[Immunophenotypic and Clinical Characteristics of SET-CAN Fusion Gene Positive Acute Leukemia Patients].

OBJECTIVE: To analyze the flow immunophenotype and clinical characteristics of leukemia patients with positive SET-CAN fusion gene. METHODS: A total of 7 newly diagnosed acute leukemia patients with SET-CAN fusion gene admitted to Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from February 2016 to February 2020 were collected. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of SET-CAN fusion gene. The immunophenotype was detected by four-color flow cytometry. The case information of 17 literatures published at home and abroad was extracted for statistical analysis. RESULTS: Among the 7 patients, 2 cases were diagnosed as mixed phenotype acute leukemia (MPAL), 2 cases as acute myeloid leukemia (AML), and 3 cases as T-acute lymphoblastic leukemia (ALL)/lymphoblastic lymphoma (LBL). Leukemia cells in bone marrow specimens of all cases expressed or partially expressed CD34, CD33 and CD7. CD5 and cytoplasmic CD3 were expressed in 5 patients except 2 patients diagnosed with AML. Bone marrow and lymph node specimens were both detected in 2 patients, and the immunophenotypes of the two specimens were not completely consistent, with differences in lineage or maturity related markers. Two patients with MPAL showed differentiated response to treatment. One AML patient gave up treatment, and another AML patient with FLT3-ITD gene mutation had a poor prognosis. All three T-ALL/LBL patients maintained a long duration of remission after induced remission, and one case underwent allogeneic hematopoietic stem cell transplantation. CONCLUSIONS: There are common characteristics of immunophenotype in patients with positive SET-CAN fusion gene. Differential expression of immunophenotype in samples from different parts is observed in some cases. The prognosis of these diseases varies.

Multicolor flow cytometric assessment of Ki67 expression and its diagnostic value in mature B-cell neoplasms.

Background: There is no unified standard data about the sensitivity and specificity regarding flow cytometry analysis of Ki67 expression during lymphoma diagnoses. Objective: This evaluated the efficacy of multicolor flow cytometry (MFC) in an estimate of the proliferative activity of B-cell non-Hodgkin lymphoma by comparing the expression of Ki67 using MFC and immunohistochemicals (IHC). Method: A total of 559 patients with non-Hodgkin B-cell lymphoma were immunophenotyped using sensitive MFC, of which 517 were newly diagnosed and 42 were transformed lymphomas. Test samples include peripheral blood, bone marrow, various body fluids, and tissues. Through MFC multi-marker accurate gating, abnormal mature B lymphocytes with restricted expression of the light chain were screened. Ki67 was added to determine the proliferation index; the positive rate of Ki67 in tumor B cells was evaluated by cell grouping and internal control. For tissue specimens, MFC and IHC analyses were performed simultaneously to assess the Ki67 proliferation index. Results: The positive rate of Ki67 by MFC was correlated with the subtype and aggressiveness of B-cell lymphoma. Ki67 could distinguish indolent lymphomas from aggressive subtypes with a cut-off value of 21.25%, and differentiate transformation from indolent lymphoma with a cut-off value of 7.65%. The expression of Ki67 by MFC (regardless of the type of samples)was highly agreement with the Ki67 proliferative index of tissue samples assessed by pathologic immunohistochemistry. MFC showed a fairly constant negative bias in evaluating tissue or bone marrow samples, compared with IHC. Conclusions: Ki67 is a valuable flow marker that can distinguish between indolent and aggressive types of lymphoma and assess whether indolent lymphomas are transformed. Using MFC to evaluate the positive rate of Ki67 is important in clinical settings. MFC has unique advantages in judging the aggressiveness of lymphoma in samples of bone marrow, peripheral blood, pleural and ascites, and cerebrospinal fluid. This is particularly important when tissue samples cannot be obtained, making it an important supplement for pathologic examination.

PD-1 combined with TRBC1 and pan-T cell antibodies for robustly monitoring angioimmunoblastic T-cell lymphoma.

