Single step multiple genotyping by MALDI-TOF mass spectrometry, for evaluation of minor histocompatibility antigens in patients submitted to allogeneic stem cell transplantation from HLA-matched related and unrelated donor

The outcome of patients underwent to allogeneic stem cell transplantation (allo- SCT) is closely related to graft versus host disease (GvHD) and graft versus leukemia (GvL) effects which can be mediated by mHAgs. 23 mHAgs have been identified and reported to be differently correlated with GVHD or GVL and the aim of this work was develop a method to genotype the mHAgs described so far. For this study we used MALDI-TOF iPLEX Gold Mass Array technology. We tested 46 donor/recipient matched pairs that underwent allo-SCT because of Philadelphia positive (Ph+) chronic myeloid leukemia (n=29) or Ph+ acute lymphoblastic leukemia (n=17). Our data show that sibling pairs had a lesser number of mHAgs mismatches compared to MUD pairs. Notably, donor/recipient genomic mismatch on DPH1 was correlated with an increased risk of acute GvHD and LB-ADIR-1R mismatch on graft versus host direction was correlated with a better RFS with no increase of GvHD risk. Our work provides a simple, accurate and highly automatable method for mHAgs genotyping and suggest the role of mHAgs in addressing the immune reaction between donor and host

Predictive value of pretransplantation molecular minimal residual disease assessment by WT1 gene expression in FLT3-positive acute myeloid leukemia

The FMS-like tyrosine kinase 3 (FLT3) mutation in acute myeloid leukemia (AML) is a negative prognostic factor and, in these cases, allogeneic stem cell transplantation (allo-SCT) can represent an important therapeutic option, especially if performed in complete remission (CR). However, it is increasingly clear that not all cytological CRs (cCRs) are the same and that minimal residual disease (MRD) before allo-SCT could have an impact on AML outcome. Unfortunately, FLT3, due its instability of expression, is still not considered a good molecular MRD marker. We analyzed the outcome of allo-SCT in a population of FLT3-positive AML patients according to molecular MRD at the pretransplantation workup, assessed by the quantitative expression evaluation of the panleukemic marker Wilms\u2019 tumor (WT1) gene. Sixty-two consecutive AML FLT3-positive patients received allo-SCT between 2005 and 2016 in our center. The median age at transplantation was 55 years. The quantitative analysis of the WT1 gene expression (bone marrow samples) was available in 54 out of 62 (87%) cases, both at diagnosis (100% overexpressing WT1 with a mean of 9747 \ub1 8064 copies) and before allo-SCT (33 WT1-negative and 21 WT1-positive cases at the pretransplantation workup). Of these cases, 33/54 (61%) were both in cCR and molecular remission (WT1-negative) at the time of transplantation, 13/54 (24%) were in cCR but not in molecular remission (WT1-positive), and 8/54 (15%) showed a cytological evidence of disease (relapsed or refractory). Both post-allo-SCT overall survival (OS) and disease-free survival (DFS) were significantly better in patients who were WT1-negative (WT1 250 copies), with a median OS and DFS not reached in the WT1-negative group and 10.2 and 5.5 months, respectively, in the WT1-positive group (OS log\u2013rank p = 0.0005; hazard ratio [HR] = 3.7, 95% confidence interval [95% CI] = 1.5\u20139; DFS log\u2013rank p = 0.0001; HR = 4.38, 95% CI = 1.9\u201310). Patients with cCR who were WT1-positive had the same negative outcome as those with a cytological evidence of disease. The relapse rate after allo-SCT was 9% (3/33) in pre-allo-SCT WT1-negative cases and 54% (7/13) in WT1-positive cases (p = 0.002). At multivariate analysis, WT1 negativity before allo-SCT and grade <2 acute graft versus host disease were the only independent prognostic factors for improved OS and DFS. These data show that pre-allo-SCT molecular MRD evaluation through WT1 expression is a powerful predictor of posttransplantation outcomes (OS, DFS, relapse rate). Patients with both cCR and a WT1-negative marker before allo-SCT have a very good outcome with very low relapse rate; conversely, patients with positive molecular MRD and refractory/relapsed patients have a negative outcome. The WT1 MRD stratification in FLT3-positive AML is a valuable tool with which to identify patients who are at high risk of relapse and that could be considered from post-allo-SCT prophylaxis with FLT3 inhibitors or other strategies (donor lymphocyte infusion, tapering of immunosuppression, azacitidine). \ua9 2017 ISEH - International Society for Experimental Hematolog

Digital PCR improves the quantitation of DMR and the selection of CML candidates to TKIs discontinuation

