CAR-T from cord blood in a patient with Ph+ acute lymphoblastic leukemia relapsing after hematopoietic stem cell transplantation.

While there is intense interest in the production of allogeneic CAR-T cells from umbilical cord units, little is known about the reactivity and persistence of CAR-T cells of umbilical origin. We report the case of a patient at our hematological center with multiple relapsing Ph+ B-ALL, notably a Blinatunomab non-responder, who underwent therapy with Brexucabtagene Autoleucel following relapse on Ponatinib post-allogeneic hematopoietic stem cell transplantation. The patient achieved a rapid CAR-T expansion and durable remission presenting in good clinical conditions 6 months post-CAR-T infusion, without manifestations of graft-versus-host disease. The case report provides insight into the reactivity and persistence of CAR-T cells of umbilical origin, confirming the potential promise of allogeneic umbilical cord-derived CAR-T cells.

Role of flow-cytometric immunophenotyping in prediction of BCR/ABL1 gene rearrangement in adult B-cell acute lymphoblastic leukemia.

We performed a retrospective analysis of 88 adult patients with B-ALL diagnosed in our center by a flow-cytometric assessment. Immunophenotypic expression of leukemic cells was explored by simultaneous evaluation of positivity, percentage of expressing cells and median fluorescence intensity (MFI). BCR/ABL1 fusion transcripts were assessed by RT-PCR analysis and were identified in 36 patients (40.9%). CD10 and CD34 were positive in the totality of BCR/ABL1-positive cases. Patients with gene rearrangement had a greater frequency of CD66c, CD13 and CD33 positivity compared with BCR/ABL1-negative cases. Moreover, BCR/ABL1-positive cases exhibited a greater median percentage and MFI values of CD13, CD33, CD66c, CD10, CD34 and CD25 expressions, but a lower median percentage and MFI values of CD38 and CD22 expressions than patients without gene rearrangement. Multivariate logistic regression analysis showed that CD10, CD38 and CD13 expressions were independent predictors for the presence of BCR/ABL1 rearrangement. Predictive probabilities of molecular occurrence based on these markers are proposed. © 2017 International Clinical Cytometry Society.

Chlorambucil plus Rituximab as Front-Line Therapy in Elderly/Unfit Patients Affected by B-Cell Chronic Lymphocytic Leukemia: Results of a Single-Centre Experience.

The current standard first line therapy for fit patients with B-CLL/SLL is based on combination of fludarabine-cyclophosphamide and rituximab. However, elderly patients or patients with comorbidities poorly tolerate purine analogue-based chemotherapy and they are often treated with Chlorambucil (Chl) only. However, complete response (CR) and overall response (OR) rates with Chl are relatively low. We now investigated whether the addition of Rituximab to Chl will improve the efficacy without impairing the tolerability in elderly and unfit patients. We included in our study 27 elderly or unfit patients that had not received prior therapy. All patients were treated with Chl (1mg/Kg per 28-day cycle for 8 cycles) plus Rituximab (375 mg/m(2) for the first course and 500 mg/m(2) for subsequent cycles until the 6(th) cycle). We obtained an OR rate of 74%. The most frequent adverse effect was grade 3-4 neutropenia, which occurred in 18.5% of the patients. Infections or grade 3-4 extra-hematological side effects were not recorded. None of the patients required reduction of dose, delay of therapy or hospitalization. Overall, these data suggest that Chl-R is an effective and well tolerated regimen in elderly/unfit patients with CLL.

The role of MTHFR and RFC1 polymorphisms on toxicity and outcome of adult patients with hematological malignancies treated with high-dose methotrexate followed by leucovorin rescue.

