosis, prognosis judgment, treatment guidance, and immune regulation in AML.

Objective: In minimal residual disease (MRD) analysis after allogeneic hematopoietic stem cell transplantation (allo-HSCT), abnormal immunophenotyping is commonly considered as evidence of a secondary recurrence or complications, leading to overtreatment. We aimed to confirm whether such phenotypic abnormality might originate from donors using multicolor flow cytometry (MFC). Materials and Methods: The MRD of bone marrow specimens of 3395 patients who had received allo-HSCT were analyzed using the conventional two-tube, eight-color MFC panel. The frequencies of abnormal immunophenotypes were also evaluated in three groups of patients without malignancies. Results: The frequency of new abnormal polymorphisms was 0.088% (3/3395) among patients who received allo-HSCT. The abnormal cells seen in three patients in complete remission were Fcγ receptor IIIB (FcγRIIIB) gene deletion (CD16- neutrophils), CD2-CD159a-CD159c+ natural killer (NK) cells, and monoclonal B lymphocytosis (MBL), respectively. In addition, abnormal T-cells (CD4+CD8+) were detected in one donor before allo-HSCT. Identical abnormalities were found in the peripheral blood of the corresponding donors of the three patients via MFC. Among the individuals without malignancies, the incidence of FcγRIIIB deletion was 0.2% (11/5256), that of NK cells with the absence of CD2 and single-positive CD159c was 0.05% (1/2000), that of monoclonal CD4/CD8 double-positive T-cells was 0.05% (1/2000), and that of MBL was 1.3% (14/1100). The frequency of NK cells with the absence of CD2 was 1.3% (1/79) and with CD8dim was 14% (11/79) in NK cell lymphoma. The following abnormalities could be identified by the two-tube, eight-color MFC panel: cκ/cλ/CD19/CD5/CD20/ CD38/CD45/CD56 (adding CD10 and CD34 as the ninth and tenth colors) and CD16+CD56/CD5/CD3/CD7/CD4/CD8/CD2/CD45 (adding CD117 as the ninth color). Conclusion: Abnormalities in recipients of allo-HSCT detected by MRD analysis may originate from their donors. Screening of donor specimens with a suitable two-tube, eight- to ten-color MFC panel may be a promising method for minimizing misdiagnoses.

Pediatric Blood & CancerEarly View e30445 LETTER TO THE EDITOR Acute monocytic leukemia with KMT2A:MLLT10 transformed to AML-M7 in a pediatric patient Ting Li, Ting Li Department of Laboratory Medicine, Beijing Ludaopei Hospital, Beijing, ChinaSearch for more papers by this authorAixian Wang, Aixian Wang Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Langfang, ChinaSearch for more papers by this authorPing Wu, Ping Wu Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Langfang, ChinaSearch for more papers by this authorMan Chen, Man Chen Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Langfang, ChinaSearch for more papers by this authorHui Wang, Corresponding Author Hui Wang [email protected] orcid.org/0000-0001-8033-5924 Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Langfang, China Correspondence Hui Wang, Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Sipulan Road, Yanjiao Development Area, Langfang, 065201, China. Email: [email protected]Search for more papers by this author Ting Li, Ting Li Department of Laboratory Medicine, Beijing Ludaopei Hospital, Beijing, ChinaSearch for more papers by this authorAixian Wang, Aixian Wang Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Langfang, ChinaSearch for more papers by this authorPing Wu, Ping Wu Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Langfang, ChinaSearch for more papers by this authorMan Chen, Man Chen Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Langfang, ChinaSearch for more papers by this authorHui Wang, Corresponding Author Hui Wang [email protected] orcid.org/0000-0001-8033-5924 Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Langfang, China Correspondence Hui Wang, Department of Laboratory Medicine, Hebei Yanda Ludaopei Hospital, Sipulan Road, Yanjiao Development Area, Langfang, 065201, China. Email: [email protected]Search for more papers by this author First published: 29 May 2023 https://doi.org/10.1002/pbc.30445Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES 1Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization classification of haematolymphoid tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022; 36(7): 1703- 1719. 