Treatment-free remission (TFR) by tyrosine kinase inhibitors (TKI) discontinuation in patients with deep molecular response (DMR) is a paramount goal in the current chronic myeloid leukemia (CML) therapeutic strategy. The best DMR level by real-time quantitative PCR (RT-qPCR) for TKI discontinuation is still a matter of debate. To compare the accuracy of digital PCR (dPCR) and RT-qPCR for BCR-ABL1 transcript levels detection, 142 CML patients were monitored for a median time of 24\ua0months. Digital PCR detected BCR-ABL1 transcripts in the RT-qPCR undetectable cases. The dPCR analysis of the samples, grouped by the MR classes, revealed a significant difference between MR4.0 and MR4.5 (P\ua0=\ua00.0104) or MR5.0 (P\ua0=\ua00.0032). The clinical and hematological characteristics of the patients grouped according to DMR classes (MR4.0 vs MR4.5-5.0 ) were superimposable. Conversely, patients with dPCR values <0.468 BCR-ABL1 copies/\ub5L (as we previously described) showed a longer DMR duration (P\ua0=\ua00.0220) and mainly belonged to MR4.5-5.0 (P\ua0=\ua00.0442) classes compared to patients with higher dPCR values. Among the 142 patients, 111 (78%) discontinued the TKI treatment; among the 111 patients, 24 (22%) lost the MR3.0 or MR4.0 . RT-qPCR was not able to discriminate patients with higher risk of MR loss after discontinuation (P\ua0=\ua00.8100). On the contrary, according to dPCR, 12/25 (48%) patients with BCR-ABL1 values 650.468 and 12/86 (14%) patients with BCR-ABL1 values <0.468 lost DMR in this cohort, respectively (P\ua0=\ua00.0003). Treatment-free remission of patients who discontinued TKI with a dPCR <0.468 was significantly higher compared to patients with dPCR\ua0 65\ua00.468 (TFR at 2\ua0years 83% vs 52% P\ua0=\ua00.0017, respectively). In conclusion, dPCR resulted in an improved recognition of stable DMR and of candidates to TKI discontinuation

Clinical presentation, outcome and risk factors of late-onset non-infectious pulmonary complication after allogeneic transplantation

The term late-onset non-infectious pulmonary complications (LONIPCs) has been used to refer to events occurring later than 3 months after allogeneic hematopoietic stem transplant (HSCT), such as bronchiolitis obliterans, bronchiolitis obliterans with organizing pneumonia, and lymphocytic or idiopathic interstitial pneumonia. The incidence of LONIPCs varies widely, ranging between 10% and 26%. Median time for LONIPC development is about 8-12 months after HSCT. Clinical symptoms may be insidious and non specific at the beginning and can be present in different types of infections. The diagnosis is made on the basis of thoracic high-resolution computed tomography and pulmonary function tests (PFT). It usually requires that standard cultures for infective agents on bronchoalveolar lavage are negative and is confirmed by transbronchial or lung biopsy, whenever possible. Total body irradiation and high doses of drugs used in the conditioning regimens , HLA disparity between donor and recipient, and chronic graft-versus-host disease (GVHD) are the main risk factors for LONIPCs. Since patients with LONIPCs have an increased risk of mortality because of infections or respiratory failure, pre- and post-transplant PFTs are strongly recommended in order to timely identify affected patients. The administration of antithymocyte globulin before unrelated donor transplants and slow taper of cyclosporine after transplant have been shown to prevent chronic GVHD and, therefore, the occurrence of LONIPC

T-cell chimerism and clinical outcome after reduced-intensity conditioning allogeneic stem cell transplantation

9nonenoneTOFFOLETTI, Eleonora; PATRIARCA, Francesca; Chiarvesio, A.; Michelutti, A.; Sperotto, A.; Cavallin, M.; Battista, M. L.; ZAJA, Francesco; FANIN, RenatoToffoletti, Eleonora; Patriarca, Francesca; Chiarvesio, A.; Michelutti, A.; Sperotto, A.; Cavallin, M.; Battista, M. L.; Zaja, Francesco; Fanin, Renat

