Clinical and biological impact of ATP-binding cassette transporter activity in adult acute myeloid leukemia.

Chemotherapy resistance is the main cause of treatment failure in acute myeloid leukemia (AML) and has been related to ATP-binding cassette (ABC) transporter activity. However, the links between ABC activity, immunophenotype, and molecular AML parameters have been poorly evaluated. Moreover, the prognostic value of ABC activity, when compared to new molecular markers, is unknown. Here we investigated the links between ABC activity, as evaluated by JC-1 +/- cyclosporine A assay, and immunophenotypic, cytogenetic, molecular, and targeted next-generation sequencing features in 361 AML patients. High ABC activity was found in 164 patients and was significantly associated with less proliferating disease, an immature immunophenotype (expression of CD34, HLA-DR, CD117, CD13), and gene mutations defining AML as belonging to secondary-type ontogenic groups. Low ABC activity was associated with more mature myeloid differentiation (CD34-, cyMPO+, CD15+, CD33+) or monocytic commitment (CD64+, CD4+weak, CD14+), with NPM1 mutations, KMT2A rearrangements, and core-binding factor gene fusions, hallmarks of the de novo-type AML ontogeny. ABC activity was one of the major factors we identified using a random forest model for early prediction of AML ontogeny. In the 230 patients evaluated at diagnosis and intensively treated, high ABC activity was a predictive factor for primary resistance, and in multivariate analysis including full molecular data, an independent factor for event-free survival (P=0.0370). JC-1 +/- cyclosporine A assay could be used at diagnosis to predict AML ontogeny and to complete prognosis evaluation in addition to new molecular markers.

Myeloid malignancies with translocation t(4;12)(q11-13;p13): molecular landscape, clonal hierarchy and clinical outcomes.

Translocation t(4;12)(q11-13;p13) is a recurrent but very rare chromosomal aberration in acute myeloid leukaemia (AML) resulting in the non-constant expression of a CHIC2/ETV6 fusion transcript. We report clinico-biological features, molecular characteristics and outcomes of 21 cases of t(4;12) including 19 AML and two myelodysplastic syndromes (MDS). Median age at the time of t(4;12) was 78 years (range, 56-88). Multilineage dysplasia was described in 10 of 19 (53%) AML cases and CD7 and/or CD56 expression in 90%. FISH analyses identified ETV6 and CHIC2 region rearrangements in respectively 18 of 18 and 15 of 17 studied cases. The t(4;12) was the sole cytogenetic abnormality in 48% of cases. The most frequent associated mutated genes were ASXL1 (n = 8/16, 50%), IDH1/2 (n = 7/16, 44%), SRSF2 (n = 5/16, 31%) and RUNX1 (n = 4/16, 25%). Interestingly, concurrent FISH and molecular analyses showed that t(4;12) can be, but not always, a founding oncogenic event. Median OS was 7.8 months for the entire cohort. In the 16 of 21 patients (76%) who received antitumoral treatment, overall response and first complete remission rates were 37% and 31%, respectively. Median progression-free survival in responders was 13.7 months. Finally, t(4;12) cases harboured many characteristics of AML with myelodysplasia-related changes (multilineage dysplasia, MDS-related cytogenetic abnormalities, frequent ASXL1 mutations) and a poor prognosis.

Multilayer intraclonal heterogeneity in chronic myelomonocytic leukemia.

The functional diversity of cells that compose myeloid malignancies, i.e., the respective roles of genetic and epigenetic heterogeneity in this diversity, remains poorly understood. This question is addressed in chronic myelomonocytic leukemia, a myeloid neoplasm in which clinical diversity contrasts with limited genetic heterogeneity. To generate induced pluripotent stem cell clones, we reprogrammed CD34+ cells collected from a patient with a chronic myelomonocytic leukemia in which whole exome sequencing of peripheral blood monocyte DNA had identified 12 gene mutations, including a mutation in KDM6A and two heterozygous mutations in TET2 in the founding clone and a secondary KRAS(G12D) mutation. CD34+ cells from an age-matched healthy donor were also reprogrammed. We captured a part of the genetic heterogeneity observed in the patient, i.e. we analyzed five clones with two genetic backgrounds, without and with the KRAS(G12D) mutation. Hematopoietic differentiation of these clones recapitulated the main features of the patient's disease, including overproduction of granulomonocytes and dysmegakaryopoiesis. These analyses also disclosed significant discrepancies in the behavior of hematopoietic cells derived from induced pluripotent stem cell clones with similar genetic background, correlating with limited epigenetic changes. These analyses suggest that, beyond the coding mutations, several levels of intraclonal heterogeneity may participate in the yet unexplained clinical heterogeneity of the disease.

