Are CML Lymphoid Blast Crisis and Ph Positive ALL Genomically Indistinguishable?

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4464-4464
Author(s):  
Colin Grace ◽  
Elisabeth P Nacheva
Keyword(s):  
Tyrosine Kinase ◽  
Dna Sequences ◽  
Acute Leukaemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Hot Spot ◽  
Blast Crisis ◽  
Disease Stage ◽  
Blast Transformation ◽  
Significance Analysis

Abstract Abstract 4464 Philadelphia positive malignant disorders are a clinically divergent group of hemoblastoses with a unique identifying feature, the BCR/ABL1 fusion gene, usually resulting from the chromosome rearrangement t(9;22)(q34;q11) or its variants, that leads to constitutive expression of an aberrant tyrosine kinase. These include chronic myeloid leukaemia (CML) and de novo acute leukaemia of both myeloid Ph(+)AML and lymphoid origin Ph(+)ALL. The latter two disorders are clinically aggressive and therapy challenging even in the era of the powerful tyrosine kinase inhibitors. CML is a multistage progressive disease, which if untreated, inevitably ends as fatal acute leukaemia, either myeloid or lymphoid. The latter is often thought to be indistinguishable from Ph(+)ALL, the most common type of ALL in adults. We have identified DNA sequences the imbalances of which appear to be significantly associated with the disease stage and lineage origin in CML and Ph(+)ALL samples. We used array CGH at a resolution of ~2kb to explore hot spot regions obtained from 102 patient samples comprising 92 CML and controls together with 10 Ph(+)ALL and show how Significance Analysis of Microarrays (1) can be used to identify differences in the genome profile of de novo Ph(+)ALL and lymphoid blast transformation of CML. We show that lymphoid blast crisis CML differs significantly from Ph(+)ALL not only due to the presence of 9p deletions but also due to genomic gains in other chromosomes. Furthermore we identify a sub group of Ph(+)ALL with a distinctive genomic profile. Having identified genome regions of potential interest, ranked in order of significance, out of the 100's of thousands of array results, it is then a challenge to design further experiments to evaluate their contribution to the biology of the BCR/ABL positive disease. 1 Tusher V, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98:5116-5121 (2001). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Significance Analysis of Microarrays (SAM) Offers Clues to Differences between the Genomes of Adult Philadelphia Positive ALL and the Lymphoid Blast Transformation of CML

2012 ◽  
Vol 11 ◽  
pp. CIN.S9258 ◽  
Author(s):  
Colin Grace ◽  
Elisabeth P. Nacheva
Keyword(s):  
De Novo ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Chromosome 9 ◽  
Comparative Genomic ◽  
Blast Transformation ◽  
Significance Analysis ◽  
Genome Data ◽  
Genome Screening ◽  
Blast Cells

Philadelphia positive malignant disorders are a clinically divergent group of leukemias. These include chronic myeloid leukemia (CML) and de novo acute Philadelphia positive (Ph(+)) leukemia of both myeloid, and lymphoid origin. Recent whole genome screening of Ph(+)ALL in both children and adults identified an almost obligatory cryptic loss of Ikaros, required for the normal B cell maturation. Although similar losses were found in lymphoid blast crisis the genetic background of the transformation in CML is still poorly defined. We used Significance Analysis of Microarrays (SAM) to analyze comparative genomic hybridization (aCGH) data from 30 CML (10 each of chronic phase, myeloid and lymphoid blast stage), 10 Ph(+)ALL adult patients and 10 disease free controls and were able to: (a) discriminate between the genomes of lymphoid and myeloid blast cells and (b) identify differences in the genome profile of de novo Ph(+)ALL and lymphoid blast transformation of CML (BC/L). Furthermore we were able to distinguish a sub group of Ph(+) ALL characterized by gains in chromosome 9 and recurrent losses at several other genome sites offering genetic evidence for the clinical heterogeneity. The significance of these results is that they not only offer clues regarding the pathogenesis of Ph(+) disorders and highlight the potential clinical implications of a set of probes but also demonstrates what SAM can offer for the analysis of genome data.