2Meyer C, Burmeister T, Groger D, et al. The MLL recombinome of acute leukemias in 2017. Leukemia. 2018; 32(2): 273- 284. 3de Rooij JD, Branstetter C, Ma J, et al. Pediatric non-Down syndrome acute megakaryoblastic leukemia is characterized by distinct genomic subsets with varying outcomes. Nat Genet. 2017; 49(3): 451- 456. 4Takita J, Motomura A, Koh K, et al. Acute megakaryoblastic leukemia in a child with the MLL-AF4 fusion gene. Eur J Haematol. 2009; 83(2): 149- 153. 5de Rooij JD, Masetti R, van den Heuvel-Eibrink MM, et al. Recurrent abnormalities can be used for risk group stratification in pediatric AMKL: a retrospective intergroup study. Blood. 2016; 127(26): 3424- 3430. 6Forlenza CJ, Zhang Y, Yao J, et al. A case of KMT2A-SEPT9 fusion-associated acute megakaryoblastic leukemia. Cold Spring Harb Mol Case Stud. 2018; 4(6):a003426. 7Qiu L, Nunez CA, Tang G, et al. A rare case of acute megakaryoblastic leukemia with t(11;17)(q23;q21) and KMT2A::MLLT6 fusion. Ann Hematol. 2022; 101(7): 1579- 1581. 8Morerio C, Rapella A, Tassano E, Rosanda C, Panarello C. MLL-MLLT10 fusion gene in pediatric acute megakaryoblastic leukemia. Leuk Res. 2005; 29(10): 1223- 1226. 9Borkhardt A, Haas OA, Strobl W, et al. A novel type of MLL/AF10 fusion transcript in a child with acute megakaryocytic leukemia (AML-M7). Leukemia. 1995; 9(10): 1796- 1797. 10Quessada J, Cuccuini W, Saultier P, Loosveld M, Harrison CJ, Lafage-Pochitaloff M. Cytogenetics of pediatric acute myeloid leukemia: a review of the current knowledge. Genes. 2021; 12(6): 924. 11Lalonde E, Rentas S, Wertheim G, et al. Clinical impact of genomic characterization of 15 patients with acute megakaryoblastic leukemia-related malignancies. Cold Spring Harb Mol Case Stud. 2021; 7(2):a005975. Early ViewOnline Version of Record before inclusion in an issuee30445 ReferencesRelatedInformation

A 17yearold male patient had been diagnosed 10 months previously with acute monocytic leukaemia, corresponding to AMLM5 according to FrenchAmericanBritish (FAB) categorisation, with KMT2A::MLLT10. After induction and consolidation chemotherapy, he achieved a complete remission (CR). Unfortunately, he relapsed after only 3 months. A bone marrow (BM) aspirate showed 89% round or oval blast cells with regular nuclei, agranular and moderately blue to grey cytoplasm, delicate lacy chromatin and prominent nucleoli (top images, ×100 objective). Immunophenotyping showed the blasts to be positive for CD34, CD33, CD13, HLADR, CD64, CD11c, CD38, CD123, CD4 (weak), CD7 (weak), CD56 (partial) and CD15 (partial), indicating monocytic differentiation. Conventional cytogenetic analysis showed 46,XY. All the evidence indicated a relapse of AMLM5. In view of the early relapse and lack of response to DCA (decitabine+cytarabine+aclarubicin) plus venetoclax, salvage allogeneic haematopoietic stem cell transplantation was performed. Nevertheless, a BM reexamination performed two months later showed 21% blasts with round, slightly irregular, or indented nuclei with fine reticular chromatin and 1– 3 nucleoli. The abundant cytoplasm was basophilic and agranular, with distinct blebs or pseudopod formation (bottom images). Flow cytometry demonstrated expression of CD33 (weak), CD42a, CD42b and CD61, with no expression of CD19, CD22, CD34, CD117, CD36, myeloperoxidase, CD11b or CD13, indicating megakaryocyte lineage (FAB classification, AML M7). KMT2A::MLLT10 was demonstrated by quantitative realtime polymerase chain reaction. The clinical course and investigations thus indicated transformation of KMT2Arearranged acute monocytic leukaemia to acute megakaryoblastic leukaemia. Of note, translocation with an 11q23 breakpoint was not observed during the course of the illness. Very few cases of AMLM7 with KMT2A::MLLT10 have been described. Of interest, this case was not a de novo AMLM7 with KMT2Arearrangement, but a particularly uncommon transformation from relapsed AMLM5. The knowledge of the fusion partner in KMT2Arearranged AML is clinically relevant as prognosis varies depending on the fusion partner. Molecular studies may be needed to identify KMT2A involvement and its fusion partner.

