Chronic Myeloid Leukemia Blast Crisis Research Articles

Clinical efficacy of second generation tyrosine kinase inhibitor and 5-azacytidine combination in chronic myelogenous leukaemia in myeloid blast crisis

Even in the tyrosine kinase inhibitors era, the prognosis of patients with chronic myeloid leukaemia in myeloid blast crisis remains dismal with few patients surviving longer than 6months. Here we report the cases of 5 patients treated with the combination of 5-azacytidine and tyrosine kinase inhibitors for myeloid blast crisis CML. All patients achieved a complete haematological response including two with a complete cytogenetic and major molecular response. Two patients underwent an allogeneic stem cell transplantation. One died from relapse 34months from diagnosis. The second is alive and free from disease at 11months from diagnosis. The other 3 patients are still in complete haematological response after 15, 24 and 33months of follow-up. These results suggest that the combination has a significant activity in myeloid blast crisis and may increase survival.

FoxO3a and nilotinib-induced erythroid differentiation of CML-BC cells

We explored the potential involvement of FoxO3a activation in erythroid and granulocytic differentiation for Ph(+) cells of chronic myeloid leukemia blast crisis (CML BC). We demonstrate that FoxO3a activation in CML blast crisis (BC) cells by overexpressing FoxO3a leads to the maturation of CML BC cells. Hemoglobin production significantly increased upon FoxO3a activation in CML BC cells. FoxO3a activation upregulated erythroid surface protein (glycophorin A, GPA), but did not significantly modulate granulocytic markers (CD11b). Additionally, FoxO3a activation reduced the mRNA and protein expression of Tal1. Similar results were observed in cells that were given nilotinib. Our results indicate that FoxO3a activation may promote erythroid differentiation of BC cells via down-regulating Tal1 expression.

Targeting of the MNK–eIF4E axis in blast crisis chronic myeloid leukemia inhibits leukemia stem cell function

Chronic myeloid leukemia responds well to therapy targeting the oncogenic fusion protein BCR-ABL1 in chronic phase, but is resistant to treatment after it progresses to blast crisis (BC). BC is characterized by elevated β-catenin signaling in granulocyte macrophage progenitors (GMPs), which enables this population to function as leukemia stem cells (LSCs) and act as a reservoir for resistance. Because normal hematopoietic stem cells (HSCs) and LSCs depend on β-catenin signaling for self-renewal, strategies to specifically target BC will require identification of drugable factors capable of distinguishing between self-renewal in BC LSCs and normal HSCs. Here, we show that the MAP kinase interacting serine/threonine kinase (MNK)-eukaryotic translation initiation factor 4E (eIF4E) axis is overexpressed in BC GMPs but not normal HSCs, and that MNK kinase-dependent eIF4E phosphorylation at serine 209 activates β-catenin signaling in BC GMPs. Mechanistically, eIF4E overexpression and phosphorylation leads to increased β-catenin protein synthesis, whereas MNK-dependent eIF4E phosphorylation is required for nuclear translocation and activation of β-catenin. Accordingly, we found that a panel of small molecule MNK kinase inhibitors prevented eIF4E phosphorylation, β-catenin activation, and BC LSC function in vitro and in vivo. Our findings identify the MNK-eIF4E axis as a specific and critical regulator of BC self-renewal, and suggest that pharmacologic inhibition of the MNK kinases may be therapeutically useful in BC chronic myeloid leukemia.

Combination of Rapamycin and Imatinib in Treating Refractory Chronic Myeloid Leukemia Myeloid Blast Crisis: a Case Report

showed CD2 0.5%, CD7 24.7%, CD11 1.2%, CD19 1.0%, CD20 1.0%, CD13 41.5%, CD33 29.9%, CD14 1.5%, CD34 49%, CD117 1.9%. Cytogenetic analysis revealed 46, XY, t(9;22)(q34;q11)/(10). Reverse transcription-polymerase chain reaction (RT-PCR) detected BCR/ABL fusion gene at 8821/10 000ablcopies. These results supported the diagnosis of CML myeloid blast crisis. Because a human leukocyte

