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Current chronic lymphocytic leukemia (CLL) International Prognostic Index (IPI) stratifies patients based on clinical, molecular, and biochemical features; however, B-cell markers also influence CLL outcomes. Here, prognostic roles of CD11c, CD38, and CD49d were first evaluated, and then an immunophenotypic score was combined with CLL-IPI for risk stratification of CLL patients.A total of 171 CLL subjects were included, and surface marker expression was assessed by flow cytometry. Levels ≥30% were chosen as cut-off of positivity to a marker; then values of 1 (for CD11c and CD38) or 3 (for CD49d) were assigned and scores determined for each patient's clone immunophenotype.CD49d positivity was significantly associated with simultaneous expression of CD11c and/or CD38, unmutated IGHV status, and higher β2-microglobulin levels compared to those with CD49d negativity. Moreover, CD49dOur results suggested clinical utility of an integrated prognostic score for better risk stratification of CLL patients. These results require further validation in prospective larger studies.

Richter’s syndrome represents the progression of chronic lymphocytic leukemia (CLL) to more aggressive diseases, most frequently diffuse large B-cell lymphoma, while Hodgkin’s lymphoma (HL) and hairy cell leukemia (HCL) are rarely described. The first case involved a 67-year-old man with a diagnosis of a high-risk stage-II CLL treated with rituximab and ibrutinib, developed a HL nodular sclerosis variant after three months of therapy for CLL. After achieving a complete remission for HL and ibrutinib cessation because of drug-related cardiotoxicity, the patient relapsed after five months off-therapy and died due to disease progression after two cycles of brentuximab-vedotin. The second case involved an 83-year-old female with a diagnosis of stage-IV CLL treated with rituximab plus bendamustine who developed a HCL eight years later. Pentostatin was unsuccessfully employed as upfront HCL therapy, and the patient was then switched to rituximab while in remission for CLL. In conclusion, Richter’s transformation risk rate might be higher in patients treated with novel targeted therapies, and multiparametric flow cytometry and lymph node biopsy at relapse could help in early identifying small clones. The treatment of predominant neoplasia is mandatory, and disease-specific drugs are administered; however, clinical efficacy might be lower in these patients.

Simple Summary Large granular lymphocyte (LGL) leukemia, a lymphoproliferative disease, is characterized by an increased frequency of large-sized lymphocytes with typical expression of T-cell receptor (TCR) αβ, CD3, CD8, CD16, CD45RA, and CD57, and with the expansion of one to three subfamilies of the TCR variable β chain reflecting gene rearrangements. Molecular analysis remains the gold standard for confirmation of TCR clonality; however, flow cytometry is time and labor saving, and can be associated with simultaneous investigation of other surface markers. Moreover, Vβ usage by flow cytometry can be employed for monitoring clonal kinetics during treatment and follow-up of LGL leukemia patients. Abstract Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder of mature T or NK cells frequently associated with autoimmune disorders and other hematological conditions, such as myelodysplastic syndromes. Immunophenotype of LGL cells is similar to that of effector memory CD8+ T cells with T-cell receptor (TCR) clonality defined by molecular and/or flow cytometric analysis. Vβ usage by flow cytometry can identify clonal TCR rearrangements at the protein level, and is fast, sensitive, and almost always available in every Hematology Center. Moreover, Vβ usage can be associated with immunophenotypic characterization of LGL clone in a multiparametric staining, and clonal kinetics can be easily monitored during treatment and follow-up. Finally, Vβ usage by flow cytometry might identify LGL clones silently underlying other hematological conditions, and routine characterization of Vβ skewing might identify recurrent TCR rearrangements that might trigger aberrant immune responses during hematological or autoimmune conditions.