Clarithromycin Targeting Autophagy Potentiates Tyrosine Kinase Inhibitor-Induced Cell Death in Resistant Chronic Myeloid Leukemia (CML) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4450-4450
Author(s):  
A. M. Carella ◽  
Gioacchino Catania ◽  
G. Beltrami ◽  
G. Pica
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Cytogenetic Response ◽  
Ifn Alpha

Abstract Abstract 4450 M-TOR is a key regulator of autophagy. Rapamycin and clarithromycin (structurally similar to rapamycin), have been demonstrated to have in vitro activity in blocking autophagy. In four patients with advanced CML, remarkable response to the combination of clarithromycin and a tyrosine kinase inhibitor was observed. Here we present the results achieved by the combination. A 43-year-old woman was diagnosed with high-risk Sokal CML in February 2000. She was treated with IFN-alpha and imatinib (400 mg/day) with persistence of 100% Ph-positive metaphases. In March 2006, WBC was no longer controlled and she was treated with nilotinib. Complete hematologic response (CHR) was achieved by the end of April 2006, but there was no cytogenetic response (CyR). She was given dasatinib (70 mg b.i.d.) without complete cytogenetic response (CCyR) and after 7 months the bcr-abl/abl ratio was 6.1% in March 2011. At that time, the patient had an infection (otits/pharyngitis) sensitive to clarithromycin, which was added to dasatinib at a dose of 500 mg b.i.d. April 2011 there was a surprising reduction in the transcript to 0.5%. As of June 2011, the value was 0.05%, and the patient continues to receive clarithromycin (500 mg/day) and dasatinib (100 mg/day). Nowadays (August 1), the patient is in CHR, CCyR and major molecular remission (MMR) (bcr-abl/abl ratio 0.001%). The patient stopped clarithromycin and he is continuing on dasatinib. A 53-year-old man was diagnosed with de novo lymphoid blast crisis CML in August 2010; bcr-abl/abl ratio was 95.2%. He had a sibling donor. In October 2010 bcr-abl/abl ratio was reduced to 0.2% after chemo + imatinib. In November 2010, bcr-abl/abl ratio was 22% and he was treated with dasatinib (70 mg b.i.d.) with WBC control and a small reduction of bcr-abl/abl ratio (18% in February 2011). Soon thereafter, he underwent allogeneic transplant. Two months after transplant (May 2011) the disease progressed and bcr-abl/abl value had increased to 47%. He was restarted on dasatinib (100 mg/day) but the transcript increased in 4 weeks to 143%. Because of our previous experience, we added clarithromycin to dasatinib on June 2, 2011. Two weeks later, bcr-abl/abl value was reduced to 3.2%, and to 1.5% after another week. We stopped clarithromycin and three weeks later under dasatinib alone the transcript increased to 20%. From one week we added newly clarithromycin to dasatinib. A 68 year old man was diagnosed with CML in October 1999. A CCyR was achieved after autografting and soon after IFN-alpha was given as maintenance. In October 2000 the patient relapsed. A second CCyR was achieved in December 2001 after imatinib (400 mg/day), which lasted for six years. In October 2006 bcr-abl/abl ratio was 4.5%. He was treated with dasatinib (70 mg. b.i.d.) with WBC control but with no CyR. In March 2011, bcr-abl/abl ratio was 42.5%. Nilotinib (600 mg. b.i.d.) was begun with no change in bcr-abl/abl ratio after 2 months. In June 2011, clarithromycin (500 mg. b.i.d.) was added; 3 weeks later, the bcr-abl/abl ratio had decreased to 17% and two weeks later (July 13, 2011) to 4%. On July 28, bcr-abl/abl is 0,00022%. A 70 year old woman was diagnosed with CML in November 1998. She was treated with IFN-alpha but only partial CyR was achieved. In January 2001, 100% Ph-positive metaphases were found in BM. She was begun on imatinib (400 mg/day) but the karyotype did not change. In May 2005 she was started on nilotinib (600 mg/daily) since bcr-abl/abl ratio was 26.5%. Blood counts were controlled but there was no change in cytogenetics. In August 2010 WBC increased to 100×103/l. Dasatinib (70 mg. b.i.d.) was begun. Because blood count control was inadequate, hydroxyurea was added. In December 2010, bcr-abl/abl ratio had increased to 140%, and E255V mutation was found. In May 2011, clarithromycin (500 mg. b.i.d.) was added. In 2 weeks, the WBC had decreased from 76×103/l to 10×103/l and bcr-abl/abl ratio was 30% (June 4, 2011). One month later (July 4, 2011) bcr-abl/abl ratio was 3% and the mutation was no longer found in bone marrow. In the last evaluation (July 13, 2011) bcr-abl/abl ratio was 0.00096%. The patient stopped clarithromycin and she is on dasatinib alone. In conclusion, no patients have gone off study for toxicity. In no case we observed grade 3–4 myelosuppression. The remarkable responses obtained in these 4 patients support the hypothesis that inhibition of autophagy may make CML cells sensitive to killing by tyrosine kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