ATM-dependent DNA damage-response pathway as a determinant in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) begins with an indolent chronic phase, and subsequently progresses to an accelerated or blastic phase. Although several genes are known to be involved in the progression to blastic phase, molecular mechanisms for the evolution toward blast crisis have not been fully identified. Oncogenic stimuli enforce cell proliferation, which requires DNA replication. Unscheduled DNA replication enforced by oncogenic stimuli leads to double strand breaks on DNA. We found the DNA damage-response pathway is activated in bone marrow of chronic-phase CML patients possibly due to an enforced proliferation signal by BCR-ABL expression. Since ataxia telangiectasia mutated (ATM) is a central player of the DNA damage-response pathway, we studied whether loss of this pathway accelerates blast crisis. We crossed Atm-knockout mice with BCR-ABL transgenic mice to test this hypothesis. Interestingly, the loss of one of the Atm alleles was shown to be enough for the acceleration of the blast crisis, which is supported by the finding of increased genomic instability as assayed by breakage–fusion–bridge (BFB) cycle formation. In light of these findings, the DNA damage-response pathway plays a vital role for determination of susceptibility to blast crisis in CML.

Bcl-xL anti-apoptotic network is dispensable for development and maintenance of CML but is required for disease progression where it represents a new therapeutic target

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug resistance. Altered expression of the anti-apoptoticBcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacological antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34(+) progenitors but not those from healthy donors, regardless of drug resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242-induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacological approach for patients undergoing blastic transformation.

Abstract 217: BCR-ABL tyrosine kinase inhibition combined with a novel pan BCL2 family inhibitor abrogates leukemia stem cell survival in a niche-dependent manner.

Abstract Leukemia stem cells (LSC) play a crucial role in initiation, maintenance, and progression of chronic myeloid leukemia (CML). Targeted therapy with a BCR-ABL targeted tyrosine kinase inhibitor (e.g. dasatinib) has improved survival of patients with chronic phase CML. However, like blast crisis (BC) CML, survival remains only 30% over 5 years, and treatment frequently fails to eliminate dormant LSC that are hypothesized to drive CML relapse. Overexpression of BCL2 family genes has been observed in human BC CML and may fuel LSC survival. Our previous data demonstrated that pro-survival long isoforms of BCL2 and MCL1 were preferentially expressed in BC CML LSC compared to normal stem cells. These data showed that the bone marrow (BM) is enriched for BCL2 and MCL1-expressing LSC and serves as a reservoir for therapeutic resistance, thereby underscoring the importance of pan-BCL2 inhibition in the BM. A potent pan-BCL2 inhibitor, sabutoclax, has been identified that inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl1-1 at nanomolar concentrations. To demonstrate whether sabutoclax or the combination of sabutoclax and dasatinib could synergize to eradicate LSC in the BM, humanized BC CML LSC mouse models were established by using 50,000 CD34+ cells selected from primary BC CML patient samples and transplanted into neonatal RAG2-/-γc-/- mice. Mice were screened for peripheral blood (PB) engraftment of human cells at 6 weeks, randomized, and dosed with (1) vehicle, (2) sabutoclax (10mg/kg, i.v., twice a week for 2 weeks), (3) dasatinib (50mg/kg, p.o., daily for 2 weeks), or (4) combination. Mice were sacrificed one day after last dose; PB, spleen (SP) and BM were collected for FACS analysis to measure engraftment and cell cycle status of LSC in different hematopoietic niches. BCL2 and MCL1 expression was measured in BM and SP by isoform specific qRT-PCR and immunohistochemistry. Treatment of CML LSC-transplanted mice with sabutoclax alone led to a significant reduction in LSC burden in the SP, but only slightly in the BM. Meanwhile, Single agent sabutoclax reduced the engraftment of quiescent LSC. The effect of sabutoclax synergized with dasatinib to further reduce LSC burden in the BM to eradicate LSC that drive therapeutic resistance. Importantly, these treatments spare normal human stem cells in both BM and SP thereby providing in vivo evidence of a reasonable therapeutic index. Sabutoclax not only reduced the expression of BCL2 and MCL1 mRNA long isoform in the BM, but also significantly reduced the number of human CD34+, BCL2+ and MCL1+ cells in both BM and SP. These results demonstrate that BM and SP niche BC CML LSC survival is driven by overexpression of multiple pro-survival BCL2 family isoforms rendering them susceptible to a novel pan-BCL2 inhibitor, sabutoclax, at doses that spare normal hematopoietic progenitors. Citation Format: Wenxue Ma, Janine Low-Marchelli, Heather S. Leu, Angela Court Recart, Jun Wei, Xianshu Huang, Maryla Krajewska, Maurizio Pellecchia, John C. Reed, Catriona H.M. Jamieson. BCR-ABL tyrosine kinase inhibition combined with a novel pan BCL2 family inhibitor abrogates leukemia stem cell survival in a niche-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2013-217