Introduction. Myelodysplastic syndromes (MDS), a group of clonal hematological diseases, are characterized by ineffective hematopoiesis, progressive peripheral blood (PB) cytopenia(s), and increased risk of developing acute myeloid leukemia (AML). Classification and risk stratification are constantly under revision for a better estimation of prognosis in those patients. Investigation of immune biomarkers is needed, because immune dysregulation also plays an important role in dysplastic hemopoiesis and immunological escape of neoplastic clones. Here, we studied frequency of low-density granulocytes (LDGs), a neutrophil subset with immunoregulatory functions, in MDS and AML at diagnosis and during treatments. Methods. A total of 17 patients (M/F, 14/12; median age, 69 years old; range, 21-84 years) and seven healthy subjects were enrolled at the Hematology and Transplant Center, University Hospital "San Giovanni di Dio e Ruggi d'Aragona", Salerno, Italy, between October 2020 and July 2021. Patients were diagnosed with AML (N = 7), or MDS (N = 10) according to the 2016 World Health Organization criteria. For immunophenotyping, fresh EDTA whole PB was stained with the ollowing antibodies: CD45; HLA-DR; CD15; CD3; CD56; CD19; CD11b; CD33; CD34; CD14; and CD16 (all from Beckman Coulter, Brea, CA). Acquisition was carried out using a Navios EX flow cytometer, and Navios software v1.3 (Beckman Coulter). Post-acquisition compensation and analysis were performed using FlowJo software (v.10.7.1, Becton Dickinson). LDGs were identified as CD3-CD56-CD19-CD11b+CD33+CD14-CD15+ cells, following previously published gating strategies (Rahman S, et al. Ann Rheum Dis. 2019). Data were analyzed using Prism (GraphPad software, La Jolla, CA). A P < 0.05 was considered statistically significant. Results. Frequencies of circulating LDGs were significantly reduced in AML patients at diagnosis compared to controls (P = 0.0018) and MDS (P = 0.0077) and were slightly decreased compared to AML in complete remission (P = 0.1605). MDS patients were then divided based on Revised International Prognostic Scoring System (IPSS-R), and very-low and low-risk MDS patients displayed significantly higher circulating LDG frequencies compared to AML at diagnosis (P = 0.0083), while no differences were described between AML at baseline and intermediate-risk MDS (P = 0.1103). Subsequently, LDGs were correlated with clinical and phenotypic features by correlation analysis showing significant negative correlations between LDGs and blasts identified by flow cytometry (r = -0.5463; P = 0.0057) but not by cytology (P = 0.1346), between LDGs and lymphocytes (r = -0.4407; P = 0.0311) or flow cytometric normalized blast count (NBC; r = -0.5283; P = 0.0096) as previously defined (Giudice V, et al. Biomedicines. 2021). A slight negative correlation was described between LDGs and WT1 expression levels (r = -0.5369; P = 0.0719), particularly evident in MDS patients (r = -0.9980; P = 0.0402), supporting our previous findings of negative prognostic impact of WT1 expression in MDS and AML. Finally, we investigated CD16 expression on LDGs, because CD16 is essential for neutrophil degranulation. Despite no differences were described between percentage of LDG subsets among patients' groups, various correlations were identified by Pearson analysis. In particular, CD16+ LDGs negatively correlated with blasts (P = 0.0229), while positively correlated with lymphocytes (P = 0.0404) detected by flow cytometry. Conversely, CD16int and CD16- LDGs negatively correlated with lymphocytes (P = 0.0109 and P = 0.0021, respectively) and positively correlated with granulocytes identified by flow cytometry (P = 0.0024 and P = 0.0008, respectively). In addition, CD16int LDGs negatively correlated with blasts detected by flow cytometry (r = -0.65; P = 0.0414). Conclusions. Our preliminary results suggested a possible role of LDGs in prognostic definition of AML and MDS patients especially when combined with other biomarkers, such as WT1 expression levels or NBC. Moreover, our data supported the hypothesis of biological heterogeneity of granulocytes, as LDG subsets variously correlated with lymphocytes and leukemic cells suggesting different roles in suppression or activation of immune responses. However, our findings need further validation in larger cohorts and in in vitro studies. Disclosures No relevant conflicts of interest to declare.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN), an extremely rare and aggressive tumor, derives from plasmacytoid dendritic cell precursors and is characterized by CD4 and CD56 positivity accompanied by the expression of isolated myeloid, B- or T-cell lineage markers. Despite the recent introduction of specific targeted therapies, prognosis is still poor with a median overall survival of one year, and allogeneic bone marrow transplantation remains the only curative treatment in eligible patients. In this series, we described two cases of adult BPDCN treated with high dose cytarabine and methotrexate and autologous hematopoietic stem cell transplantation, or fludarabine, cytarabine, and idarubicin achieving the first a complete lasting remission, while the second only a transient improvement in skin lesions.

