Long-term molecular remission in a patient with acute myeloid leukemia harboring a new NUP98-LEDGF rearrangement.

A large variety of molecular rearrangements of the NUP98 gene have been described in the past decades (n = 72), involving fusion partners coding for different transcription factors, chromatin modifying enzymes, as well as various cytosolic proteins. Here, we report the case of an AML-M2 patient with a variant NUP98-LEDGF/PSIP1 gene fusion (N9-L10). In this patient, three different NUP98-LEDGF fusion mRNAs were characterized due to alternative splicing in LEDGF exon 11. Targeted high-throughput sequencing revealed the presence of IDH1, SRSF2, and WT1 additional pathogenic mutations. To improve the therapeutic monitoring, quantification of NUP98-LEDGF mRNA by real-time PCR was developed. Because of poor response to conventional chemotherapy, allogeneic stem cell transplantation was performed, followed by 20 cycles of azacitidine-based preemptive treatment of relapse. More than 31 months after diagnosis, corresponding to 25 months post SCT and 4 months after the last cycle of azacytidine, the patient is in complete molecular remission (undetectable NUP98-LEDGF mRNA transcripts). This study highlights the considerable variability in breakpoint location within both NUP98 and LEDGF, associated with alternative splicing affecting LEDGF. It also emphasizes the need to fully characterize the breakpoints within the two genes and the identification of all fusion mRNAs, particularly for the development of a molecular monitoring assay. All these data seem critical for the optimal management of NUP98-LEDGF + hematological malignancies commonly associated with a poor prognosis.

Additional cytogenetic abnormalities and variant t(9;22) at the diagnosis of childhood chronic myeloid leukemia: The experience of the International Registry for Chronic Myeloid Leukemia in Children and Adolescents.

BACKGROUND: In the adult population with newly diagnosed chronic myeloid leukemia (CML), variant translocations are usually not considered to be impairing the prognosis, whereas some additional cytogenetic abnormalities (ACAs) are associated with a negative impact on survival. Because of the rarity of CML in the pediatric population, such abnormalities have not been investigated in a large group of children with CML. METHODS: The prognostic relevance of variant t(9;22) and ACAs at diagnosis was assessed in 301 children with CML in the chronic phase who were enrolled in the International Registry for Chronic Myeloid Leukemia in Children and Adolescents. RESULTS: Overall, 19 children (6.3%) presented with additional cytogenetic findings at diagnosis: 5 children (1.7%) had a variant t(9;22) translocation, 13 children (4.3%) had ACAs, and 1 had both. At 3 years, for children with a classic translocation, children with ACAs, and children with a variant t(9;22) translocation who were treated with imatinib as frontline therapy, the probability of progression-free survival (PFS) was 95% (95% confidence interval [CI], 91%-97%), 100%, and 75% (95% CI, 13%-96%), respectively, and the probability of overall survival (OS) was 98% (95% CI, 95%-100%), 100% (95% CI, 43%-98%), and 75% (95% CI, 13%-96%), respectively. No statistical difference was observed between the patients with classic cytogenetic findings and those with additional chromosomal abnormalities in terms of PFS and OS. CONCLUSIONS: In contrast to adults with CML, additional chromosomal abnormalities observed at diagnosis do not seem to have a significant prognostic impact. Cancer 2017;123:3609-16. © 2017 American Cancer Society.

Treatment and molecular monitoring update in chronic myeloid leukemia management.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from the t(9;22)(q34;q11) translocation. It is characterized by the presence of the BCR-ABL1 fusion gene encoding the BCR-ABL oncoprotein characterized by a deregulated tyrosine kinase activity. Targeted therapies using tyrosine kinase inhibitors (TKI) such as imatinib, dasatinib, nilotinib, bosutinib, or ponatinib have profoundly changed the natural history of the disease with a major impact on survival. Indeed, most patients diagnosed today can enjoy a near normal life expectancy. The efficacy of TKI treatment can be accurately evaluated by a molecular monitoring based on the quantification of BCR-ABL1 mRNA transcripts and the detection of resistance mutations in the BCR-ABL kinase domain. International recommendations for an optimal management of CML using biological parameters are regularly published. They were designed to evaluate the response to the treatment and to consider, if necessary, a switch to another TKI. A sustained and deep molecular response is obtained in a significant percentage of patients. Clinical trials of TKI discontinuation were performed in such a population, and half of patients do not relapse. In the remaining patients, a rapid appearance of the malignant clone was observed, undoubtedly the consequence of the persistence of residual leukemic stem cells (LSCs). How to discriminate patients who may safely stop TKI? How to target residual LSCs, and do we have to eradicate all these cells? Additional research investigation and clinical trials are needed to answer these questions in order to consider a potential cure of CML.