Response to Tyrosine Kinase Inhibitor Therapy In Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation for Advanced Phase Chronic Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3515-3515
Author(s):  
Ailsa Holroyd ◽  
Elizabeth Phillips ◽  
Richard Szydlo ◽  
David Marin ◽  
Letizia Foroni ◽  
...  
Keyword(s):  
Overall Survival ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Tyrosine Kinase ◽  
Cell Transplantation ◽  
Research Funding ◽  
Blast Crisis ◽  
Disease Stage ◽  
Advanced Phase ◽  
Historical Group

Abstract Abstract 3515 With the advent of tyrosine kinase inhibitors (TKI), allogeneic stem cell transplantation (allo-SCT) is largely reserved for patients with CML who do not achieve durable cytogenetic responses to TKIs or patients with advanced phase (Adv) disease. Data relating to the outcome of transplant in Adv-CML is limited. We have allografted 43 patients (median age 40.8 yrs) for Adv phase disease who had received prior treatment with one or more TKI. The indications for allo-SCT included progression from CP to accelerated phase (AP) (n=16) or blast crisis (BC) (n=11) on TKI and presentation in accelerated phase (AP) (n=9) or blast crisis (BC) (n=7). The median duration of TKI therapy prior to transplantation was 5.5 months (range 1–51 months); 42 patients received imatinib, 9 received dasatinib (8 following imatinib failure), 2 received nilotinib (following imatinib and dasatinib failure). 35 patients were transplanted from HLA-identical siblings and 36 patients received myeloablative conditioning. The status at transplant was CP>1 in 17 patients, AP in 24, and BC in 2. In patients in whom CP was restored prior to transplant (n=17), this had been achieved using a TKI only in 6 and with combination chemotherapy in 11. There was no difference in disease-free survival (DFS) or overall survival (OS) between the TKI only group and the group that received chemotherapy in addition. Among the 43 patients in the TKI-treated cohort, 13 died without relapse, 3 from graft versus host disease (GVHD), 8 from sepsis, pneumonitis and multiorgan failure, and one each from graft failure and VOD. The estimated probabilities of non-relapse mortality (NRM) at 100 days and 1 year were 17.3% and 43.3%, Grade 2–4 acute GVHD was seen in 24% and extensive cGVHD in 54%. The estimated 1- and 3-year DFS rates were 23% and 16%. The 1 year and 3 year estimates of overall survival according to disease stage at allo-SCT were as follows: AP (54.2% and 50%), CP >1 (49.4% and 29.6%) and BC disease 0% and 0%. The impact of maximal disease stage was examined, documented as either AP (23/43 patients) or BP (20/43 patients) at any time prior to allo-SCT. The probability of 3 year OS for patients who were in AP at maximal disease stage was 61% compared to 33% of patients who had at one time been in BC (p=0.04). Post allo-SCT, patients were monitored for relapse by RQ-PCR. Eleven patients received TKIs, 5 for molecular relapse, 1 for cytogenetic relapse, 4 for hematological relapse and 1 for GvHD. Three of the 11 remain alive, 2 of whom received a TKI for molecular relapse.We compared the outcome of these 43 patients with that of 158 patients who were transplanted for Adv-CML but who had been treated before TKI became available. The disease status at time of transplant was AP (n=90), CP>1 (n=41) and BC (n=27). The two groups were matched for type of donor, conditioning regimens and time from diagnosis to transplant but the historical group were younger at allo-SCT with a median age of 33.3 yrs (p=0.001). There were no significant differences in the incidences of acute and chronic GvHD, NRM, DFS or OS between the two groups. The 1 year and 3 year estimates of OS for the historical cohort were 46.4% and 38.5% in AP, 53.7% and 24.3% for CP >1 and 7% and 0% in BC. For the total group of 201 patients the outcome of transplant defined as 3yr OS was 40.9% for AP 25.7% for CP>1 and 0% for BC. In conclusion, we found that patients receiving transplant for advanced phase disease after prior treatment with a TKI have similar outcomes to a historical group of advanced phase patients transplanted prior to the advent of TKI therapy. Our data strongly support the influence of disease stage in prediction of allo-SCT survival. Allo-SCT may be valuable for CML patients who have never progressed to BC. Overt BC is a predictor of poor allo-SCT outcome, so attempts should be made to restore CP prior to allo-SCT. Close monitoring of patients still classifiable as AP who are responding poorly to TKI should permit identification of those who may do well if offered allo-SCT before their disease has progressed further. Disclosures: Marin: Novartis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding.