Treatment of acute myeloid leukemia (AML) in elderly is still challenging. Indeed, high-dose chemotherapy followed by hematopoietic stem cell transplantation with myeloablative regimens is not always feasible because patients are often unfit and have several comorbidities; however, they frequently show multiple negative prognostic factors and have a worse overall survival compared to younger adults. Venetoclax, the first-in-class Bcl-2 antagonist and first approved for treatment of chronic lymphocytic leukemia, inhibits the anti-apoptotic functions of Bcl-2 inducing apoptosis and tumor growth arrest. Venetoclax is also used in combination with azacytidine, or decitabine, or low-dose cytarabine for treatment of elderly newly diagnosed AML. However, several mechanisms of resistance have already been described, such as increased expression of other anti-apoptotic proteins by the leukemic clone. In this case series, we investigated hematopoiesis and immune cell perturbations during venetoclax plus azacytidine treatment in elderly AML patients. A total of six AML patients (M/F, 2/4; median age, 71 years old; range, 63-79 years) were retrospectively evaluated at the Hematology and Transplant Center, University Hospital "San Giovanni di Dio e Ruggi d'Aragona", Salerno, Italy. Patients received a diagnosis of AML based on the 2016 World Health Organization (WHO) criteria and chemotherapy with azacytidine 75 mg/m 2 daily for 7 days per cycle and venetoclax 70 mg/daily. Two patients were NPM1 mutated (one of them also had mutated IDH1, VAF 27.2%), while all subjects had FLT3 wild type. Based on the 2017 European LeukemiaNet risk classification, two patients had favorable risk and four intermediate. Median follow-up was 10.1 months (range, 4.9-16.6 months), and all patients were in partial or complete remission at the time of writing. Flow cytometry immunophenotype, complete blood counts (CBCs), and WT1 expression levels were performed at diagnosis and after every cycle of therapy as per our institutional guidelines. In our case series, leukemic cells were already decreased after the first cycle of therapy (blasts by flow cytometry + SD, 54.7+39.9% vs 4.2+5.4%, diagnosis vs post I cycle; P = 0.0671; paired t-test performed), while normal granulocytes detected by flow cytometry recovered only after the third cycle of therapy (20.7+23.7% vs 53+6.6%, diagnosis vs post III cycle; P = 0.1396; uncorrected Fisher's mixed model performed). Treated patients also displayed a contextual decreased in WT1 expression levels (normalized WT1 copy number + SD, 1810+2723 copies vs 201+132.9 copies, diagnosis vs post I cycle; P = 0.2660; paired t-test performed). Platelet count tended to increase after the first cycle (P = 0.0680); however, at the end of the second cycle, half of patients were again thrombocytopenic (platelets < 100 x 10 3/µL). Interestingly, percentage of lymphocytes detected by flow cytometry were significantly increased after the second cycle of azacytidine plus venetoclax compared to baseline and after the first cycle of therapy (mean+SD, 13.5+13.3% vs 48+8.7%, diagnosis vs post II cycle; P = 0.0167; and vs 28+11.3%, vs post III cycle; P = 0.0480), likely because an increase in Natural Killer (NK) cell frequency peaking after the second cycle (mean+SD, 19.4+4.4% vs 32.5+15.1%, diagnosis vs post II cycle; P = 0.1383). Moreover, five out of six patients displayed expansion of plasma cells detected by flow cytometry in the bone marrow after the first cycle: in particular, one case patient had expansion of aberrant CD45-/dimCD38++CD138++CD56+CD19- plasma cells, while one subject showed only a transient appearance of clonal plasma cells after the second cycle. No differences in bone marrow monocyte frequencies were described during treatment. Our preliminary results added evidence to efficacy and safety of the combination of venetoclax and azacytidine in treatment of elderly AML in a real-world setting. These drugs might synergistically function on hematopoiesis by inducing apoptosis of neoplastic cells while favoring differentiation of other lineages, as suggested by the expansion of plasma cells, or triggering NK-mediated immunosurveillance. However, prognostic and clinical significance of plasma cell and NK cell expansion in the setting of AML treatment needs to be further explored in larger prospective cohorts. Disclosures No relevant conflicts of interest to declare.