PURPOSE: In the last years, the influence of different genes involved in metabolism of chemotherapeutic agents has been studied. Methotrexate (MTX) is a key compound of chemotherapeutic regimens used in the treatment of acute lymphoblastic leukemia (ALL), primary central nervous system lymphoma (PCNSL) and Burkitt's lymphomas (BL). This study aims to evaluate the role of MTHFR C677T and A1298C polymorphisms and G80A reduced folate carrier gene (RFC1) in a cohort of adult patients with lymphoproliferative malignancies submitted to high-dose MTX followed by leucovorin rescue. METHODS: We performed the analysis of these polymorphisms on genomic DNA with RFLP-PCR. RESULTS: Patients carrying MTHFR A1298C variant showed decreased hepatic and hematological toxicity (P = 0.03). Overall survival (OS) and progression-free survival (PFS) between homozygous wild-type and variant patients for the RFC1 G(80)A were significantly different (P = 0.035 and P = 0.02, respectively). A significant correlation between hematological toxicity and age (P = 0.003) was observed. There was no significant influence of MTHFR C677T genotype on toxicity, OS and PFS. CONCLUSIONS: Leucovorin rescue given after high-dose MTX probably accounts for the lack of influence of C677T polymorphism. To better define a role of RFC1 polymorphism on patients outcome, it would be worthwhile to perform a study on intracellular MTX level and RFC1 substrate binding affinities in different genotypes.

Comparison between oral and intravenous fludarabine plus cyclophosphamide regime as front-line therapy in patients affected by chronic lymphocytic leukaemia: influence of biological parameters on the clinical outcome.

The fludarabine plus cyclophosphamide (FC) regimen was reported to be superior to chlorambucil or fludarabine alone in terms of complete response (CR), overall response (OR) and progression-free survival (PFS) in previously untreated patients with chronic lymphocytic leukaemia (CLL). In the present study, we compared the efficacy and toxicity of FC administered through oral and intravenous route in 65 untreated patients affected by advanced CLL. No statistical differences were noticed between the two routes of administration in terms of OR, PFS, time to re-treatment (TTR) and overall survival (OS) of analysed patients. We also assessed the influence on the clinical outcome of the mutation status of the immunoglobulin variable region heavy chain (IgVH) gene, of the cytogenetic abnormalities and of the expression of ZAP70 and CD38 in patients' primary samples. Among the 58 evaluable patients, 31 (53%) achieved a CR and 18 (31%) a partial response. The median PFS was 35 months, median TTR was 42 months and median OS was not reached after 45 months (range, 1-161). A significantly lower OR rate was noticed in patients with high-risk cytogenetic abnormalities (del 17p, del 11q). In this study, high-risk cytogenetic abnormalities and unmutated IgVH genes were independent predictors of TTR. These results underline the importance of biological stratifications in front-line treatment of CLL patients. We confirm that FC is an effective regimen with mild toxicities; it could be recommended for patients with low-risk biological parameters who represent, in our experience, about 30% of the total.

The proliferative response to CpG-ODN stimulation predicts PFS, TTT and OS in patients with chronic lymphocytic leukemia.

We recently reported that leukemic cells from IgVH-unmutated/progressive CLL more frequently proliferate in response to CpG-ODN stimulation than their corresponding counterparts. Here we evaluated the prognostic impact of this proliferative response in 91 CLL patients. The proliferative response was highly predictive of PFS, TTT and OS in the whole series and refined prognosis in patients with M-CLL. BCR stimulation modulated the response to CpG-ODN, suggesting that the proliferative capacity of the leukemic cells is related to antigen-encounter history. These data support the hypothesis that the capacity of the leukemic cells to respond to external stimuli influences disease progression in CLL.

MTHFR polymorphisms involved in vitamin B12 deficiency associated with atrophic gastritis.