The Rho-ROCK pathway as a new pathological mechanism of innate immune subversion in chronic myeloid leukaemia.

CD1d-restricted invariant natural killer T (iNKT) cells are believed to play a key role in cancer immune surveillance, and are functionally deficient in patients with chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CD1d or its mRNA transcripts, consistent with intracellular retention. In vitro treatment of CML mDCs with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 partially restored both cell surface CD1d expression and CD1d-mediated antigen presentation, whereas it had no effect on HD mDCs. An inhibitor of BCR-ABL tyrosine kinase (TK), imatinib mesylate (IM), had no such activity. Similar recovery of CD1d expression occurred with fasudil, another ROCK inhibitor that is commonly used in clinical trials. Our data support the conclusion that BCR-ABL-dependent ROCK, but not TK, is involved in CD1d downregulation. We propose that ROCK, which is most likely activated by the DH/PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CD1d expression on CML mDCs. Our study reveals the ROCK-mDC axis as a new potential target to restore immune surveillance in patients with CML, offering new therapeutic perspectives for CML treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Extensive analysis of the T315I substitution and detection of additional ABL mutations in progenitors and primitive stem cell compartment in a patient with tyrosine kinase inhibitor-resistant chronic myeloid leukemia.

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML). However, resistance is occasionally observed, mainly due to mutations within the BCR-ABL kinase domain. The T315I substitution confers complete resistance to all TKIs commonly used in clinical practice. To date, the hierarchical level of stem cells in which this mutation initially appears has not been studied. The aim of this study was to evaluate the behavior of T315I mutated cells and to study the presence of potential additional mutations in progenitors and stem cells from a patient with CML. A comprehensive analysis of BCR-ABL(315I) mRNA expressing cells in mononuclear cells, in CD34+ cell populations, and in their primitive long-term culture-initiating cell (LTC-IC) derived progenitors was performed. We show that the T315I substitution arises in primitive Ph1 stem cells without altering their myeloid and erythroid terminal differentiation potential. Leukemic cells expressing a T315I mutated BCR-ABL display a progressive decline in LTC-IC assays as described for non-mutated CML cells at diagnosis. Finally, in our experiments, additional non-315 ABL mutations were identified in hematopoietic stem cell colonies. This observation is suggestive of genetic instability affecting CML progenitors.

Gain of the short arm of chromosome 2 (2p) is a frequent recurring chromosome aberration in untreated chronic lymphocytic leukemia (CLL) at advanced stages.

Using array-based CGH, we identified 2p gain in 22/78 (28%) untreated Binet stages B/C CLL, which was the second most frequent copy number change after 13q deletion. It never occurred as a sole abnormality and was associated with other changes (6q deletion; 1p gain). The region of 2p gain frequently included two oncogenes, REL and MYCN. All patients with gain of REL were unmutated for IGHV (p=0.03). Gain of MYCN was associated with increased mRNA expression (p=0.005), suggesting a pathogenic role for MYCN. Gain of 2p appears to be a marker of progression and may contribute to the poor prognosis.

Successful second allogeneic stem cell transplantation in second remission induced by dasatinib in a child with Philadelphia chromosome positive acute lymphoblastic leukemia.

We report on the use of dasatinib, a second-generation bcr-abl kinase inhibitor, in a child in early relapse of Philadelphia chromosome positive acute lymphoblastic leukemia after hematopoietic stem cell transplantation. This patient benefited from the use of dasatinib obtaining of a complete molecular response which allowed a second successful transplant. Moreover, dasatinib was well tolerated in this heavily pretreated patient.

Quantitative monitoring of the T315I mutation in patients with chronic myeloid leukemia (CML).

Tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment of chronic myeloid leukemia (CML). However, resistances are occasionally observed, mainly due to mutations within the BCR-ABL kinase domain. The T315I substitution confers complete resistance to TKIs commonly used in clinical practice. In the present study, we used an allele-specific quantitative-RT-PCR to perform a molecular follow-up of BCR-ABL transcripts harboring the T315I mutation. We retrospectively quantified BCR-ABL315I mRNA in five patients who acquired the T315I mutation. Our results highlight the relevance of allele-specific Q-RT-PCR experiments for the monitoring of mutated BCR-ABL transcripts and suggest that the kinetics of emergence of T315I mutant mRNA is influenced by the stage of the disease and the presence of previous BCR-ABL kinase domain mutations.

