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The standardization of multiparameter flow cytometry-based measurable residual disease (MFC-MRD) assessment in acute myeloid leukemia (AML) lacks clear criteria to define leukemia-associated immunophenotypes (LAIPs). In addition, the most specific/sensitive aberrations used to define LAIPs are often partially expressed by the leukemic clone at diagnosis, raising questions about their reliability for accurate MRD quantification. To address this, we investigated whether the quantification of LAIP+ cells reflects residual disease in cases of partial LAIP expression. The following two MFC-MRD approaches were evaluated by comparing their results to RT-qPCR for NPM1 mutations: (1) the LAIP-method, wherein all cells within the patient-specific template created at diagnosis are counted without further gating; (2) the LAIP-based different-from-normal (DfN)-method, wherein cells+ for LAIP-specific aberrant markers are further selected. A total of 125 bone marrow samples from 25 NPM1-mutated AML patients were studied. Our data demonstrate that the LAIP-based DfN-method improves the MFC-MRD accuracy and comparability with molecular MRD. ROC analysis identified cut-offs of 0.034% and 0.095% to discriminate positive/negative results in patients receiving intensive chemotherapy and hypomethylating agents, respectively. We also found distinct accuracy degrees based on the LAIP-specific aberrant markers used for MRD assessment. These results refine the MFC-MRD method and highlight the importance of therapy-specific MRD cut-offs and LAIP classification based on specificity and sensitivity.

Mature T-cell neoplasms arise from the neoplastic transformation of a single T lymphocyte, and all cells in a neoplastic clone share the same V segment in the beta chain of the T-cell receptor (TCR). These segments may represent an innovative target for the development of targeted therapies. A specific V segment of the TCR beta chain (TRBV5-1) was analyzed using bioinformatic tools, identifying three potential antigenic peptides. One of these peptides, selected for synthesis, was used to screen a library of human single-chain variable fragments (scFv) through phage display. One fragment demonstrated high affinity and specificity for the antigen and was used to produce a human monoclonal antibody of the IgG1 class. Surface plasmon resonance (SPR) studies confirmed the high affinity of the monoclonal antibody for the antigen in the nanomolar range. Flow cytometry analysis on patients' samples demonstrated that the antibody, conjugated with a fluorochrome, selectively binds to tumor T lymphocytes expressing TRBV5-1, without binding to other lymphocytes or blood cell components. The development of fully human IgG1 monoclonal antibodies targeting specific V segments of the TCR beta chain represents a potential therapeutic option for patients with mature T-cell neoplasms.

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked to CD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell–mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments.

Background ZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method). Methods Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set). Results The optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70 + cases, respectively. Univariate analyses resulted in a better separation of ZAP-70 + vs. ZAP-70 - CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70 + cases, respectively. ZAP-70 + patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70 - CLL, and to cases classified ZAP-70 + by the T-method only. Conclusions We suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70 + (T/B Ratio less than 3.0) from ZAP-70 - (T/B Ratio more/equal than 3.0) cases.

Conventional cytology (CC) of cerebrospinal fluid (CSF) fails to demonstrate malignant cells in up to 45 % of patients with acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LL) in whom occult leptomeningeal disease is present. Flow cytometry (FCM) is considered more sensitive than CC, but clinical implications of CC negativity/CC positivity are not yet established. CSF samples from 38 adult patients with newly diagnosed ALL/LL were examined. Five (13 %) and nine (24 %) specimens were CC positive-FC positive (FCM pos /CC pos ) and CC negative-FC positive (CC neg /FCM pos ), respectively. The remaining 24 (63 %) samples were double negative (CC neg /FCM neg ) ( p  = 0.001). CC neg /FCM pos patients showed a significantly shorter overall survival (OS) compared to CC neg /FCM neg ones. In multivariate analysis, the status of single FCM positivity was demonstrated to affect independently duration of OS ( p  = 0.005). In conclusion, FCM significantly improves detection of leptomeningeal occult localization in ALL/LL and appears to anticipate an adverse outcome. Further prospective studies on larger series are needed to confirm this preliminary observation.

Acute myeloid leukemia (AML) with FLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3-ITD-positivity. The aim of this study was to characterize the distribution of FLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3-ITDmut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3-ITDmut AML (n = 12). A higher FLT3-ITDmut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/−,CD25−,CD99low/−) (p = 0.0005) and mononuclear cells (MNCs) (p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higher FLT3-ITDmut burden was also observed in LPCs of AML patients with a small FLT3-ITDmut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3-ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3-ITDmut AML.

