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.

Abstract Background: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is the most prevalent adult leukemia, and its incidence continues to rise year after year. Rapid and precise diagnosis is an essential element in effective case management, however, the clinical diagnosis, treatment, and prognosis of CLL/SLL are not fully elucidated. Case presentation: we report the case of a 66-year-old man with atypical CLL/SLL. The white blood cell (WBC) count (842.0 × 109/L), platelet count (30.6 × 109/L), and abnormal lymphocytes were increased in peripheral blood. Flow cytometry showed 98.34% of nucleated cells were malignant monoclonal mature B cell. Peripheral blood smear found the leukocytes and lymphocytes with abnormal morphology were increased. Fluorescence in situ hybridization showed CCND1 (11q23)/IGH (14q32) and abnormal chromosome 12 were invisible, 91%-93% of interphase nuclei presented D13S319 and TP53, 17p13.1 loss. Histopathology analysis of bone marrow observed the proliferation centers with immunoblasts. Immunohistochemistry showed that bone marrow was positive for PAX-5, CD20, CD23, and CD5, negative for CD3, cyclinD1, and sox11, and partial positive for Ki67. The patient was diagnosed as CLL/SLL based on above clinical and laboratory findings. The patient was managed with oral 50 mg Vinetoc, fluid replacement, hydration and alkalinization, and the symptoms were significantly relieved. Conclusions: This report further expands the knowledge of clinical diagnosis and treatment of atypical CLL/SLL.

Introduction Multicolor flow cytometry (MFC) has been frequently adopted as a method for minimal residual disease (MRD) detection, and is also a promising technique to detect post-transplant lymphoproliferative disorder. Some abnormal donor origin cells might be found when detecting MRD following an allogeneic hematopoietic stem cell transplantation (allo-HSCT). To minimize the effects from donor cells, using MFC prior to allo-HSCT to screen donor peripheral blood (PB) or bone marrow (BM) might be feasible. Methods We performed 3395 allo-HSCTs between January 2013 and December 2019 at Lu Daopei Hospital in Langfang, China. MRD was detected in recipients' BMs according to a conventional two-tube 8 or 9-color MFC panel. Abnormal cells were observed in BMs from three patients in complete remission (CR) one to four months post allo-HSCT. Abnormal neutrophils lacking CD16 expression were found in a patient with secondary acute myeloid leukemia (AML) that developed from a myelodysplastic/ myeloproliferative neoplasm (MDS/MPN). After ruling out MDS and paroxysmal nocturnal hemoglobinuria (PNH), we hypothesized that an Fcγ receptor IIIB (FcγRIIIB) gene deletion was the most likely reason. Abnormal natural killer (NK) cells were detected in the BM from an allo-HSCT recipient with T-cell acute lymphoblastic leukemia (ALL), and monoclonal B lymphocytosis (MBL) in allo-HSCT recipient with B-cell ALL. These three patinets' PBs were detected using MFC after the new finding to decide the cell origin. Besides, 4.54%(in WBC) CD4+ and CD8+ double positive T- cells which were monoclonal cells of the TCRVβ repertoire were detected in a PB sample from a donor prior to allo-HSCT. To evaluate the incidence rate The immunophenotypings were studied in the BMs from 79 NK lymphoma patients. Results Identical phenotypes were recognized in PBs obtained from the three respective donors. The fourth donor did not donate her cells for allo-HSCT, yet. The incidence rate of abnormal cells in donor samples was 0.1% (4/3395 cases), but this rate might be underestimated because MFC screening was not a routine procedure for donors. Additionally, only abnormal immunophenotyping related to patient diagnosis might have been found using an MRD panel as this panel only included markers related to diagnosis. Among general population, the incidence rate of suspicious FcγRIIIB deletion was 0.2% (11/5256 cases), the incidence rate of NK cells without CD2+ and homogeneously expressed CD159c was 0.05% (1/2000 cases) and none among the 79 NK lymphoma samples. The rate of MBL was 0.75% (15/2000 cases) and 1.36% in older than 40 years old people and the rate of monoclonal CD4/CD8 DP T-cells was 0.05% (1/2000 cases). All of these abnormal cells or polymorphism could be analyzed using a two tube MFC panel-- ckappa/clambda/(CD34)/CD19/ CD5/CD20/CD38 /CD45/CD56 and CD16/(CD117)/CD3/ CD4/CD5/ CD8/CD56/CD45/CD2. Conclusion Donor original abnormal cells or phenotypic polymorphisms could have an effect on MFC-based MRD or PTLD detection of recipients following allo-HSCT. These patients might be mis-diagnosed as being MRD positive or having PTLD if the technician lacks experience. To avoid mis-diagnosis and minimize the risk of allo-HSCT, it might be promising to utilize a suitable MFC panel to screen donor PB or BM samples prior to transplantation. Disclosures No relevant conflicts of interest to declare.