Genetic polymorphisms affecting methylentetrahydrofolate reductase (MTHFR) activity may influence hematological and neurological dysfunction in cobalamin-deficient patients. We studied the prevalence of C677T and A1298C polymorphisms by analyzing genomic DNA in 30 cobalamin-deficient patients. No significant difference was found in 677 and 1298 genotype distribution with respect to hematological parameters, B12 and folate levels, and neurological symptoms. The two MTHFR polymorphisms were not protective against anemia or neurological dysfunction in patients with cobalamin deficiency; however, we found evidence of a significant increase in atrophic gastritis in the 677TT group (P = 0.009) but not for the 1298CC genotype. Based on observations that inadequate cobalamin intake and reduced MTHFR activity might be significant risk factors for gastric cancer, and the increased risk of gastric cancer shown in patients affected by atrophic gastritis, we speculate that concomitant atrophic gastritis and impaired MTHFR function could have a role in the development of gastric cancer.

Comparison of ZAP-70/Syk mRNA levels with immunoglobulin heavy-chain gene mutation status and disease progression in chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: The protein tyrosine kinase ZAP-70 has recently emerged as a major prognostic indicator in chronic lymphocytic leukemia (CLL). ZAP-70 is structurally and functionally homologous to Syk, a key mediator of B-cell receptor signaling. We therefore evaluated ZAP-70 expression in CLL B cells using Syk as an intracellular standard. DESIGN AND METHODS: The relative amounts of ZAP-70 and Syk were determined in purified B cells from 92 CLL patients using a novel reverse transcriptase/polymerase chain reaction (RT-PCR) procedure that co-amplifies both transcripts with equal efficiency. The ZAP-70/Syk mRNA ratio was correlated with VH gene mutation status, median treatment-free survival and FACS analysis of ZAP-70 expression. RESULTS: ZAP-70 was expressed in the majority of cases with unmutated VH genes (88%), but also at lower levels in a substantial fraction of cases with mutated VH genes (44%). High levels of ZAP-70, defined as ZAP-70/Syk mRNA ratios above 0.25, were observed mainly in cases with unmutated VH genes and correlated with short treatment-free survival. In contrast, no difference was observed in the median treatment-free survival between patients with low ZAP-70/Syk ratios (0.05-0.25) and patients with no or negligible ZAP-70 expression (ZAP-70/Syk<0.05). In 73 cases ZAP-70 expression was investigated by RT/PCR and FACS analysis; concordance with VH gene mutation status was 86% and 71%, respectively. INTERPRETATION AND CONCLUSIONS: ZAP-70 is frequently expressed in CLL B cells, but only high levels correlate with unmutated VH gene status and progressive disease. Expression of ZAP-70 can be accurately assessed by analysis of the ZAP-70/Syk mRNA ratio, thus providing an alternative to FACS analysis.

Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells.

The clinical course of chronic lymphocytic leukemia (CLL) differs significantly between patients with mutated (M-CLL) and unmutated (U-CLL) immunoglobulin (Ig) variable heavy-chain (V(H)) genes, implying a role for B-cell receptor (BCR) signaling in the pathogenesis of this disease. We have now investigated activation of downstream BCR signaling pathways in U-CLL and M-CLL B cells using soluble anti-IgM (sol-IgM) and immobilized anti-IgM (imm-IgM) antibodies as models for antigenic stimulation. Ligation of the BCR with sol-IgM induced incomplete responses in both CLL subsets, resembling the pattern described for tolerant B cells. This response was characterized by transient phosphorylation of extracellular signal-related kinase (ERK) and Akt (protein kinase B [PKB]), lack of activation of c-JUN NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and variable activation of phospholipase Cgamma2 (PLCgamma2) and nuclear factor-kappaB (NF-kappaB). Stimulation with imm-IgM elicited a more complete BCR signal and significantly prolonged phosphorylation of ERK and Akt, indicating persistent or repetitive BCR signaling. Moreover, this type of stimulation increased the levels of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) and protected from chemotherapy-induced apoptosis, whereas induction of apoptosis and down-regulation of Mcl-1 was observed following stimulation with sol-IgM. These data demonstrate that only sustained BCR signaling can promote survival of CLL B cells and indicate that the main difference between CLL with mutated and unmutated V(H) genes may reside in the availability of such stimulation.