Genome-Wide Analysis of Genetic Alterations in Chronic Myelogenous Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1089-1089 ◽  
Author(s):  
Charles G Mullighan ◽  
Ina Radtke ◽  
Jinghui Zhang ◽  
Letha A. Phillips ◽  
Xiaoping Su ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Copy Number ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Blast Crisis ◽  
Genetic Alterations ◽  
Myelogenous Leukemia ◽  
Disease Stage ◽  
Genome Wide Analysis ◽  
Genome Wide

Abstract Expression of BCR-ABL1 is the hallmark of chronic myelogenous leukemia (CML) and a subset of de novo acute lymphoblastic leukemia (ALL), but the factors determining disease lineage, and progression of CML to myeloid or lymphoid blast crisis, are incompletely understood. We recently reported deletion of IKZF1 (encoding the lymphoid transcription factor Ikaros) in 85% of de novo pediatric and adult BCR-ABL1 ALL, and in lymphoid blast crisis in a small cohort of CML cases (Nature2008;453:110), suggesting that IKZF1 deletion is important in the pathogenesis of BCR-ABL1 lymphoid leukemia. To identify genetic determinants of disease stage and blast crisis lineage in CML, we have now performed high-resolution, genome wide analysis of DNA copy number abnormalities (CNA) and loss-of heterozygosity (LOH) and candidate gene resequencing in a cohort of 90 CML patients that included 64 samples obtained at chronic phase (CP), 15 samples at accelerated phase (AP), 9 lymphoid blast crisis (LBC) and 22 myeloid blast crisis (MBC) samples. Importantly, 25 patients had sequential samples (CP and/or AP, as well as blast crisis samples) enabling analysis of lesions acquired at progression to blast crisis. All blast crisis samples were flow sorted to at least 90% purity prior to DNA extraction. Germline samples for 28 cases obtained at remission or by flow sorting of blast crisis samples were also examined. Affymetrix SNP 6.0 arrays, interrogating over 1.87 million genomic loci, were used for 85 samples, and 500K arrays for the remainder. Identification of tumor-specific (somatic) copy number analysis was performed by directly comparing CML samples to matched germline samples were available, or by filtering results against databases of inherited copy number variants for samples lacking germline material. Genomic resequencing of IKZF1, PAX5 and TP53 was performed for all AP, LBC and MBC samples. There were few CNAs in CP-CML (mean 0.27 deletions and 0.07 gains per case), with no recurring lesions identified apart from deletions or gains at the chromosomal breakpoints of BCR and ABL1 (3 cases each). Notably, the size of these translocation associated deletions was highly variable, ranging from 6kb (one ABL1 deletion) and 15 kb (one BCR deletion) to deletions extending to the telomeres of chromosomes 9 and 22. No significant increase in lesion frequency was identified in AP cases (0.14 deletions and 0.9 gains per case), however the number and cumulative extent of genomic aberrations was significantly higher in both lymphoid and myeloid blast crisis samples. LBC cases had a mean of 8.1 deletions/case (P<0.0001v CP) and 2.8 gains/case (P=0.0024), where as MBC had fewer alterations with only an average of 2.8 deletions/ case (P=0.028 v CP) and 2.2 gains/case (P=0.0018). Similarly, the cumulative extent of DNA altered by CNAs was higher in both LBC (200 Mb/case) and MBC (257 Mb/case) than CP-CML (4.1 Mb/case). There were striking differences in the type of CNAs in MBC and LBC samples. Seven of 9 LBC cases had focal CNAs targeting genes regulating normal B-lymphoid development, including IKZF1 (6 cases, 2 homozygous), PAX5 (4 cases), and EBF1 (1 case with focal homozygous deletion restricted to the EBF1 locus). Thus, of these 7 cases, two had a single CNA in this pathway, three had two lesions, and two cases had three lesions. In contrast, only 4 of 22 MBC cases had lesions in this pathway, most commonly from whole or sub chromosomal deletions involving chromosomes 7 and 9. Deletion of the CDKN2A/B locus (encoding the tumor suppressors and cell cycle regulators INK4A, ARF and INK4B) was seen in 6 (67%) LBC samples, but only 2 (9%) MBC cases, and never in CP or AP CML. Other lesions commonly seen in de novo BCR-ABL1 ALL were also observed in LBC samples, including deletions of MEF2C, C20orf94, and the HBS1L gene immediately upstream of the oncogene MYB. Apart from acquisition of new or more complex abnormalities involving BCR and ABL1, the only recurring mutation observed in MBC was deletion (4 cases) or splice-site point mutations (2 cases) of TP53. These data demonstrate a lack of genomic instability with few genetic alterations in CP or AP CML. Lymphoid blast crisis samples have similar genetic alterations to those seen in de novo BCR-ABL1 ALL, whereas myeloid blast crisis displays completely distinct patterns of mutation, most commonly targeting P53. These results indicate that genomic abnormalities are important determinants of lineage and disease progression in BCR-ABL1 leukemia.