Background: The diagnosis of AITL is challenging. It may be delayed or even missed due to critical clinical conditions and its histologic and immunophenotypic overlap with other neoplastic and reactive lymphoid proliferations. Objective: The key objective is to obtain an efficient diagnosis, sensitive disease monitoring and treatment efficacy assessment of AITL using multiparameter flow cytometry (MFC). Methods: In total, 167 de novo AITL patients were immunophenotypically profiled using sensitive MFC. We precisely identified the aberrant T-cell populations of AITL and performed an in-depth description of their phenotypic characteristics in comparison with their residual normal CD4+ T cells. A comparison of Programmed death receptor-1 (PD-1) expression was performed among AITL and other T-cell lymphomas. Results: MFC detected a neoplastic T-cell population in 94.1% (80/85) of tissue, 71.5% (108/151) of bone marrow (BM), 100% (8/8) of peripheral blood (PB) and 78.6% (11/14) of body fluid samples. The most frequent immunophenotypic aberrations included the absence and diminished expression of CD3 (71.25% in tissues, 71.3% in BM, 75% in PB, 81.8% in hydrothorax and ascites specimens), followed by the loss or partial loss of CD7 (71.25% in LN, 67.6% in BM, 50% in PB, 81.8% in hydrothorax and ascites specimens). The immunophenotyping of neoplastic T-cell populations showed a high degree of similarity among different sites of the same patient and they might change over time but were relatively stable. Bright PD-1 expression showed high sensitivity and specificity in differentiating AITL from other T-cell lymphomas. In 14 AITL patients, neoplastic T-cell populations were initially missed by T-cell screening tube but were successfully discovered by bright PD-1 expression. Conclusion: T-cell screening tube can reliably screen neoplastic T-cell populations in AITL patients with typical immunophenotyping, such as loss of surface CD3 and loss of CD7 with a relatively high ratio. Bright PD-1 expression is essential for identifying aberrant T cells in almost all AITLs. The clonality assessment antibody TRBC1 is efficient for robustly and cheaply assessing T-cell clonality. Using PD-1 and TRBC1 combined with pan-T cell antibodies can make a precise diagnosis of AITL and also sensitively monitor minimal residual disease regardless of the antigenic drift of the neoplastic T cells.

The association of complex genetic background with the prognosis of acute leukemia with ambiguous lineage.

Acute leukemia with ambiguous lineage (ALAL) is a rare and highly aggressive malignancy with limited molecular characterization and therapeutic recommendations. In this study, we retrospectively analyzed 1635 acute leukemia cases in our center from January 2012 to June 2018. The diagnose of ALAL was based on either EGIL or 2016 WHO criteria, a total of 39 patients were included. Four patients diagnosed as acute undifferentiated leukemia (AUL) by both classification systems. Among the patients underwent high-throughput sequencing, 89.5% were detected at least one mutation and the median number of gene mutation was 3 (0-8) per sample. The most frequently mutated genes were NRAS (4, 21%), CEBPA (4, 21%), JAK3 (3, 16%), RUNX1 (3, 16%). The mutations detected in mixed-phenotype acute leukemia (MPAL) enriched in genes related to genomic stability and transcriptional regulation; while AUL cases frequently mutated in genes involved in signaling pathway. The survival analysis strongly suggested that mutation burden may play important roles to predict the clinical outcomes of ALAL. In addition, the patients excluded by WHO criteria had even worse clinical outcome than those included. The association of the genetic complexity of blast cells with the clinical outcomes and rationality of the diagnostic criteria of WHO system need to be evaluated by more large-scale prospective clinical studies.

The Early Diagnostic Dilemma in Angioimmunoblastic T Cell Lymphoma with Excessive Plasma Cells Proliferation.

BACKGROUND: Angioimmunoblastic T cell lymphoma (AITL) is an aggressive Epstein-Barr virus-associated T cell lymphoma. Clinical syndromes of AITL are not confined to fever and lymphadenopathy, and patients may initially present with polyclonal plasma cell proliferation, which may obscure the underlying disease of AITL, delaying diagnosis. Case Presentation. Here, we report two AITL patients with excessive plasma cell proliferation in the bone marrow, peripheral blood, and ascites even mimicking plasma cell leukemia. Both of them had poor endings. CONCLUSIONS: Our report emphasizes the complexity of the clinical manifestations of AITL, which aims to increase the alertness of physicians and improve the rate of early diagnosis. Integrated diagnostic approaches such as histopathology, flow cytometry, cytogenetics, and molecular biology are essential for accurate diagnosis and precise therapy.

A new score including CD43 and CD180: Increased diagnostic value for atypical chronic lymphocytic leukemia.