Abstract 3454: Bcl-xL anti-apoptotic network is dispensable for emergence and maintenance of CML but required for disease progression, and represents an alternative target for halting survival of blast crisis CML progenitors.

Abstract The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug-resistance and disease progression. We generated an inducible mouse model in which simultaneous expression of p210 BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect the development of the chronic phase-like myeloproliferative disease; however, none of the deficient mice progressed to an advanced phenotype, suggesting the importance of the anti-apoptotic Bcl-xL signaling network for the progressing early progenitor cell survival. Indeed, pharmacologic inhibition of Bcl-xL with the Bcl-xL/Bcl-2 antagonist ABT-263, combined with activation of the Bcl-xL-regulator BAD, through the PP242-mediated inhibition of mTORC1/2, markedly augmented apoptosis of BCR-ABL1+ cell lines and primary CD34+ progenitors from CML-BC but not from healthy donors, regardless of the drug-resistance effect induced by bone marrow stromal cell-generated signals. Accordingly, shRNA-mediated downregulation of BAD and the BCR-ABL1-target hnRNP A1, which promotes translation of Bcl-xL but not Bcl-2, impaired the ability of PP242 to potentiate the effect of ABT-263, and mimicked the anti-leukemic activity of ABT-263, respectively, in primary leukemic CD34+ progenitors and/or CML-BC cell lines. Thus, suppression of the antiapoptotic potential of Bcl-xL together with activation of Bad represents a novel potential pharmacologic approach for treatment of those patients undergoing blastic transformation. Citation Format: Jason Harb, Paolo Neviani, Justin E. Ellis, Gregory J. Ferenchak, Brenda I. Chyla, Peter Hokland, Denis Claude Roy, Michael A. Caligiuri, Guido Marcucci, Claudia S. Huettner, Danilo Perrotti. Bcl-xL anti-apoptotic network is dispensable for emergence and maintenance of CML but required for disease progression, and represents an alternative target for halting survival of blast crisis CML progenitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3454. doi:10.1158/1538-7445.AM2013-3454

Abstract 247: ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia.

Abstract The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. While DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity may also be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole transcriptome sequencing of normal, chronic phase (CP) and serially transplantable blast crisis (BC) chronic myeloid leukemia (CML) progenitors revealed increased interferon-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the interferon responsive ADAR1 p150 isoform and a propensity for increased A-to-I RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promoted expression of the myeloid transcription factor PU.1 and induced malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a mis-spliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of BC CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies. Citation Format: Qingfei Jiang, Leslie A. Crews, Christian L. Barrett, Angela Court-Recart, Daniel Goff, Anil Sadarangani, Jessica Rusert, Sheldon Morris, Lawrence Goldstein, Hye-Jung Chun, Marco Marra, Kelly Fraser, Kim-Hien Dao, Mark Minden, Catriona Jamieson. ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 247. doi:10.1158/1538-7445.AM2013-247

Does BCR/ABL1 positive Acute Myeloid Leukaemia Exist?

The BCR/ABL1 fusion gene, usually carried by the Philadelphia chromosome (Ph) resulting from t(9;22)(q34;q11) or variants, is pathognomonic for chronic myeloid leukaemia (CML). It is also occasionally found in acute lymphoblastic leukaemia (ALL) mostly in adults and rarely in de novo acute myeloid leukaemia (AML). Array Comparative Genomic Hybridization (aCGH) was used to study six Ph(+)AML, three bi-lineage and four Ph(+)ALL searching for specific genomic profiles. Surprisingly, loss of the IKZF1 and/or CDKN2A genes, the hallmark of Ph(+)ALL, were recurrent findings in Ph(+)AML and accompanied cryptic deletions within the immunoglobulin and T cell receptor genes. The latter two losses have been shown to be part of 'hot spot' genome imbalances associated with BCR/ABL1 positive pre-B lymphoid phenotype in CML and Ph(+)ALL. We applied Significance Analysis of Microarrays (SAM) to data from the 'hot spot' regions to the Ph(+)AML and a further 40 BCR/ABL1(+) samples looking for differentiating features. After exclusion of the most dominant markers, SAM identified aberrations unique to de novo Ph(+)AML that involved relevant genes. While the biological and clinical significance of this specific genome signature remains to be uncovered, the unique loss within the immunoglobulin genes provides a simple test to enable the differentiation of clinically similar de novo Ph(+) AML and myeloid blast crisis of CML.