Impact of cytogenetic abnormalities in adults with Ph-negative B-cell precursor acute lymphoblastic leukemia.

Multiple cytogenetic subgroups have been described in adult Philadelphia chromosome (Ph)-negative B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), often comprising small numbers of patients. In this study, we aimed to reassess the prognostic value of cytogenetic abnormalities in a large series of 617 adult patients with Ph-negative BCP-ALL (median age, 38 years), treated in the intensified Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/2005 trials. Combined data from karyotype, DNA index, fluorescence in situ hybridization, and polymerase chain reaction screening for relevant abnormalities were centrally reviewed and were informative in 542 cases (88%), allowing classification in 10 exclusive primary cytogenetic subgroups and in secondary subgroups, including complex and monosomal karyotypes. Prognostic analyses focused on cumulative incidence of failure (including primary refractoriness and relapse), event-free survival, and overall survival. Only 2 subgroups, namely t(4;11)/KMT2A-AFF1 and 14q32/IGH translocations, displayed a significantly worse outcome in this context, still observed after adjustment for age and after censoring patients who received allogeneic stem cell transplantation (SCT) in first remission at SCT time. A worse outcome was also observed in patients with low hypodiploidy/near triploidy, but this was likely related to their higher age and worse tolerance to therapy. The other cytogenetic abnormalities, including complex and monosomal karyotypes, had no prognostic value in these intensive protocols designed for adult patients up to the age of 60 years.

Precision and prognostic value of clone-specific minimal residual disease in acute myeloid leukemia.

The genetic landscape of adult acute myeloid leukemias (AML) has been recently unraveled. However, due to their genetic heterogeneity, only a handful of markers are currently used for the evaluation of minimal residual disease (MRD). Recent studies using multi-target strategies indicate that detection of residual mutations in less than 5% of cells in complete remission is associated with a better survival. Here, in a series of 69 AMLs with known clonal architecture, we design a clone-specific strategy based on fluorescent in situ hybridization and high-sensitivity next generation sequencing to detect chromosomal aberrations and mutations, respectively, in follow-up samples. The combination of these techniques allows tracking chromosomal and genomic lesions down to 0.5-0.4% of the cell population in remission samples. By testing all lesions in follow-up samples from 65 of 69 evaluable patients, we find that initiating events often persist and appear to be, on their own, inappropriate markers to predict short-term relapse. In contrast, the persistence of two or more lesions in more than 0.4% of the cells from remission samples is strongly associated with lower leukemia-free and overall survivals in univariate and multivariate analyses. Although larger prospective studies are needed to extend these results, our data show that a personalized, clone-specific, MRD follow up strategy is feasible in the vast majority of AML cases.

Characteristics and outcome of early-onset, severe forms of Wiskott-Aldrich syndrome.

On the basis of a nationwide database of 160 patients with Wiskott-Aldrich syndrome (WAS), we identified a subset of infants who were significantly more likely to be attributed with an Ochs score of 5 before the age of 2 (n = 26 of 47 [55%], P = 2.8 × 10(−7)). A retrospective analysis revealed that these patients often had severe refractory thrombocytopenia (n = 13), autoimmune hemolytic anemia (n = 15), and vasculitis (n = 6). One patient had developed 2 distinct cancers. Hemizygous mutations predictive of the absence of WAS protein were identified in 19 of the 24 tested patients, and the absence of WAS protein was confirmed in all 10 investigated cases. Allogeneic hematopoietic stem cell transplantation (HSCT) was found to be a curative treatment with a relatively good prognosis because it was successful in 17 of 22 patients. Nevertheless, 3 patients experienced significant disease sequelae and 4 patients died before HSCT. Therefore, the present study identifies a distinct subgroup of WAS patients with early-onset, life-threatening manifestations. We suggest that HSCT is a curative strategy in this subgroup of patients and should be performed as early in life as possible, even when a fully matched donor is lacking.

Chromosomal aberrations and their prognostic value in a series of 174 untreated patients with Waldenström's macroglobulinemia.