scholarly journals Co-expression of the CBFβ-MYH11 and BCR-ABL fusion genes in chronic myeloid leukaemia / Coexistenţa genelor de fuziune CBFβ-MYH11 şi BCR-ABL în leucemia mieloidă cronică

2015 ◽  
Vol 23 (2) ◽  
Author(s):  
Roxana Popescu ◽  
Angela Dăscălescu ◽  
Cătălin Dănăilă ◽  
Doramina Ghiorghiu ◽  
Mihaela Zlei ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Philadelphia Chromosome ◽  
Tyrosine Kinase Domain ◽  
Blast Transformation ◽  
Monocytic Differentiation ◽  
Blast Phase ◽  
Therapeutic Decisions

AbstractThe coexistence of t(9;22) and inv(16) has been described in a very limited number of cases of CML, de novo or therapy-related AML. We report a patient with CML who presented both inversion of chromosome 16 and Philadelphia chromosome and evolved towards the blast phase under treatment with Imatinib. Laboratory diagnosis and monitoring was made by flow cytometry, conventional cytogenetics and molecular genetics techniques. The inv(16), detected by karyotyping in the Philadelphia chromosome positive clone at the moment of the blast transformation, was retrospectively assessed by means of real-time PCR, and was proved to have been present since diagnosis. The bone marrow biopsy performed in the blast phase of CML confirmed the presence of blasts belonging to the myeloid lineage, with indications of monocytic differentiation, frequently associated with inv(16). Moreover, the case also associated a F359V tyrosine kinase domain mutation, resulting in intermediate resistance to Imatinib and Nilotinib, which imposed therapy-switch to Dasatinib. In our case the evolution was progressive, followed by death due to lack of response to tyrosine kinase inhibitors, 18 months after diagnosis. The coexistence of t(9;22) and inv(16) in CML seems to be associated with an aggressive clinical evolution and resistance to tyrosine kinase inhibitor therapy. Due to the very small number of cases described in literature, therapeutic decisions are still difficult for patients displaying these abnormalities

Estradiol Induces Gene Proximity and MLL-MLLT3 Fusion In An AICDA-Mediated Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 806-806
Author(s):  
Andrew T Vaughan ◽  
Rebecca Wright ◽  
Katrina Slemmons
Keyword(s):  
Acute Leukemia ◽  
Dna Cleavage ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Hot Spot ◽  
Clinical Samples ◽  
Common Mechanism ◽  
Mll Gene ◽  
Increased Risk ◽  
Translocation Breakpoints