Background Post-transplant lymphoproliferative disorder (PTLD) is a serious complication that can occur following an allogenic hematopoietic stem cell transplant (allo-HSCT). PTLD occurs in approximately 0.8% to 20% of patients following an allo-HSCT and is associated with Epstein-Barr virus (EBV) infection in about 60% to 80% of patients. EBV positive (EBV+) PTLD generally arises early, several months following transplantation. The routine methods to diagnose EBV+ PTLD are clinical symptoms, EBV copy number, imageological examination, and pathological diagnosis, of which pathological diagnosis is the gold standard. Yet obtaining a biopsy is not possible with some patients and is not applicable for post-therapy monitoring. Therefore, there is an urgent need to find a simple, highly efficient, feasible, sensitive, and specific diagnostic and monitoring tool. Flow cytometry (FCM) has been established as a highly cost-effective method to diagnose lymphoma over the past several decades, and particularly for screening monoclonal B and/or plasma cells (MC B/P). Several recent publications as well as our own published and unpublished data have found that MC B/P are present in the peripheral blood of most PTLD patients. To this end, FCM detection of MC B/P in PB is a promising screening and monitoring method for EBV(+) PTLD. Objective To investigate the effectiveness of detecting MC B/P in PB by FCM for EBV+ PTLD screening and monitoring. Methods A total of 1470 patients received allo-HSCT at the Hebei Yanda Ludaopei Hospital, China from January 2018 through December 2019. We conducted a retrospective study of 481 patients with fever and lymphadenopathy following allo-HSCT. Patient PB was extracted for FCM MC B/P analysis. Plasma EBV viral loads were detected by PCR. The median fellow-up time was 6 months (range: 2 days to 21 months). The relationships of PB MC B/P, EBV load and clinical EBV-associated PTLD symptoms were investigated. Results: MC B/Ps were detected in the PB of 47 patients, of which 29 cases had monoclonal B cells, 14 cases had detectable co-existence of monoclonal B cells and monoclonal plasma cells, and 4 cases had detectable monoclonal plasma cells. The median time to PTLD onset following allo-HSCT was 70 days (range: 33 days to 491 days). The median percentage of monoclonal B cells was 0.43% (range: 0.1% to 23.41%. The median percentage of monoclonal plasma cells was 0.25%). Forty of 47 patients (85.1%) were finally diagnosed with PTLD using clinical comprehensive examination of symptoms. The incidence of clinical EBV+ PTLD was 2.9% (44 of 1470 cases). By FCM, MC B/Ps were observed in the PB of 91% of the patients (40 of 44 cases). No MC B/Ps were found in the PB of 4 patients. MC B/P detection in PB by FCM screening could effectively predict the incidence of EBV+ PTLD with a sensitivity of 90.91% and specificity of 98.40%. The positive predictive value was 85.17% and the negative predictive value was 99.08%. There was no significant correlation between EBV viral copy number and percentage of MC B/P. The median fellow-up time was 6 months (range: 2 days to 21 months) for all patients. The therapeutic response rate was 87.5% (35 of 40 patients) for MC B/P positive EBV+ PTLD patients. In those patients with a therapeutic response, clinical symptoms improved, lymph nodes significantly shrank, EBV viral loads decreased, MC B/Ps decreased or vanished, and CD20+ cell proportion decreased or vanished. Among the 7 patients who had MC B/Ps in PB but were not diagnosed with PTLD , their clinical symptoms recovered after treatment with immunosuppressive therapy. Conclusion By detecting PB MC B/Ps using FCM, we can successfully screen for EBV+ PTLD and monitor patient therapeutic responses in most cases. FCM to detect MC B/Ps in PB is a new, promising method for the diagnosis and monitoring of EBV+ PTLD. Compared to a biopsy, this appears to be a more simple, easier and more applicable tool to diagnose and monitor EBV+ PTLD. Disclosures No relevant conflicts of interest to declare.

Flow cytometry (FCM) uses flow cytometer to detect the expression of immunological markers on cells based on antigen-antibody binding and laser activating fluorescence. FCM plays a very important role in clinical diagnosis especially in acute leukemia diagnosis and subclassification. However, due to the difficulty in acquiring the sample and the existence of normal lymphocytes in the background, its application and improvement should be further strengthened in the diagnosis of mature lymphoid neoplasm. This paper focuses on the similarity and differences between FCM and pathology immunohistology, immune makers in lymphoma detection, immunophenotypic features of common mature lymphoid neoplasm, common issues and key points in FCM detection, to study the diagnostic application of flow cytometry in mature lymphoid neoplasm. This will help clinicians understand this technic, and make a correct diagnosis by FCM combining with pathology. Key words: Flow cytometry; Mature lymphoid; Neoplasm; Diagnosis

Background: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is the most prevalent adult leukemia, and its incidence continues to rise year after year. Rapid and precise diagnosis is an essential element in effective case management, however, the clinical diagnosis, treatment, and prognosis of CLL/SLL are not fully elucidated. Case presentation: we report the case of a 66-year-old man with atypical CLL/SLL. The white blood cell (WBC) count (842.0 × 10 9 /L), platelet count (30.6 × 10 9 /L), and abnormal lymphocytes were increased in peripheral blood. Flow cytometry showed 98.34% of nucleated cells were malignant monoclonal mature B cell. Peripheral blood smear found the leukocytes and lymphocytes with abnormal morphology were increased. Fluorescence in situ hybridization showed CCND1 (11q23)/IGH (14q32) and abnormal chromosome 12 were negative, 91%~93% of interphase nuclei presented D13S319 and TP53, 17p13.1 loss. Histopathology analysis of bone marrow observed the proliferation centers with immunoblasts. Immunohistochemistry showed that bone marrow was positive for PAX-5, CD20, CD23, and CD5, negative for CD3, cyclinD1, and sox11, and partial positive for Ki67. The patient was diagnosed as CLL/SLL based on above clinical and laboratory findings. The patient was managed with oral 50 mg Vinetoc, fluid replacement, hydration and alkalization, and the symptoms were significantly relieved. Conclusions: This report further expands the knowledge of clinical diagnosis and treatment of atypical CLL/SLL.