Measurable residual disease (MRD) is increasingly employed as a biomarker of quality of complete remission (CR) in intensively treated acute myeloid leukemia (AML) patients. We evaluated if a MRD-driven transplant policy improved outcome as compared to a policy solely relying on a familiar donor availability. High-risk patients (adverse karyotype, FLT3-ITD) received allogeneic hematopoietic cell transplant (alloHCT) whereas for intermediate and low risk ones (CBF-AML and NPM1-mutated), alloHCT or autologous SCT was delivered depending on the post-consolidation measurable residual disease (MRD) status, as assessed by flow cytometry. For comparison, we analyzed a matched historical cohort of patients in whom alloHCT was delivered based on the sole availability of a matched sibling donor. Ten-years overall and disease-free survival were longer in the MRD-driven cohort as compared to the historical cohort (47.7% vs. 28.7%, p = 0.012 and 42.0% vs. 19.5%, p = 0.0003). The favorable impact of this MRD-driven strategy was evident for the intermediate-risk category, particularly for MRD positive patients. In the low-risk category, the significantly lower CIR of the MRD-driven cohort did not translate into a survival advantage. In conclusion, a MRD-driven transplant allocation may play a better role than the one based on the simple donor availability. This approach determines a superior outcome of intermediate-risk patients whereat in low-risk ones a careful evaluation is needed for transplant allocation.

Background The level of measurable residual disease (MRD) is one of the most important features correlating depths of response and long‐term outcomes in multiple myeloma (MM) and MRD evaluation is currently the gold standard tool for assessing treatment response. Nevertheless, reproducibility across laboratories is a major concern, as discrepancies among results make comparability impractical. Aims: herein, we report preliminary results from the “Italian MM‐MRD network” project. Patients & Methods MRD in bone marrow (BM) samples have been measured from newly diagnosed MM patients using next‐generation flow‐cytometry (NGF) or next‐generation sequencing (NGS) approaches in different laboratories. Results The NGF workgroup (7 laboratories) implemented the Euro‐Flow Standard‐Operating‐Protocol to reach minimum 1 × 10−5 sensitivity. The inter‐operator retrospective study (Stage 1) showed high inter‐center concordance in monoclonal plasma cells detection (ICC = 0.90, p < 0.001), whereas moderate concordance was observed in the inter‐laboratory correlation (Stage 2) in in‐vivo samples (ICC = 0.63, p < 0.001), reaching a median limit‐of‐detection (LOD) and limit‐of‐quantification (LOQ) of 8 × 10−6 and 2 × 10−5, respectively. Greater variability was also observed in the analysis of other BM cell populations. The NGS workgroup (4 laboratories) employed a targeted amplicon‐based approach to detect clonotypic IGH/IGK gene rearrangements in diagnostic samples, subsequently used to track MRD in mock samples. The experimental design was divided into three quality‐control (QC) rounds, focused on finding a shared strategy for clonotype identification (QC1: 100% concordance among centers), or quantifying MRD in mock samples (concordance: 81% [QC2]; 91% [QC3]). The 10−5‐sensitivity level was successfully reached in most of tested dilutions (QC2: 19/20 = 95%; QC3: 19/23 = 83%). Conclusion Overall, this pilot study provided preliminary data for MRD harmonization across Italian centers, paving the way for an expanded network, aiming at reducing variability, improving comparability, and enabling broader use of MRD‐monitoring in clinical practice.

Diagnosis and prognostic stratification of myelodysplastic syndromes (MDS) have been complemented by new techniques, including flow cytometry and NGS. To analyze the relationship between molecular and cytofluorimetric data, we enrolled in this retrospective study, 145 patients, including 106 diagnosed with MDS and 39 controls. At disease onset, immunophenotypic (IF), cytogenetic tests, and cytomorphological (CM) examination on bone marrow were carried out in all patients, while NGS was performed in 58 cases. Ogata score presented a specificity of 100% and a sensitivity of 59%. The detection of at least two phenotypic aberrancies in Ogata negative patients increased the sensitivity to 83% and specificity to 87%. Correlations were identified between IF aberrancies and mutations, including positive Ogata>2 and mutations in SRSF2 (p=0.035), CD15 and U2AF1 (0.032), CD56 and DNMT3A (p=0.042), and CD38 and TP53 (p=0.026). In multivariate analysis, U2AF1 mutations, associated with del(20q) and/or abnormalities of chromosome 7 (group 4 as defined by the EuroMDS score), significantly correlated with an inferior overall survival (p=0.019). These parameters and Ogata score>2 also showed a significant correlation with inferior event-free survival (p=0.023 and p=0.041, respectively). Both CM and FC features correlated with prognosis and mutational patterns. In an integrated MDS work-up, these tools may guide indications for mutational screening for optimal risk stratification.