Abstract Translocation breakpoints involving the MLL gene linked to Infant Acute Leukemia (IAL) and therapy related acute leukemia (tAL) are tightly clustered between MLL exons 8 and 12. Exon 12 also marks the location of a well-described cleavage hotspot that is synchronous with a sharp decline in total MLL fusions observed in clinical samples. Though multiple MLL fusion partners have been identified, fusions to MLLT3 (AF9) and AFF1 (AF4) comprise 56% of all clinical rearrangements so far assayed. Epidemiological data has linked maternal exposure to birth control formulations with an increased risk of IAL involving MLL gene rearrangements. Subsequent in-vitro studies suggested a role of estradiol (E2) in the generation of such rearrangements. In order to probe the action of E2 in generating these lesions, the ability of E2 to impact MLL rearrangement formation was studied, focusing on the exon 12 hotspot and using immortalized but not transformed TK6 lymphoblastoid cells. Real-time PCR studies showed that in this cell line, transient exposure to 10 nM E2 enhanced transcription of MLL eight fold over controls. E2 treatment also increased transcription of MLL partner genes, MLLT3 and AFF1, through to a lesser degree. To determine if the process of transcription led to gene co-localization, chromatin conformation capture (3C) experiments were performed. Here, brief exposure to 10 nM E2 led to the co-localization of MLL with MLLT3, using primer sets targeting both MLL introns 9 and 13 and MLLT3 introns 4 and 8. These data indicated contact between these two genes, over a substantial region, consistent with their occupation of an operationally defined “transcription factory”. Surprisingly, low levels of E2 also stimulated the generation of de-novo MLL- MLLT3 fusion transcripts, without the application of any genotoxic stressors. To identify the process whereby each gene is fragmented, an essential precursor to any rearrangement, RNAi knockdown of activation induced cytidine deaminase (AICDA) was studied. AICDA activity is normally associated with class switch recombination and somatic hypermutation, but has recently been identified as the fragmenting agent associated with c-myc/IgH fusions in Burkitts lymphoma. RNAi knockdown of AICDA suppressed the induction of MLL-MLLT3 fusion transcript formation observed with E2 treatment, suggesting AICDA involvement in MLL and partner gene fragmentation. To probe this association in more detail, a ChIP analysis was performed targeting AICDA recruitment to MLL intron 11 (hot spot for rearrangements) or intron 12 (few MLL rearrangements). Here, E2 dependent localization of AICDA was noted upstream of the DNA cleavage hotspot and within the region of elevated MLL fusions in intron 11, but not in a region showing few rearrangements. Combined, these studies show that concentrations of E2 that occur during pregnancy, or during use of oral contraceptives, have the potential to initiate MLL fusions through an endogenous AICDA-mediated mechanism, that is enhanced by gene proximity associated with synchronous transcription of both MLL and partner genes. Further, the link between transcription-induced co-localization and MLL rearrangements may identify a common mechanism of MLL fusion gene formation relevant to a wider range of clinical diagnoses. If correct, then this mechanism may be a target for manipulation, particularly in controlled settings such as the delivery of potentially leukemogenic therapeutics. Disclosures: No relevant conflicts of interest to declare.

Bcr-Abl Tyrosine Kinase Domain Mutations Both Pre-Exist and Develop during Imatinib Treatment and Are Responsible for Resistance in Patients with Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ALL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 639-639 ◽  
Author(s):  
Heike Pfeifer ◽  
Barbara Wassmann ◽  
Anna Pavlova ◽  
Lydia Wunderle ◽  
Patrick Brueck ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
De Novo ◽  
Point Mutations ◽  
Kinase Domain ◽  
Disease Stage ◽  
Tyrosine Kinase Domain ◽  
Imatinib Treatment ◽  
Newly Diagnosed ◽  
Imatinib Resistance ◽  
Prior Chemotherapy