Waldenström's macroglobulinemia is a disease of mature B cells, the genetic basis of which is poorly understood. Few recurrent chromosomal abnormalities have been reported, and their prognostic value is not known. We conducted a prospective cytogenetic study of Waldenström's macroglobulinemia and examined the prognostic value of chromosomal aberrations in an international randomized trial. The main aberrations were 6q deletions (30%), trisomy 18 (15%), 13q deletions (13%), 17p (TP53) deletions (8%), trisomy 4 (8%), and 11q (ATM) deletions (7%). There was a significant association between trisomy of chromosome 4 and trisomy of chromosome 18. Translocations involving the IGH genes were rare (<5%). Deletion of 6q and 11q, and trisomy 4, were significantly associated with adverse clinical and biological parameters. Patients with TP53 deletion had short progression-free survival and short disease-free survival. Although rare (<5%), trisomy 12 was associated with short progression-free survival. In conclusion, the cytogenetic profile of Waldenström's macroglobulinemia appears to differ from that of other B-cell lymphomas. Chromosomal abnormalities may help with diagnosis and prognostication, in conjunction with other clinical and biological characteristics.

Clinical profile and genetic basis of Wiskott-Aldrich syndrome at Chandigarh, North India.

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency disorder characterized by thrombocytopenia with small sized platelets, eczema, and recurrent infections. There is paucity of information on WAS from the Indian subcontinent. We describe the clinical and molecular profile of 8 patients with WAS as seen in the Pediatric Immunodeficiency Clinic at the Advanced Pediatrics Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, India. METHODS: A detailed analysis of the clinical profiles, investigations and outcome of the 8 children diagnosed with WAS during the period 2006- 2010 was performed. Confirmation of the genetic diagnosis was done at the Service d'Hématologie, d'Immunologie et de Cytogénétique, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France and the National Defense Medical College, Saitama, Japan. RESULTS: 8 patients were diagnosed as WAS in 5 years. The ages at diagnosis ranged from 13 weeks to 9 years while the mean age of onset of the symptoms was 117 days +/- 136 days. The diagnosis was established within a mean period of 31 months (ranging 1-108 months) from the onset of symptoms. Recurrent infections and diarrhea were seen in 6 and 7 out of the 8 patients, respectively, while eczema was variable. Autoimmunity manifestations were observed in 2 children. Thrombocytopenia and small platelet size was the hallmark of the disease and the main clinical clue to diagnosis in our patients. Mutations in the WASP gene were seen in 8 children, out of which 2 were novel mutations. While one child successfully underwent bone marrow transplantation, two children are doing well on immunoglobulin replacement and cotrimoxazole prophylaxis. Out of 8 children 4 children in our cohort died--all had high WAS scores and could not be offered hematopoietic stem cell transplantation. CONCLUSION: WAS should be suspected clinically in any male infant with persistent unexplained thrombocytopenia and especially if the platelet size is small. Clinical presentation can be very variable and it is therefore important to recognize the entire spectrum of the disease. Understanding the molecular basis has important implications for the diagnosis, treatment, and genetic counseling of patients with WAS.

Diagnosis of Burkitt lymphoma using an algorithmic approach--applicable in both resource-poor and resource-rich countries.

Distinguishing Burkitt lymphoma (BL) from B cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma (DLBCL) and BL (DLBCL/BL), and DLBCL is challenging. We propose an immunohistochemistry and fluorescent in situ hybridization (FISH) based scoring system that is employed in three phases - Phase 1 (morphology with CD10 and BCL2 immunostains), Phase 2 (CD38, CD44 and Ki-67 immunostains) and Phase 3 (FISH on paraffin sections for MYC, BCL2, BCL6 and immunoglobulin family genes). The system was evaluated on 252 aggressive B-cell lymphomas from Europe and from sub-Saharan Africa. Using the algorithm, we determined a specific diagnosis of BL or not-BL in 82%, 92% and 95% cases at Phases 1, 2 and 3, respectively. In 3·4% cases, the algorithm was not completely applicable due to technical reasons. Overall, this approach led to a specific diagnosis of BL in 122 cases and to a specific diagnosis of either DLBCL or DLBCL/BL in 94% of cases that were not diagnosed as BL. We also evaluated the scoring system on 27 cases of BL confirmed on gene expression/microRNA expression profiling. Phase 1 of our scoring system led to a diagnosis of BL in 100% of these cases.

The c.273+11dup genetic change in the WAS gene is a functionally neutral polymorphism.