Imatinib failure and response to dasatinib in a patient with chronic myeloid leukemia in blast crisis and a novel, nine-nucleotide BCR-ABL insertion mutation

Imatinib failure and response to dasatinib in a patient with chronic myeloid leukemia in blast crisis and a novel, nine-nucleotide BCR-ABL insertion mutation

A “complicated” fracture: a Philadelphia chromosome-positive myeloid sarcoma of the bone

Dear Editor, In April 2009, a 19-year-old male presented with progressive pain on his left upper arm. A MRI scan of the arm showed a large tumor of the distal left humerus (Fig. 1a). Because an osteosarcoma was suspected, a biopsy was performed. After the biopsy, the patient presented with a fracture of his arm as a result of minor trauma. This fracture was managed conservatively with an upper arm cast. The biopsy revealed necrotic bone fragments with an infiltration of myeloblasts admixed with immature eosinophils; hence, the diagnosis of a myeloid sarcoma (MS) was established. Besides the pain in his arm, the patient had no complaints, especially no fever, night sweats, weight loss, hemorrhage, or anemia. On physical examination, neither lymphadenopathy and hepatosplenomegaly nor neurological signs were noticed. His peripheral blood counts were normal. A PET scan did not show any other bone lesion. On histological examination, the MS consisted of myelo/monoblasts with expression of lysozyme, partly weak myeloid peroxidase (MPO), and focally CD34 and CD117. A bone marrow aspirate was performed to exclude acute myeloid leukemia (AML). Morphology of the bone marrow did not support a diagnosis of AML or chronic myeloid leukemia (CML), although with flow cytometric immunophenotyping (IPT), a small population (2 %) of aberrant myeloblasts expressing CD15, CD34, and CD117 were found. Intriguingly, cytogenetic analysis of the bone marrow aspirate showed a Philadelphia chromosome (t(9;22)(q34;q11.2)) in 2 of 13 analyzed cells. Subsequent fluorescence in situ hybridization on the tumor biopsy showed that the blasts harbored the BCR-ABL fusion gene (Fig. 1b). PCR performed on peripheral blood and bone marrow showed BCR-ABL levels of 3.3×10 and 1.5×10 copies/ml, respectively. Because of the bad prognosis, the patient was treated with intensive chemotherapy followed by allogeneic stem cell transplantation (SCT) with peripheral blood stem cells from a matched unrelated donor. The therapy was complemented with local radiotherapy of his left arm. During his treatment, the left arm recovered swiftly, and at the time of SCT, he was no longer depending on the cast. Now 2 years after SCT, he is still in remission with an undetectable BCR-ABL level. MS is a rare disease that is reported in 2.5–9.0 % of patients with AML. It can occur concomitantly, following or, rarely, antedating the onset of systemic AML [1]. MS can also occur in the presence of other hematological diseases such as myelodysplastic syndrome and myeloproliferative neoplasia, including CML [2]. Certain known cytogenetic abnormalities, in particular t(8;21), have been associated with a higher incidence of MS [1, 2]. However, the presence of a Philadelphia chromosome t(9;22) is extremely rare and virtually no reports exist on cases in AML with the remainder almost exclusively occurring during CML blast crisis [2]. The phenotype of MS is mostly monoblastic or myelomonocytic, often expressing CD34, CD68, CD99, CD117, MPO, and TdT [2]. In our patient, the MS had this common L. F. J. van Groningen (*) :W. J. F. M. van der Velden Department of Hematology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, the Netherlands e-mail: l.vangroningen@aig.umcn.nl