Abstract Background: Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL are an important cause of resistance to imatinib (IM) in pts. with CML and Ph+ ALL. The significantly inferior response to IM in Ph+ALL pts. who failed prior chemotherapy compared to those with de novo Ph+ALL suggests that treatment with cytotoxic drugs may promote the development of TKD mutations. However, it is not known whether the frequency and pattern of TKD mutations at the time of treatment initiation with TK inhibitors are related to disease stage or prior anti-leukemic therapy. Moreover, the potential of combined treatment with IM and multi-agent chemotherapy to influence the development of mutational resistance, as compared to IM alone, has not been determined. Patients and methods: 51 pts. with newly diagnosed Ph+ALL (>55 yrs.) enrolled in a GMALL study of combined IM and chemotherapy, and 68 Ph+ALL pts. who had failed prior chemotherapy and received single-agent IM as salvage therapy were analysed for the occurrence of point mutations within the TKD. Bone marrow samples collected pre-treatment, during therapy and at relapse were examined by denaturing high-performance liquid chromatography (D-HPLC) and cDNA sequencing. Results: The frequency of TKD mutations pre-IM was 44% in newly diagnosed Ph+ALL and 53% (34/64) in pts. with advanced Ph+ALL. At relapse after combination therapy (n=19), the frequency of de novo ALL pts. harbouring a TKD mutation had increased to 89% (P-loop 47%, T315I 29%, A-loop 24%), 2 pts. (11%) showed wild-type BCR-ABL. The frequency of TKD mutations in pts. with advanced disease who relapsed after IM was 55% (P-loop 73%, T315I 23%, A-loop 4 %). In both patient groups, the D-HPLC pattern showed concordance between the mutation detected in pre-therapeutic specimens and the dominant mutation detected at relapse. The CR rate in de novo pts. receiving IM induction was 90 % irrespective of detectable mutations pre-study. Bcr-abl transcripts became undetectable during the course of therapy in 40% of pts. with and 37% of pts. without a mutation. Median remission duration in pts. with a T315I mutation (n=4) was 130 d (range: 53–319d), in contrast to 526 d (range: 504–549d) with activation loop and 411 d (range: 106–745d) with P-loop mutations. To date, 7 pts. with an initially detected mutation remain in CR after median FU of 12.8 mo (range 2.4–24.5 mo.). Conclusions: Bcr-abl TKD mutations are detectable prior to first imatinib exposure in approximately 50% of Ph+ALL patients. Clinical imatinib resistance is in most cases associated with the identical mutation detected pre-IM, which is not eradicated by the combination of chemotherapy and IM. Identification and elimination of TKD mutations during early stages of treatment is essential to improve treatment.

Argentine Registry of Hematologic Disease (RAEH) Chronics Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4455-4455
Author(s):  
Alicia Inés Enrico ◽  
Beatriz Moiraghi ◽  
Graciela Klein ◽  
Maria del Rosario Cabrejo ◽  
Renee Crisp ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Target Population ◽  
First Year ◽  
Disease Epidemiology ◽  
Environmental Causes

Abstract Abstract 4455 Introduction: The CML Registry was developed by the Argentine Society of Hematology as a part of the centralized RAEH. Results from epidemiologic and clinical data related to CML, will enable to determine the geographic distribution of the target population, to establish associated environmental causes, and mainly to rationalize resources supply. Objective: a) To analyze characteristics of CML patients registered in the RAEH; b) To evaluate the CML Registry performance through its first year. Materials and Methods: Patients with CML registered in the RAEH from January 1st, 2011 up to July 31st, 2011. The protocol allowed to enroll de novo patients as well as patients diagnosed from 2000 on. Result: Data reported by 15 hospitals were included: 224 patients were registered. Mean age was 50 (18–86 y) and gender distribution was female: 102, male: 122. Occupational data showed no a characteristic pattern. 96.5% of patients were diagnosed in chronic phase, while 3.5% were diagnosed in accelerated phase/blast crisis. In 6% of patients cytogenetic tests detected 8 abnormalities besides t(9,22): double Philadelphia chromosome and monosomy 12 were the most frequent findings. FISH tests were recorded for 18% of patients at the time of diagnosis. Bone marrow biopsy was reported as a diagnostic procedure in 51%. Qualitative BCR/ABL was recorded for 31% of patients at the time of diagnosis. Molecular RQ-PCR tests for follow-up of treatment response were reported for 51% of patients. Of the registered population, 21% received interferon as previous therapy to Imatinib (IM); 89% received IM 400mg daily; 6% required dose increase. Second line treatment with dasatinib or nilotinib was recorded in 34% and 14% of patients, respectively. At 60 months mean follow-up 8% of the registered population had developed blast crisis and 6% had died. Conclusions. The RAEH’s first year of performance in CML was assessed. This only reflects the experience with 15 sites. Data registered will allow us in the following years to learn about disease epidemiology and available resources to improve patient accessibility. Disclosures: No relevant conflicts of interest to declare.