Several pediatric patients showing symptoms consistent with the Wiskott-Aldrich syndrome (WAS) were referred to us and turned out to display the c.273+11dup change in the WAS gene. It consisted of the insertion of one C in an unusual tract of 7C near the intron 2 donor splicing site of the WAS gene. In the patients, non-synonymous WAS mutations were found twice only and one mutation was elucidated in RUNX1. In the absence of a non-synonymous mutation in the WAS gene, the c.273+11dup change affected neither the levels nor the sequence of WAS mRNA. In the presence of a non-synonymous WAS mutation, the c.273+11dup alteration failed to worsen the expected phenotype. Minor splicing abnormalities concerning exon 10 were observed both in WAS patients, and in healthy individuals carrying or not carrying the c.273+11dup. The c.273+11dup change was encountered four times in 107 normal male and female controls (172 alleles tested: 2.3%), and eight times in a series of 248 male patients (248 alleles tested: 3.2%). We conclude that the presence of the additional C in the WAS gene is a functionally neutral polymorphism.

Chromosomal abnormalities in transformed Ph-negative myeloproliferative neoplasms are associated to the transformation subtype and independent of JAK2 and the TET2 mutations.

Evolution to myelofibrosis (MF), acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) may occur over time in myeloproliferative neoplasms (MPN) patients most likely due to the acquisition of additional mutations. The Groupe Francophone de cytogenetique hematologique (GFCH) has collected and reviewed 82 patients with transformation of MPN (66 AML/MDS and 16 MF). JAK2V617F and TET2 mutations were searched for in 40 and 32 patients, respectively. Significantly more -7/del(7q) (P = 0.004) and -5/del(5q) (P = 0.03) were found in AML/MDS with a higher incidence of dup1q (P = 0.01) in MF. Some specific chromosomal abnormalities occurred together, for example -5/del(5q) and -17/del(17p) (P = 0.0007). In multivariate analysis, two factors were independently associated with an inferior overall survival (OS); AML/MDS transformation (P < 0.0001) and -5/del(5q) abnormality (P = 0.02). Although both giving rise to loss of 7q, der(1;7) differed from other 7q deletions in terms of distribution (lower frequency of AML/MDS, P = 0.02), association with chromosomal abnormalities (absence of -5/del(5q), P = 0.003; increased del(20q), P = 0.05), and longer OS (P = 0.0007). We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in samples following transformation, ranging from wild-type to mutated forms of both genes. The mutated and wild-type forms of the genes were not found to be associated with a specific chromosomal abnormality. There was no evidence that JAK2 or TET2 mutations were associated with the type of MPN transformation, whereas the type of cytogenetic abnormalities were strongly linked, perhaps indicating that they play a specific role in the transformation process.

[Elderly patients with myelodysplastic syndrome with del 5q receiving lenalidomide: two case reports with poor prognosis]. / Patients âgés atteints de syndrome myélodysplasique avec anomalie 5q- traités par lénalidomide : à propos de deux observations avec évolution défavorable.

We report two cases of myelodysplastic syndrome (MDS) with del(5q) isolated cytogenetic abnormality in elderly patients: AREB-1 in Patient 1, "5q syndrome" in Patient 2. A first line of treatment including hematopoietic growth factors (darbepoetin alone or associated with G-CSF) failed after several months and a treatment with lenalidomide was initiated in both cases. The treatment was poorly tolerated (myelosuppression) in Patient 1 without an improvement of the quality of life; a progression of the disease was observed with an increase of the bone marrow blastosis and a new acquired karyotypic abnormality (t13;17), leading to the prescription of 5-azacytidine. Patient 2 had a good response to lenalidomide for several months with an increase in the haemoglobin level and a transfusion independence. The patient's prognosis suddenly worsened with the occurrence of a blood blastosis (10%): 5-azacytidine was thus initiated. These two case reports illustrate the recent advances in the management of patients with MDS with del(5q), including the use of lenalidomide.

Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene-Driven Myeloid Leukemia.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631.

Synchronous FIP1L1-PDGFRA-positive chronic eosinophilic leukemia and T-cell lymphoblastic lymphoma: a bilineal clonal malignancy.

Several reports of successful empirical treatment of idiopathic hypereosinophilic syndrome with imatinib led to the recent identification of the FIP1L1-PDGFRA fusion gene rearrangement, which characterizes a distinctive group of chronic eosinophilic leukemias. This fusion gene can be detected in eosinophils, neutrophils, mast cells, T cells, B cells and monocytes in FIP1L1-PDGFRA-positive hypereosinophilic patients suggesting a multilineage involvement. Furthermore, the same FIP1L1-PDGFRA rearrangement was identified in patients with hypereosinophilia and atypical mast cell proliferations, raising the question of a disease with two concomitant lines of differentiation. In addition, a recent report noted two cases with the association of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia and T-cell lymphoblastic lymphoma (T-LBL). We report here the only third case of synchronous chronic eosinophilic leukemia and T-LBL, both associated with a FIP1L1-PDGFRA fusion transcript, confirming the occurrence of such disease and suggesting a clonal proliferation with two lines of differentiation probably arising from a primitive multipotent medullary stem cell.

HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production.

Human immunodeficiency virus (HIV) infection is associated with B-cell malignancies in patients though HIV-1 is not able to infect B-cells. The rate of B-cell lymphomas in HIV-infected individuals remains high even under the combined antiretroviral therapy (cART) that reconstitutes the immune function. Thus, the contribution of HIV-1 to B-cell oncogenesis remains enigmatic. HIV-1 induces oxidative stress and DNA damage in infected cells via multiple mechanisms, including viral Tat protein. We have detected elevated levels of reactive oxygen species (ROS) and DNA damage in B-cells of HIV-infected individuals. As Tat is present in blood of infected individuals and is able to transduce cells, we hypothesized that it could induce oxidative DNA damage in B-cells promoting genetic instability and malignant transformation. Indeed, incubation of B-cells isolated from healthy donors with purified Tat protein led to oxidative stress, a decrease in the glutathione (GSH) levels, DNA damage and appearance of chromosomal aberrations. The effects of Tat relied on its transcriptional activity and were mediated by NF-κB activation. Tat stimulated oxidative stress in B-cells mostly via mitochondrial ROS production which depended on the reverse electron flow in Complex I of respiratory chain. We propose that Tat-induced oxidative stress, DNA damage and chromosomal aberrations are novel oncogenic factors favoring B-cell lymphomas in HIV-1 infected individuals.

Cryptic 6p21.3 duplications and triplication involving HMGA1 partially masked by add 6p in four cases of myelodysplasia.

Rearrangements of 6p are frequent in both myeloid and lymphoid malignant hematological disorders. High-mobility group AT-hook 2 (HMGA2) rearrangements have been described in myelofibrosis with myeloid metaplasia (MMM) and also in myelodysplasia. High-mobility group A proteins are nonhistone nuclear proteins that bind DNA and regulate the transcriptional activity of many genes. We used FISH, with bacterial artificial chromosome RP11-513I15 probe, to study 16 cases of myeloid malignancies with chromosome 6 short arm rearrangements, most of them following myeloproliferative disorders. Among these we found two 6p21.3 duplications and one 6p21.3 triplication involving HMGA1 in four cases of myelodysplasia with and without myelofibrosis. In these four cases, duplications and triplication were partially masked at the cytogenetic level by a derivative chromosome 6 resulting from translocation with another chromosome. HMGA1 proteins have been recently found overexpressed in human leukemias, but to our knowledge this is the first reported duplication of HMGA1.

Recurrent rearrangement of the Ewing's sarcoma gene, EWSR1, or its homologue, TAF15, with the transcription factor CIZ/NMP4 in acute leukemia.

Fusions of the TET-proteins (TLS/FUS, EWSR1, and TAF15/RBP56) to different transcription factors are involved in various malignancies including Ewing's sarcoma, primitive neuroectodermal tumors, and acute myeloid leukemia. These are thought to arise through transcriptional deregulation, with the transcription factor defining the tumor phenotype. We show that, as result of a t(12;17)(p13;q11) or its variant t(12;22)(p13;q12), the transcription factor gene CIZ/NMP4 is recurrently involved in acute leukemia through fusion with either EWSR1 or TAF15. The fusions possess transforming properties in NIH3T3 cells but do not affect the expression of CIZ target genes, suggesting a contribution to oncogenesis that is independent of the transactivating properties of the fusion protein. These results also extend the involvement of TET-protein fusions to acute lymphoblastic leukemia and suggest a role for CIZ/NMP4 in lymphoid and myeloid development.

Cytogenetics in the management of Philadelphia-negative myeloproliferative neoplasms: an update by the Groupe francophone de cytogénétique hématologique (GFCH).

The recent years have witnessed tremendous progress in the molecular characterization of Philadelphia-negative myeloproliferative neoplasms (MPN). Beside a better understanding of pathophysiology, these abnormalities often constitute very useful diagnostic markers in diseases where exclusion of reactive states used to be the strongest argument. However, conventional and molecular cytogenetics keep a major interest in MPN, either as a second line exploration, in cases where no molecular marker is available, for differential diagnosis or as a proof of clonality or in first line for cases with hyperleukocytosis, for differential diagnosis (CML), to evidence druggable targets (ABL1, RET, PDGFR…) or as a prognosis marker. In this article, we will review the interest of cytogenetic techniques in myeloproliferative neoplasms.