Fangchinoline induces G0/G1 arrest by modulating the expression of CDKN1A and CCND2 in K562 human chronic myelogenous leukemia cells

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by the oncoprotein BCR-ABL, which exhibits a constitutive tyrosine kinase activity. Imatinib mesylate (IM), an inhibitor of the tyrosine kinase activity of BCR-ABL, has been used as a first-line therapy for CML. However, IM is less effective in the accelerated phase and blastic phases of CML and certain patients develop IM resistance due to the mutation and amplification of the BCR-ABL gene. Fangchinoline, an important chemical constituent from the dried roots of Stephaniae tetrandrae S. Moore, exhibits significant antitumor activity in various types of cancers, including breast, prostate and hepatocellular carcinoma. However, the effects and the underlying mechanisms of fangchinoline in CML remain unclear. In the present study, we identified that fangchinoline inhibits cell proliferation in a dose- and time-dependent manner in K562 cells derived from the blast crisis of CML. Additional experiments revealed that fangchinoline induces cell cycle arrest at the G0/G1 phase and has no effect on apoptosis, which is mediated through the upregulation of cyclin-dependent kinase (CDK)-N1A and MCL-1 mRNA levels, as well as the downregulation of cyclin D2 (CCND2) mRNA levels. These findings suggest the potential of fangchinoline as an effective antitumor agent in CML.

Philadelphia positive chronic myeloid leukemia mimicking acute lymphoblastic leukemia in a child

Chronic myeloid leukemia (CML) is a rare hematological malignancy in children. The more common acute lymphoblastic leukemia (ALL) needs to be excluded in cases with lymphoid blast crisis phase of childhood CML. A 12-year-male child with generalized lymphadenopathy and hepatosplenomegaly had a crisis phase of presentation instead of the usual chronic phase of onset of disease. Hematological tests and molecular study were performed. Peripheral blood, bone marrow and lymph node fine-needle aspiration cytology smear showed features of CML in blast crisis. Blast cells were myeloperoxidase negative. Philadelphia chromosome with major BCR-ABL fusion product and B-cell lineage immunomarkers indicated CML origin of blasts rather than de novo ALL. Usually myeloid blast crisis is common in CML but the present case had lymphoid blast crisis which is less commonly documented. We report a case of ‘adult form of CML in the juvenile age group with lymphoid blast crisis and extramedullary blast proliferation.’

Pediatric chronic myeloid leukemia with B-cell lymphoid blast crisis at presentation

TO THE EDITOR: Chronic Myeloid Leukemia (CML) is a rare hematological malignancy accounting for less than 3% of pediatric and adolescent leukemias, with an annual incidence of approximately 1 per million children and young people aged 20% blasts in the marrow or the presence of extra-medullary blast proliferation [3, 4]. Blast transformation of CML is lymphoid in 30% of cases and myeloid in the remaining 70%. We report the case of a 10-year-old boy who presented with a 6-week history of weight loss and left-sided upper abdominal pain. On examination, he was found to have substantial splenomegaly extending into the right iliac fossa. His full blood count showed a hemoglobin level of 6.6 g/dL, a platelet count of 148×109/L, and a total white cell count of 575×109/L. A differential count showed the following: blasts, 30%; promyelocytes, 6%; myelocytes, 24%; metamyelocytes, 11%; neutrophils, 7%; eosinophils, 9%; basophils, 3%; monocytes, 1%; and lymphocytes, 10% (Fig. 1). Flow cytometry of the blast population showed that the blasts expressed CD19, cyCD79a, CD10, HLA-DR, CD34, and TdT surface antigens. Chromosome analysis showed a t(9;22)(q34;q11) translocation consistent with a Philadelphia chromosome (Fig. 2A). Interphase fluorescence in situ hybridization (FISH) showed BCR-ABL1 fusion signals in 90% of the nuclei (Fig. 2B). A minor breakpoint cluster region (210 kDa) was confirmed using RT-PCR. In the absence of a documented CML-CP, distinguishing between lymphoid blast crisis of CML and a Philadelphia chromosome-positive ALL can be difficult. A diagnosis of CML in B-cell lymphoid blast crisis rather than de novo precursor B-cell ALL was made because of the concurrent presence of basophilia, a predominance of metamyelocytes and myelocytes, and the p210 BCR-ABL transcript. The patient did not have any additional chromosomal anomalies associated with advanced phase CML and was negative for IgH rearrangement on FISH analysis. Fig. 1 Blood film demonstrating chronic myeloid leukemia in lymphoid blast transformation. Fig. 2 (A) Karyotype showing t(9;22)(q34;q11) Philadelphia translocation. (B) Interphase FISH, Vysis dual-fusion probe set. Green: BCR; red: ABL1; yellow: fused BCR and ABL signals corresponding to der(9) and der(22) translocation products. The patient achieved a complete hematological response and a minor cytogenetic response following induction therapy comprising dexamethasone, vincristine, daunorubicin, asparaginase, and imatinib. Nonetheless, he subsequently died of idiopathic pneumonitis after allogeneic stem-cell transplantation. The progression from CML-CP to CML-BC in the pre-tyrosine kinase era is well described, but CML-BC at presentation in children is extremely rare. The case reported here adds to the literature on the simultaneous presence of features of both lymphoblastic transformation and CML at presentation in the absence of cytogenetic clonal evolution. Further research into the biology of the aggressive phase of CML is required to develop novel targets to improve outcomes.