Objective:To investigate the significance of multicolor flow cytometry (MFC) monitoring of minimal residual disease (MRD) in the course of allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CD19-chimeric antigen receptor(CAR)-T cell immunotherapy for patients with refractory, relapsed B-cell acute lymphoblastic leukemia (r/r B-ALL).Methods:37 patients with r/r B-ALL admitted to Hebei Yanda Lu Daopei Hospital from January to July 2019, aged 15 (6, 19) years old, including 24 males and 13 females, were treated with CD19-CAR-T cell immunotherapy bridging allo-HSCT. MFC with cytoplasmic CD79a antibody to set up B-cell gates was used to monitor patients′ bone marrow (BM), cerebrospinal fluid (CSF), and tissue samples on day 0 (prior to the CAR-T cell immunotherapy), day 15, day 28 post CAR-T cell immunotherapy, and post transplantation.The MRD values of these samples were analyzed to evaluate the residual tumor cells and metastasis. The killing effect of the CAR-T cells was evaluated by the recovery of CD19+B cells before transplantation and the period between the timepoint when CD19+B cells was recovered and the timepoint when CAR-T cells were infused. Peripheral blood CAR-T cells were counted at different time points. Statistic analysis was performed by Kaplan-Meie assay and Log-rank test to analyze the difference of univariate cumulative survival.Results:(1)Among the 37 patients, 8 died and 29 survived. 5 patients relapsed after transplantation, of which 4 relapsed patients died and 1 survived. (2)MFC MRD negative remission rate of the death group was lower than that of the survival group at the following time points: post-CAR-T therapy and prior to transplantation (5/8 vs. 28/29, χ 2=7.540, P=0.006); day 15 of the CAR-T cell reinfusion (3/8 vs. 24/29, χ 2=6.512, P=0.011); day 28 of the reinfusion (3/8 vs. 276/29, χ 2=10.065, P=0.002). The probability of extramedullary MFC MRD positive tumor infiltration in the death group was higher than that in the survival group(7/8 vs. 14/29, χ 2=3.931, P=0.047). After CAR-T cell immunotherapy, the recovery period of CD19-positive cells in the death group, or the time for CAR-T cells to kill CD19-positive cells, was shorter than that in the survival group [42.00 days(30.00,49.00) vs. 55.00 days(41.50,73.50), Z=0.022, P=0.020]. Conclusion:The positive results of MRD by MFC at the following timepoints may predict unfavorable outcomes, such as post-CAR-T therapy and prior to transplantation, day 15 and 28 of the CAR-T cell immunotherapy, which may provide some guidance for clinical management.

International Journal of Laboratory HematologyVolume 45, Issue 1 p. e10-e14 LETTER TO THE EDITOR Full spectral flow cytometry analysis of the bone marrow immune cells in patients with myelodysplastic syndrome Man Chen, Man Chen Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorMinjing Fu, Minjing Fu Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorWei Zhao, Wei Zhao Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorAixian Wang, Aixian Wang Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorXueying Wu, Xueying Wu Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorMeiwei Gong, Meiwei Gong Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorJunyi Zhen, Junyi Zhen Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorGuanlan Yue, Guanlan Yue Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorHui Wang, Corresponding Author Hui Wang ldpwanghui@163.com orcid.org/0000-0002-3470-4159 Hebei Yanda Lu Daopei Hospital, Langfang, China Correspondence Hui Wang, Hebei Yanda Lu Daopei Hospital, Hebei, Langfang 065201, China. Email: ldpwanghui@163.comSearch for more papers by this author Man Chen, Man Chen Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorMinjing Fu, Minjing Fu Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorWei Zhao, Wei Zhao Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorAixian Wang, Aixian Wang Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorXueying Wu, Xueying Wu Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorMeiwei Gong, Meiwei Gong Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorJunyi Zhen, Junyi Zhen Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorGuanlan Yue, Guanlan Yue Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorHui Wang, Corresponding Author Hui Wang ldpwanghui@163.com orcid.org/0000-0002-3470-4159 Hebei Yanda Lu Daopei Hospital, Langfang, China Correspondence Hui Wang, Hebei Yanda Lu Daopei Hospital, Hebei, Langfang 065201, China. Email: ldpwanghui@163.comSearch for more papers by this author First published: 11 August 2022 https://doi.org/10.1111/ijlh.13945Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Open Research DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author, Hui Wang, upon reasonable request. Volume45, Issue1February 2023Pages e10-e14 RelatedInformation

Minimal residual disease (MRD) detection is an important prognostic parameter in patients with refractory or relapsed B-cell acute lymphoblastic leukemia (R/R B-ALL). CD79a has been reported to exhibit a high degree of linage-specificity for B-cell differentiation, with a specificity of 88% and a sensitivity of 100%. In this study, we investigated the efficiency and prognostic role of cytoplasmic CD79a (cCD79a) antibody-gated multicolor flow cytometry (MFC) in MRD detection in patients with B-ALL who received CD19-targeted chimeric antigen receptor (CAR) T-cell therapy bridging to allogeneic hematopoietic stem cell transplantation (allo-HSCT). The retrospective analysis was carried on to 59 patients who accepted allo-HSCT after CD19-CAR-T infusion from June 2016 to May 2017. The MFC MRD statuses before and after allo-HSCT were both strongly correlated with the transplantation prognosis, the MFC panel with cCD79a gating can effectively monitor MRD after CD19 CAR T-cell therapy and predict the prognosis after allo-HSCT. Trial registration: ClinicalTrials#: ChiCTR-IIh-16008711.gov: NCT03173417. Registered 30 May 2017 - retrospectively registered, https://www.clinicaltrials.gov/.