Moreau score has been used to differentiate chronic lymphocytic leukemia (CLL) from other mature B-cell neoplasms. However, it showed limitations in Asian patients. Therefore, we conducted a new score system replacing CD5 and CD23 with CD43 and CD180 to evaluate its diagnostic value of CLL. 237 untreated samples diagnosed with mature B-cell neoplasms were collected and were randomly divided into an exploratory and a validation cohort by a 2:1 ratio. The expression of CD5, CD19, CD20, CD23, CD43, CD79b, CD180, CD200, FMC7, and surface immunoglobulin (SmIg) were analyzed among all the samples. A proposed score was developed based on the logistic regression model. The sensitivity and specificity of the proposed score were calculated by ROC curves. CD43/CD180, CD200, FMC7, and CD79b were included in our new CLL score, which showed a sensitivity of 91.8% and a specificity of 83.1%. These results were confirmed in a validation cohort with a sensitivity of 90.5% (p = 0.808) and a specificity of 79.5% (p = 0.639). In CD5 negative or CD23 negative CLL group, the new CLL score displayed improved sensitivity of 79.4% compared to Moreau score and CLLflow score (41.2% and 47.1%, respectively). In atypical CLL group, the new CLL score showed improved sensitivity of 84.2% compared to Moreau score and CLLflow score (61.4% and 64.9%, respectively). This proposed atypical CLL score helped to offer an accurate differentiation of CLL from non-CLL together with morphological and molecular methods, particularly in Chinese patients with atypical immunophenotype.

CAR T-Cell Therapy Is Effective but Not Long-Lasting in B-Cell Lymphoma of the Brain.

Advanced central nervous system (CNS) lymphoma is an exclusion criterion for most chimeric antigen receptor (CAR) T-cell studies due to the associated levels of neurotoxicity. In this study, we described five patients with chemorefractory B-cell CNS lymphoma who received CAR19 and CAR22 T-cell "Cocktail" therapy and follow-up for 6-16 months. All patients experienced cytokine release syndrome (CRS). Two patients experienced CAR T-cell-related encephalopathy syndrome (CRES), which was controllable. The best response was observed in two patients, who successfully achieved complete remissions (CR), and the other three patients achieved partial remissions (PR). Four patients had progressive disease (PD) after remission. In addition, one CR patient and one PD patient accepted CAR T-cell infusion following hematopoietic stem cell transplantation therapy in the 3rd month and were in ongoing remission for 14 and 6 months of follow-up, respectively. The targeted antigens in two patients were still positive, and CAR T-cells were reboosted in the cerebrospinal fluid (CSF) after PD, but a small number of CD3-positive T-cells were observed to infiltrate into the tumor. Our study indicates the efficacy of CAR T-cell therapy for CNS lymphoma with an acceptable safety profile; however, the remission did not last long, perhaps due to the tumor immunosuppressive microenvironment (TME) of the CNS. CAR T-cell therapy should be combined with other treatments to help improve the TME of cerebral lymphoma.

Inflammatory signatures for quick diagnosis of life-threatening infection during the CAR T-cell therapy.

BACKGROUND: Chimeric antigen receptor-modified (CAR) T-cell immunotherapy is a novel promising therapy for treatment of B-cell malignancy. Cytokine release syndrome (CRS) and infection are the most common adverse events during CAR T-cell therapy. Similar clinical presentation of concurrent CRS and infection makes it difficult to differentially diagnose and timely treat the condition. METHODS: We analyzed the features of infection events during the first 30 days after CAR T-cell infusion (CTI) in 109 patients from three clinical trials (ChiCTR-OPN-16008526, ChiCTR-OPC-16009113, ChiCTR-OPN-16009847). Based on the dynamic changes of interleukin (IL)-6 and ferritin, we proposed the "double peaks of IL-6" pattern as a feature of life-threatening infection during the first 30 days after CTI. Meanwhile, we screened candidate biomarkers from 70-biomarker panel to establish a prediction model for life-threatening infection. RESULTS: In this study, 19 patients (17.4%) experienced a total of 19 infection events during the first 30 days after CAR T-cell infusion. Eleven patients (10.1%) had grade 4-5 infection, which were all bacterial infection and predominantly sepsis (N = 9). "Double peaks of IL-6" appeared in 9 out of 11 patients with life-threatening infection. The prediction model of three-cytokines (IL-8, IL-1ß and interferon-γ) could predict life-threatening infection with high sensitivity (training: 100.0%; validation: 100.0%) and specificity (training: 97.6%; validation: 82.8%). On base of the aforementioned methods, we proposed a workflow for quick identification of life-threatening infection during CAR T-cell therapy. CONCLUSIONS: In this study, we worked out two diagnostic methods for life-threatening infection during CAR T-cell therapy by analyzing inflammatory signatures, which contributed to reducing risks of infection-induced death.