Changes in Megakaryocytes in Cases of Thrombocytopenia: Bone Marrow Aspiration and Biopsy Analysis

Thrombocytopenia (platelet counts less than 150,000/μl) is commonly encountered in various hematological disorders including myelodysplastic syndromes as well as various non-myelodysplastic hematological conditions. The present study was undertaken to calculate the prevalence of various conditions associated with thrombocytopenia and to record the megakaryocytic alterations in various cases of thrombocytopenia. Apart from this by means of statistical analysis it was tried to analyze whether a significant difference existed in megakaryocytic alteration noted in myelodysplastic versus non- myelodysplastic conditions. A prospective series of 60 bone marrow aspirations along with concomitant bone marrow biopsies was conducted in a tertiary care centre catering to both urban as well as rural population in north India. The distribution of morphological changes in cases of non myelodysplastic conditions and myelodysplastic were compared using Chi-Square test. A p-value less than 0.05 was considered significant. The commonest cause of thrombocytopenia for which bone marrow examination was sought was dimorphic anaemia (18 cases, 30%), followed by myelodysplastic syndrome (06 cases, 10%) which was followed equally by acute lymphocytic leukemia and blast crisis of chronic myeloid leukemia (CML). Of all the non-MDS conditions apart from dimorphic anaemia, idiopathic thrombocytopenic purpura and chronic myeloid leukemia (blast crisis); megakaryocytic dysplastic forms were not noted in any other condition. In cases of myelodysplasia; dysplastic forms, bare megakaryocytic nuclei, hypogranular forms and micromegakaryocytes were seen. Comparison between frequencies of normal, high and low number of nuclear lobes among MDS (n=9) and non MDS (n=68) conditions were found to be statistically significant. Further studies on the evaluation of megakaryocytic alteration and their contribution to thrombocytopenia can provide growing knowledge to the pathogenesis of numerous hematopoietic disorders that may identify broader clinical applications of the newer strategies to regulate platelet count and functioning.

Biological and Clinical Implications of Exon 8 P53 (R282W) Gene Mutation in Relation to Development and Progression of Chronic Myeloid Leukaemia Patients in India Population

Background: TP53, located on chromosome 17p13, is one of the most mutated genes affecting many types of human cancers .To establish an association between the incidence of exon 8 p53 (R282W) gene and progression of the disease in CML and also to correlate the presence of mutation with the clinicopathological features of the disease. Methods: p53 status was investigated by studying mutations in the p53 gene at exon 8 region after confirming the diagnosis by BCR-ABL. 100 CML samples were analyzed using the Allele-Specific Oligonucleotide PCR assay. Mutations occurred in 58% of the cases in exon 8 codon 282 region of the p53 gene. C : T transitions occurred at a high frequency with a statistically significant result (p=0.03). Results: Of the 100 clinically confirmed specimens, 58% tested positive for the mutation. Also, the mutation was found to be higher in the progressed stages (88.2% in accelerated phase and 60.0% in blast crisis) of CML compared to the chronic stage (35.2%). A statistically significant association (p=0.001) was found between the occurrence of p53 R282W mutation and the clinical phase of CML with chronic, accelerated and blast crisis phases. The mutation was detected in a vast majority (88.2%) of patients in the accelerated and the blast crisis phase (60.0%) indicating that this mutation might play a critical role in predicting the progression of disease in CML. Clinicopathological correlation with TLC, platelet count and the haematological response elicited a significant association with patients harboring the mutation with (p=0.01), (p=0.001) and (p=0.01) respectively. Conclusion: Our study suggests that p53 mutations in the exon 8 region might have a strong influence on disease progression and poor response of imatiib (Tyrosine kinase inhibitor) in CML patients.