Targeting kinase-activating genetic lesions to improve therapy of pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children, characterized by an abnormal proliferation of immature lymphoid cells. Thanks to risk-adapted combination chemotherapy treatments currently used, survival at 5 years has reached 90%. ALL is a heterogeneous disease from a genetic point of view: patients' lymphoblasts may harbor in fact several chromosomal alterations, some of which have prognostic and therapeutic value. Of particular importance is the translocation t(9;22)(q34;q11.2) that leads to the formation of the BCR-ABL1 fusion gene, encoding a constitutively active chimeric tyrosine kinase (TK): t(9;22)(q34;q11.2) that is present in ~3% of pediatric ALL patients with B-immunophenotype and is associated with a poor outcome. This type of ALL is potentially treatable with specific TK inhibitors, such as imatinib. Recent studies have demonstrated the existence of a subset of BCR-ABL1 like leukemias (~10-15% of B-immunophenotype ALL), whose blast cells have a gene expression profile similar to that of BCR-ABL1 despite the absence of t(9;22)(q34;q11.2). The precise pathogenesis of BCR-ABL1 like is still to be defined, but they are mainly characterized by the activation of constitutive signal transduction pathways due to chimeric TKs different from BCR-ABL1. BCR-ABL1 like ALL patients represent a group with unfavorable outcome and are not identified by current risk criteria. In this review, we will discuss the design of targeted therapy for patients with BCR-ABL1 like ALL, which could consider TK inhibitors, and discuss innovative approaches suitable to identify the presence of patient's specific chimeric TK fusion genes, such as targeted locus amplification or proteomic biosensors.Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children, characterized by an abnormal proliferation of immature lymphoid cells. Thanks to risk-adapted combination chemotherapy treatments currently used, survival at 5 years has reached 90%. ALL is a heterogeneous disease from a genetic point of view: patients' lymphoblasts may harbor in fact several chromosomal alterations, some of which have prognostic and therapeutic value. Of particular importance is the translocation t(9;22)(q34;q11.2) that leads to the formation of the BCR-ABL1 fusion gene, encoding a constitutively active chimeric tyrosine kinase (TK): t(9;22)(q34;q11.2) that is present in ~3% of pediatric ALL patients with B-immunophenotype and is associated with a poor outcome. This type of ALL is potentially treatable with specific TK inhibitors, such as imatinib. Recent studies have demonstrated the existence of a subset of BCR-ABL1 like leukemias (~10-15% of B-immunophenotype ALL), whose blast cells have a gene expression profile similar to that of BCR-ABL1 despite the absence of t(9;22)(q34;q11.2). The precise pathogenesis of BCR-ABL1 like is still to be defined, but they are mainly characterized by the activation of constitutive signal transduction pathways due to chimeric TKs different from BCR-ABL1. BCR-ABL1 like ALL patients represent a group with unfavorable outcome and are not identified by current risk criteria. In this review, we will discuss the design of targeted therapy for patients with BCR-ABL1 like ALL, which could consider TK inhibitors, and discuss innovative approaches suitable to identify the presence of patient's specific chimeric TK fusion genes, such as targeted locus amplification or proteomic biosensors

Targeting kinase-activating genetic lesions to improve therapy of pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children, characterized by an abnormal proliferation of immature lymphoid cells. Thanks to risk-adapted combination chemotherapy treatments currently used, survival at 5 years has reached 90%. ALL is a heterogeneous disease from a genetic point of view: patients' lymphoblasts may harbor in fact several chromosomal alterations, some of which have prognostic and therapeutic value. Of particular importance is the translocation t(9;22)(q34;q11.2) that leads to the formation of the BCR-ABL1 fusion gene, encoding a constitutively active chimeric tyrosine kinase (TK): BCR-ABL1 that is present in ~3% of pediatric ALL patients with B-immunophenotype and is associated with a poor outcome. This type of ALL is potentially treatable with specific TK inhibitors, such as imatinib. Recent studies have demonstrated the existence of a subset of BCR-ABL1 like leukemias (~10-15% of Bimmunophenotype ALL), whose blast cells have a gene expression profile similar to that of BCR-ABL1 despite the absence of t(9;22)(q34;q11.2). The precise pathogenesis of BCR-ABL1 like ALL is still to be defined, but they are mainly characterized by the activation of constitutive signal transduction pathways due to chimeric TKs different from BCR-ABL1. BCR-ABL1 like ALL patients represent a group with unfavorable outcome and are not identified by current risk criteria. In this review, we will discuss the design of targeted therapy for patients with BCR-ABL1 like ALL, which could consider TK inhibitors, and discuss innovative approaches suitable to identify the presence of patient's specific chimeric TK fusion genes, such as targeted locus amplification or proteomic biosensors

Clinical impact of CD200 expression in patients with acute myeloid leukemia and correlation with other molecular prognostic factors

CD200, a protein belonging to the immunoglobulin superfamily, has been associated with a poor prognosis in lymphoproliferative disorders and in acute leukemia. We studied the expression of CD200 in a series of 244 patients with diagnosis of acute myeloid leukemia (AML), to evaluate its impact on outcome and its possible association with other known prognostic factors. CD200 was found in 136/244 (56%) patients, in 41 of whom (30%) with high intensity of expression (MFI ≥ 11). CD200 was more frequent in secondary compared to de novo leukemia (p = 0.0006), in CD34 positive cases (p = 0.00001), in Bcl2 overexpressing cases (p = 0.01), in those wild-type Flt3 (p = 0.004) and with favorable or unfavorable compared to intermediate karyotype (p = 0.0003). CD200+ patients have a two-fold lower probability to attain complete remission, both in univariate (p = 0.006) and multivariate (p = 0.04) analysis. The negative impact of CD200 was found also in overall survival (p = 0.02) and was correlated with the intensity of expression of the molecule (p = 0.024). CD200 has an additive negative impact on survival in patients with unfavorable cytogenetic (p = 0.046) and in secondary leukemia (p = 0.05), and is associate with a worsening of outcome in patients with favorable biological markers, such as mutated NPM (p = 0.02), wild-type Flt3 (p = 0.034), negativity of CD34 (p = 0.03) and of CD56 (p = 0.03). In conclusion, CD200 is emerging as both a prognostic factor and a potential target of novel therapeutic approaches for AML, aiming to reverse the “do not eat me” signal of CD200 or to manipulate the suppressive immune microenvironment induced by CD200 binding to its receptor