Acute myeloid leukemia (AML) is the most common heterogeneous hematopoietic malignancy in adults, accounting for 80% of acute leukemia cases.1 Chromosomal translocation is the most explored in hematological malignancies, which results in rearrangement of proto-oncogenes or key transcription sites often forming new fusion proteins. The AML1-ETO and PML-RARA, respectively, result from the chromosomal translocation t(8;21)(q22;q22) and t(15;17)(q24;q21), and they are the common chromosomal translocations in AML; however, simultaneous occurrence of both translocations in one patient is rare. Here, we first report a case of concurrent AML1-ETO and PML-RARA using flow cytometry sorting (FACSorting) flowed by morphology, immunophenotyping, fluorescence in situ hybridization (FISH), and molecular technique. A 17-year-old boy was admitted to our hospital with fever, vertigo, and scattered bleeding petechiae on the skin and mucous membranes over the whole body in April 2018. The patient had been diagnosed with AML by another hospital. Routine blood examination revealed white blood cells count of 14.68 × 109/L, hemoglobin concentration of 79.5 g/L, and platelet count of 34.1 × 109/L. The karyotype 2 showed 45,X,-Y,add(1)(p36.1),der(8)ins(8;21)(q22;q22q22)inv(8)(p23q22),del(11)(p13p14-15),t(15;17)(q24;q21),der(21)ins(8;21)(q22;q22q22) (Figure 1A). Among the 500 interphase nuclei analyzed, nuc ish (ETO, AML1)×3(ETO con AML1×1)[474]/(ETO×2, AML1×3)(ETO con AML1×1)[25]/(ETO, AML1)×2[1] (Figure 1B,C). Combined with the chromosome results of Figure 1A, it is suggested that insertion translocation of chromosome 8 and chromosome 21 was occurred followed by inversion (Figure 1B,C). Combined with the chromosome results of Figure 1A, it is suggested that insertion translocation of chromosome 8 and chromosome 21 was occurred followed by inversion (Figure 1B,C). Bone marrow smears observed 90% immature myeloid cells with two kinds of morphological characteristics. The majority AML patients possessed the features of PML-RARA (Red arrow), with irregular nuclei, visible distortion or folding, many azurophilic granules, and visible internal and external plasma (Figure 1D). The minority AML patients had the morphological features with AML1-ETO fusion gene (Green arrow), containing many azurophilic granules, and obvious superficial staining areas in the nuclear folds (Figure 1D). PML-RARA and AML1-ETO fusion genes were detected by RT-qPCR of 15.3% and 228.33%, respectively. WT1 and ASXL2 gene mutations were found by next generation sequencing. Flow cytometric analysis revealed two distinct aberrant cell populations: (a) CD117- immature myeloid cells and (b) CD117+ myeloid blasts (Figure 1E). These results together confirmed the previous AML diagnosis, and the co-expression of AML1-ETO and PML-RARA fusion genes. Moreover, the patient was treated with 10 mg arsenious acid for 2 days, 10 mg NIT for 3 days, 4 mg omacetaxine for 3 days, 100 mg cytarabine for 7 days, 3.25 g refined realgar for 7 days, and 20 mg tretinoin for 28 days. One month later, reexamination revealed 50% of bone marrow involvement; PML-RARA and AML1-ETO fusion genes were detected at 221.9% and 27.3%, respectively, indicating that chemotherapy was ineffective. Transplant was indicated for the patient; however, the patient died prior to transplant due to severe infection. After FACSorting, the CD117+/CD123− and CD117−/CD123+ malignant cells were sent to detect morphology, FISH, and PCR. Hybridization showed yellow signals for both PML-RARA and AML1-ETO fusion genes in malignant myeloblasts, as well as in leukemic promyelocytes (Figure 2A-D). In aberrant promyelocytes, PML-RARA and AML1-ETO fusion transcripts were detected at 150.00% and 37.49%, respectively (Figure 2A-D). Additionally, 44.73% PML-RARA and 205.40% AML1-ETO fusion transcripts were detected in malignant myeloblasts. In terms of cell morphology, the leukemic promyelocytes possessed the characteristic morphological features of PML-RARA (Figure 2E). Malignant myeloblasts had the characteristic morphological features of AML with AML1-ETO fusion gene (Figure 2F). The cell characteristic morphological features of PML-RARA and