Tumor necrosis factor α knockout impaired tumorigenesis in chronic myeloid leukemia cells partly by metabolism modification and miRNA regulation.

PURPOSE: Half of the chronic myeloid leukemia (CML) patients with sustained deep molecular response suffer from relapse after discontinuation mainly because tyrosine kinase inhibitors (TKIs) cannot eradicate leukemia stem cells (LSCs). In addition, tumor necrosis factor α (TNF-α) is highly detected in CML patients. Our aim was to explore whether TNF-α is a potential target for LSC elimination. MATERIALS AND METHODS: We applied a CRISPR/Cas9 gene editing technique, colony-forming cell assay, subcutaneous tumor models, miRNA-seq and liquid chromatography-mass spectroscopy (LC-MS) on metabonomics to explore the feasibility and mechanism of TNF-α as a new therapeutic target for CML. RESULTS: We demonstrated that TNF-α knockout remarkably decreased the proliferative, colony-forming and in vivo tumorigenesis capacities of the CML K562 cell line. The apoptosis was increased when TNF-α knockout cells were cultured with imatinib. The mechanisms involved in the abovementioned phenomena were that TNF-α knockout inhibited the citrate cycle and increased starch, sucrose, amino sugar and nucleotide sugar metabolism. In addition, differentially expressed miRNAs between TNF-α knockout and control cells were involved in the cell cycle, CML, P13K-Akt and pathways in cancer. CONCLUSION: We identified that TNF-α may serve as a new target therapy for CML and described the metabolic pathways associated with TNF-α in CML cells for the first time.

Deletion of MBD2 inhibits proliferation of chronic myeloid leukaemia blast phase cells.

Aberrant methylation of tumour suppressor genes is associated with the progression to a blast crisis in chronic myeloid leukaemia (CML). Methyl-CpG-binding domain protein 2 (MBD2) has been studied as a "reader" of DNA methylation in many cancers, but its role in CML is unclear. We constructed cell models of a homozygous deletion mutation of MBD2 using gene-editing technology in K562 cells and BV173 cells. Here, we demonstrated that the deletion of MBD2 inhibited cell proliferation capacity in vitro. MBD2 deletion also significantly inhibited K562 cell proliferation in a xenograft tumour model in vivo. Additionally, the JAK2/STAT3 signalling pathway, which is abnormally active in CML, was inhibited by MBD2 deletion, and MBD2 deletion could up-regulate the expression of SHP1. In conclusion, our findings suggest that MBD2 is a candidate therapeutic strategy for the CML blast phase.

A clinical observation of Chinese chronic myelogenous leukemia patients after discontinuation of tyrosine kinase inhibitors.

Whether tyrosine kinase inhibitors (TKIs) can be safely discontinued is a key focus of chronic myelogenous leukemia (CML) at present. We report a clinical observation of TKIs cessation in Chinese CML patients and a probable connection between CML leukemia stem cells (LSCs) and relapse. In all, 22 of 1057 patients consented to participate in this observation. The average time of complete molecular response was 12.73 months after TKI withdrawal. LSCs could be flow cytometrically detected in most of the patients. However, the number of LSCs did not differ between the relapsers and non-relapsers. We evaluated the leukemogenetic ability of the LSCs by transplanting bone marrow into irradiated NOD/SCID mice. The results indicated that part of the bone marrow from the relapsers lead to leukemogensis in the mice. Besides, we found that LSCs-derived microvesicles might serve as a novel factor for the stratification of undetectable minimal residual disease and an early warning sign of relapse. In summary, post-TKI cessation relapse seems to show none association with the number of LSCs. A mouse xenograft model would provide a novel and useful method of analyzing LSCs function and predicting relapse. Microvesicles may provide important information about optimal molecular monitoring schedules in TKI discontinuation strategies.