Expression of the cell cycle regulators p14(ARF) and p16(INK4a) in chronic myeloid leukemia.

Expression of p14(ARF) and p16(INK4a) tumor suppressor genes was investigated in 109 patients with chronic myeloid leukemia (CML). The p14(ARF) and p16(INK4a) mRNA levels were significantly low in patients in chronic phase (CP) at presentation and high in patients treated with interferon-alpha (IFN-alpha), especially in non-responders. A moderate overexpression of p14(ARF) with a normal expression of p16(INK4a) was observed in imatinib-resistant patients. Although protein expression did not consistently match mRNA levels, a role for the two cell cycle regulators in the IFN-alpha signaling pathway is suggested as well as a relation with the resistance to IFN-alpha or imatinib therapy.

Therapy-related acute lymphoblastic leukemia with MLL rearrangement following treatment of Burkitt's leukemia.

The occurrence of therapy-related acute lymphoblastic leukemia (ALL) is rare and, to our knowledge, is not reported in patients treated for Burkitt's leukemia. We report on a child with ALL with translocation t(4;11)(q21;q23) involving the MLL gene, 13 months after chemotherapy for Burkitt's leukemia. This present observation indicates that caution should be exercised in using high cumulative doses of DNA topoisomerase II inhibitors in such patients.

Comparison of M-FISH and conventional cytogenetic analysis in accelerated and acute phases of CML.

FISH and multicolor FISH (M-FISH) techniques have greatly enhanced the resolution of conventional cytogenetic analysis, thus enabling the identification of novel regions of rearrangement in hematological malignancies. We report on the analysis of cells from 24 chronic myelogenous leukemia (CML) patients, in either accelerated phase (14 cases) or blast crisis (10 cases) aimed at searching for previously unidentified additional abnormalities related to disease evolution. Indeed, in 6 of 24 cases (25%) M-FISH allowed a more precise description of chromosomal aberrations, the finding of cryptic rearrangements, characterization of markers, identification of additional material and a better interpretation of complex aberrations. However, new recurrent aberration did not emerge from M-FISH analysis.

p190(BCR-ABL) rearrangement as a secondary change in a case of acute myelo-monocytic leukemia with inv(16)(p13q22).

The simultaneous occurrence of two specific primary chromosomal changes in hematological malignancies is rare. We report on a patient with acute myelo-monocytic leukemia and both inv(16)(p13q22) and t(9;22)(q34;q11) with a p190(BCR-ABL) rearrangement. The t(9;22)(q34;q11) translocation appears to be a secondary change. Similar secondary BCR-ABL rearrangements have already been described and, in most cases, the chimeric protein was of the p190(BCR-ABL) type as in our case. A complete remission was obtained by conventional chemotherapy followed with imatinib mesylate maintenance therapy. At relapse, the BCR-ABL transcripts were undetectable, which suggests that imatinib mesylate could be an effective adjuvant treatment in acute leukemia with a secondary t(9;22)(q34;q11).

E2A/HLF fusion gene in an acute lymphoblastic leukemia patient with disseminated intravascular coagulation and a normal karyotype.

INTRODUCTION: Disseminated intravascular coagulation (DIC) is a rare event in acute lymphoblastic leukemia (ALL). However, it has been described in a few cases of pre-B ALL with translocation t(17;19)(q22;p13) which results in the fusion of E2A gene with sequences of HLF gene. Here, we report a case of pre-B ALL with DIC and an apparently normal karyotype by R banding. MATERIALS AND METHODS: Fluorescent in situ hybridization (FISH) studies were performed on bone marrow cells from the patient at presentation and after three months of therapy. RT-PCR was used to detect the E2A-HLF transcript. The type of rearrangement was characterized by sequencing. RESULTS: The t(17;19)(q22;p13) was detected by FISH analysis. The fusion E2A-HLF was amplified by RT-PCR and sequenced, giving a type I rearrangement with a long insertion (146 nucleotides) between E2A exon 13 and HLF exon 4. CONCLUSION: While translocation t(17;19) is undetectable by R-banding technique, it can be detected with FISH and amplified with RT-PCR. Therefore, systematic molecular investigations should be conducted for all patients with pre-B ALL associated with DIC, in order to appreciate the incidence and the prognostic value of this rare abnormality.