Activation Of p53 By MDM2-Inhibition Sensitizes Quiescent Blast Crisis CML Stem/Progenitor Cells To Bcl-2 Inhibitor- and Tyrosine Kinase Inhibitor-Induced Cell Death

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1476-1476
Author(s):  
Bing Z Carter ◽  
Po Yee Mak ◽  
Duncan H Mak ◽  
Marina Konopleva ◽  
Jorge Cortes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Annexin V ◽  
Apoptosis Resistance ◽  
Protein Levels

Abstract The transcriptional activation of pro-apoptotic Bcl-2 family members is a major mechanism of p53-mediated cell death. p53 can also directly contribute to mitochondrial mediated apoptosis by interacting with Bcl-2 family proteins. A number of anti-apoptotic Bcl-2 proteins such as Mcl-1 and Bcl-xL are regulated by the Bcr-Abl tyrosine kinase and confer apoptosis resistance to CML cells. It is therefore not surprising that p53 gene alterations may play a role in the disease progression of CML. The reported p53 mutation rate of 30% suggests potential therapeutic benefit of p53 activation by MDM2 inhibition alone or as sensitization of CML to therapeutic agents. Tyrosine kinase inhibitors (TKIs), the front-line therapy for patients with chronic phase CML, are less effective in patients with blast crisis (BC) CML and inactive against quiescent CML stem/progenitor cells. Here we examine the effects of nutlin3a, which activates p53 by inhibiting MDM2, and its combinations with the Bcl-2 inhibitor ABT-737 and the TKI nilotinib on the viability of proliferating and quiescent CD34+CML stem/progenitor cells obtained from patients with BC CML. Mononuclear cells from patients with BC CML who were resistant to multiple TKIs were stained with the cell division tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM) derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells are distinguishable by flow cytometry (within 7 to 12 days), the cells were treated with nutlin3a, ABT-737, nilotinib, nutlin3a plus ABT-737, or nutlin3a plus nilotinib for 48 hours with or without MSC co-culture. The cells were then stained with a CD34 antibody, and apoptosis (annexin V staining) was determined by flow cytometry. Apoptosis in proliferating and quiescent progenitor cells was defined as percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbrightcells, respectively. Nutlin3a alone was able to decrease viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50 = 2.50 ± 0.92 μM and 2.54 ± 0.60 μM, respectively) and quiescent (IC50 = 3.70 ± 1.22 μM and 4.27 ± 0.77 μM, respectively) CD34+ CML stem/progenitor cells. Although not very active by itself, ABT-737, when combined with nutlin3a (n = 6), induced apoptosis synergistically in proliferating (CI = 0.24 ± 0.12) and more so in quiescent (CI = 0.09 ± 0.10) CD34+CML stem/progenitor cells, even when cells were co-cultured with MSCs (CI = 0.22 ± 0.09 for proliferating and CI = 0.03 ± 0.05 for quiescent stem/progenitor cells). The combination of nutlin3a with nilotinib (n = 4) was also highly synergistic in proliferating stem/progenitors (CI = 0.02 ± 0.04 without and CI = 0.05 ± 0.04 with co-culture) and in quiescent progenitors (CI = 0.07 ± 0.29 without and CI = 0.03 ± 0.06 with co-culture). Treatment of primary CML blast cells with nultin3a increased p53, and pro-apoptotic Bax and Puma levels in all samples examined, indicating activation of transcriptional activity of p53. In addition, nutlin3a treatment decreased the protein levels of anti-apoptotic Bcl-xL and/or Mcl-1. Nilotinib also decreased the expression, both at the RNA and protein levels, of Mcl-1 and Bcl-xL, even in patients who had not responded to nilotinib treatment clinically. Conclusions Here we demonstrate that activation of p53 induces apoptosis in proliferating and quiescent BC CML stem/progenitor cells and sensitizes to Bcl-2 inhibitor- and TKI-induced cell death by, at least in part, regulating the expression of Bcl-2 family proteins. Results suggest a potential for utilizing this strategy in the treatment of BC CML, and also for the elimination of quiescent CML stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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