Inactivation of RIZ1 Gene by Promoter Hypermethylation is Associated with Disease Progression and Resistance to Imatinib in Indian Chronic Myelogenous Leukemia Patients, First Study from India

Background: The epigenetic impact of DNA methylation in chronic myelogenous leukemia (CML) is not completely understood. RIZ1 expression and activity are reduced in many cancers. In CML, blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located. RIZ1 is a PR domain methyltransferase that methylates histone H3 lysine 9, a modification important for transcriptional repression. In CML blast crisis cell lines RIZ1 represses insulin-like growth factor-1 expression and autocrine signaling. Together these observations suggest that RIZ1 may have a role in the chronic phase to blast crisis transition in CML. Methods: To examine whether promoter methylation is involved in the disease development and progression of CML, we investigated promoter methylation status of RIZ1 gene in 100 chronic myeloid leukemia’s (CML) patients and 50 controls by MSP method. Results: The RIZ1 methylation was studied in 100 CML patients, 9 were cases were methylation positive cases, six of nine were in blastic phase, 2 in chronic phase and one patient in accelerated phase. It was seen that RIZ1 methylation was increased significantly from early to advanced phase. The higher frequency of RIZ1 methylation was reported in haematologically resistant cases (42% vs 2%) and molecularly resistant cases (16.77% vs 1.92%) than the responders. The higher frequency of RIZ1 methylation was found in CML patients who were treated with interferon initially followed by imatinib treatment. Also RIZ1 hypermethylation was associated with faster disease progression p<0.003 than the non methylated cases. No correlation was found between RIZ1 gene methylation with age, thrombocytopenia, types of bcr/abl transcripts of CML patients. Conclusion: We conclude that epigenetic silencing of RIZ1 gene is associated with CML progression and imatinib resistance. Early detection of RIZ1 methylation could be a predictive marker for imatinib resistance and disease progression in CML.

Extramedullary blast crisis of chronic myelogenous leukemia as an initial presentation

Extramedullary blast crisis of chronic myelogenous leukemia (CML) is defined as the development of extramedullary disease caused by the infiltration of blasts regardless of proliferation of blasts in the bone marrow. The onset of extramedullary blast crisis in the newly diagnosed patients is known to be extremely rare. Here, we present a case of extramedullary blast crisis of CML as an initial presentation in a 17-year-old female presenting with pain in the left femur tumor. This case was treated successfully with dasatinib and allogeneic hematopoietic stem cell transplantation, with achievement of long-term remission.

Rapid Evolution to Blast Crisis Associated with a Q252HABL1Kinase Domain Mutation in e19a2BCR-ABL1Chronic Myeloid Leukaemia

A minority of chronic myeloid leukaemia (CML) patients express variant transcripts of which the e19a2 BCR-ABL1 fusion is the most common. Instances of tyrosine kinase inhibitor (TKI) resistance in e19a2 BCR-ABL1 CML patients have rarely been reported. A case of e19a2 BCR-ABL1 CML is described in whom imatinib resistance, associated with a Q252H ABL1 kinase domain mutation, became apparent soon after initiation of TKI therapy. The patient rapidly transformed to myeloid blast crisis (BC) with considerable bone marrow fibrosis and no significant molecular response to a second generation TKI. The clinical course was complicated by comorbidities with the patient rapidly succumbing to advanced disease. This scenario of Q252H-associated TKI resistance with rapid BC transformation has not been previously documented in e19a2 BCR-ABL1 CML. This case highlights the considerable challenges remaining in the management of TKI-resistant BC CML, particularly in the elderly patient.