AML1-ETO were consistent with the description in Figure 1D. Up to now, there are few reports of AML with the coexistence of AML1-ETO and PML-RARA fusion genes. Charrin et al3 first reported such a case in 1992, wherein three distinct clones were observed in the bone marrow of a patient with leukemia: t(15;17), t(8;21), t(15;17)/t(8;21). Other cases of coexistence of t(8;21) or t(15;17) in leukemia have been described.4, 5 Although cases of PML-RARA co-expression with AML1-ETO are rare, studies on the proximity of nonrandomly associated translocation genes involved in leukemia subtypes to interphase genomic location suggest that PML and RARA genes are relatively close to each other in hematopoietic cells, as are AML1 and ETO,6 suggesting increased probability of AML1-ETO and PML-RARA fusion gene occurrence. Researchers examined AML cases defined as acute leukemia with malignant myeloblasts accompanied by leukemic promyelocytes and found that both PML-RARA and AML1-ETO fusion genes coexisted in a few cases.7 In this study, the cells in the bone marrow sample were divided into significant difference two cell populations and the expression level of the PML-RARA fusion gene in leukemic promyelocytes was higher than that in malignant myeloblasts, while the reverse was true for the AML1-ETO fusion gene. Therefore, a correlation between differentiation and related fusion gene expression could not be made. A single effect does not directly contribute to the development of AML in patients with AML1-ETO fusion gene, but may require a second-hit factor like the appearance of the PML-RARA fusion gene or other relevant gene mutations.8 The study found that the AML1-S291fs300X mutation was introduced in CD34 stem/progenitor cells and human induced pluripotent stem cells, confirming that the AML1 mutation induced bone marrow differentiation arrest at the myeloblasts stage.9 Among patients with leukemia having both the AML1-ETO fusion and C-KIT gene mutations, most possessed both phenotypic changes, whereas in three patients in complete remission, only the AML1-ETO fusion gene was expressed.8 Therefore, we suspected the C-KIT gene mutation may develop subsequently from the event of the t(8;21), indicating that a stepwise model occurs in leukemogenesis. That is, AML1-ETO represents the first genetic hit to initiate leukemia, while the C-KIT gene mutation may be the second hit for overall leukemia development. Our results indicated that the C-KIT receptor (CD117) was highly expressed in malignant myeloblasts, while the C-KIT gene mutation was negative, ruling out the possibility of a second genetic hit in the C-KIT pathway. Previous reports have implicated ASXL2 gene mutations in AML1-ETO fusion gene pathogenesis,10 but failed to detect WT1 and ASXL2 gene mutations in sorted cells due to the limited sample size. Charrin et al3 asserted that t(8;21) may be the first mutation in early bone marrow stem cells, while t(15;17) is acquired by the patient during the course of the disease. Whether the PML-RARA fusion gene is a secondary genetic hit for the simultaneous occurrence of PML-RARA and AML1-ETO fusion genes in AML needs to be investigated in greater depth. In summary, the coexistence of PML-RARA and AML1-ETO fusion genes is rare in AML. Although further studies with a greater number of patients are needed, this case is the first one applying FACSorting technique combining with morphology, FISH, and PCR techniques to accurately define the leukemogenesis in patients with concurrent AML1-ETO and PML-RARA fusion genes in AML. The authors declare that they have no competing interest. Hui Wang, corresponding author, designed the research and revised paper. Man Chen, first author, analyzed data and wrote the paper. Minjing Fu, Qing Du, and Meiwei Gong involved in test samples and gather clinical data. Junyi Zhen, Ping Wu, Tong Wang, and Hongxing Liu reported results. Xueying Wu and Aixian Wang drew the figure. All procedures were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from the patient for publication of this case. The data that support the findings of this study are available from the corresponding author upon reasonable request.