Concurrent Diagnosis of Chronic Myeloid Leukemia and Follicular Lymphoma: An Unreported Presentation

Lymphadenopathy in chronic myeloid leukemia (CML) is usually due to extramedullary involvement with accelerated or blast phases of the disease. The occurrence of non-Hodgkin lymphoma (NHL) as a synchronous malignancy with CML is rare. We report a case of a 73-year-old male who presented with dyspnea and right-sided lower extremity edema in the setting of leukocytosis. Bone marrow evaluation indicated a chronic phase chronic myeloid leukemia (CML), confirmed by molecular testing. Imaging of the chest for persistent dyspnea revealed supraclavicular and mediastinal lymphadenopathy. Biopsy of the cervical node showed expanded lymphoid follicles with atypical germinal centers that were positive for CD10, BCL-2, and BCL-6, consistent with follicular lymphoma (FL). Nodal PCR demonstrated clonal IGH and IGK gene rearrangements, and FISH analysis was positive for IGH-BCL-2 fusion. Together, these tests supported the diagnosis of FL. Additionally, the lymph node showed paracortical expansion by maturing pan-hematopoietic elements, no blastic groups, and positive RT-PCR analysis for BCR-ABL1, indicating concomitant involvement by chronic phase-CML. To our knowledge, this is the first reported case of a patient with a concurrent diagnosis of CML and FL.

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Variable transcription of BCR-ABL by Ph+ cells arising from hematopoietic progenitors in chronic myeloid leukemia [see comments]

Blood ◽

10.1182/blood.v83.7.1744.bloodjournal8371744 ◽

1994 ◽

Vol 83 (7) ◽

pp. 1744-1749 ◽

Cited By ~ 1

Author(s):

A Keating ◽

XH Wang ◽

P Laraya

Keyword(s):

Chronic Myeloid Leukemia ◽

Internal Control ◽

Myeloid Leukemia ◽

Critical Role ◽

Pcr Amplification ◽

Chronic Phase ◽

Pcr Analysis ◽

Rt Pcr ◽

Polymerase Chain ◽

Aberrant Gene

Recent studies suggest that the BCR-ABL gene plays a critical role in the pathogenesis of Ph+ chronic myeloid leukemia (CML). We investigated the hematopoietic colonies derived from the marrows of 12 patients with Ph+ CML in chronic phase by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of BCR-ABL mRNA and by cytogenetics. Colonies were individually harvested and each colony divided into two portions, one for cytogenetics and the other for isolation of total RNA for PCR of BCR-ABL transcripts and for an RNA internal control. We found that 23% +/- 18% (mean +/- SD, range 0% to 60%) of Ph+ colonies did not transcribe the aberrant gene. In each case when BCR-ABL transcription was not detected, normal ABL mRNA was present. The data suggest that hitherto unknown mechanisms may regulate BCR-ABL expression in some Ph+ cells and indicate that caution should be exercised in the interpretation of results using RT-PCR analysis of hematopoietic colonies from clinical specimens and from experiments with antisense oligonucleotides directed at the BCR-ABL gene. These data also raise the notion of a transitional Ph+ precursor cell in which BCR-ABL may become upregulated and lead to a fully expressed phenotype. We conclude that further studies correlating the frequency of Ph+ PCR- progenitors with prognostic clinical variables are warranted.

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Variable transcription of BCR-ABL by Ph+ cells arising from hematopoietic progenitors in chronic myeloid leukemia [see comments]

Blood ◽

10.1182/blood.v83.7.1744.1744 ◽

1994 ◽

Vol 83 (7) ◽

pp. 1744-1749 ◽

Cited By ~ 42

Author(s):

A Keating ◽

XH Wang ◽

P Laraya

Keyword(s):

Chronic Myeloid Leukemia ◽

Internal Control ◽

Myeloid Leukemia ◽

Critical Role ◽

Pcr Amplification ◽

Chronic Phase ◽

Pcr Analysis ◽

Rt Pcr ◽

Polymerase Chain ◽

Aberrant Gene

Abstract Recent studies suggest that the BCR-ABL gene plays a critical role in the pathogenesis of Ph+ chronic myeloid leukemia (CML). We investigated the hematopoietic colonies derived from the marrows of 12 patients with Ph+ CML in chronic phase by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of BCR-ABL mRNA and by cytogenetics. Colonies were individually harvested and each colony divided into two portions, one for cytogenetics and the other for isolation of total RNA for PCR of BCR-ABL transcripts and for an RNA internal control. We found that 23% +/- 18% (mean +/- SD, range 0% to 60%) of Ph+ colonies did not transcribe the aberrant gene. In each case when BCR-ABL transcription was not detected, normal ABL mRNA was present. The data suggest that hitherto unknown mechanisms may regulate BCR-ABL expression in some Ph+ cells and indicate that caution should be exercised in the interpretation of results using RT-PCR analysis of hematopoietic colonies from clinical specimens and from experiments with antisense oligonucleotides directed at the BCR-ABL gene. These data also raise the notion of a transitional Ph+ precursor cell in which BCR-ABL may become upregulated and lead to a fully expressed phenotype. We conclude that further studies correlating the frequency of Ph+ PCR- progenitors with prognostic clinical variables are warranted.

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Discontinuation of tyrosine kinase inhibitors in chronic myeloid leukemia: when is this a safe option to consider?

Hematology ◽

10.1182/asheducation-2013.1.184 ◽

2013 ◽

Vol 2013 (1) ◽

pp. 184-188 ◽

Cited By ~ 6

Author(s):

Kendra Sweet ◽

Vivian Oehler

Keyword(s):

Chronic Myeloid Leukemia ◽

Imatinib Mesylate ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Deep Level ◽

Chronic Phase ◽

Cytogenetic Response ◽

Response To Therapy ◽

Molecular Response ◽

Rt Pcr

Abstract Mrs G is a 54-year-old woman with a diagnosis of chronic-phase chronic myeloid leukemia dating back 8 years. She had a low-risk Sokal score at diagnosis and was started on imatinib mesylate at 400 mg orally daily within one month of her diagnosis. Her 3-month evaluation revealed a molecular response measured by quantitative RT-PCR of 1.2% by the International Scale. Within 6 months of therapy, she achieved a complete cytogenetic response, and by 18 months, her BCR-ABL1 transcript levels were undetectable using a quantitative RT-PCR assay with a sensitivity of ≥ 4.5 logs. She has maintained this deep level of response for the past 6.5 years. Despite her excellent response to therapy, she continues to complain of fatigue, intermittent nausea, and weight gain. She is asking to discontinue imatinib mesylate and is not interested in second-line therapy. Is this a safe and reasonable option for this patient?

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The t(8;22) in chronic myeloid leukemia fuses BCR to FGFR1: transforming activity and specific inhibition of FGFR1 fusion proteins

Blood ◽

10.1182/blood.v98.13.3778 ◽

2001 ◽

Vol 98 (13) ◽

pp. 3778-3783 ◽

Cited By ~ 137

Author(s):

Asuman Demiroglu ◽

E. Joanna Steer ◽

Carol Heath ◽

Kerry Taylor ◽

Mark Bentley ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Messenger Rna ◽

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Growth Factor Receptor ◽

Chronic Phase ◽

Mek Inhibitor ◽

Farnesyltransferase Inhibitors ◽

Rt Pcr ◽

Comparable Rate

Abstract This report describes 2 patients with a clinical and hematologic diagnosis of chronic myeloid leukemia (CML) in chronic phase who had an acquired t(8;22)(p11;q11). Analysis by fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR) indicated that both patients were negative for the BCR-ABL fusion, but suggested that the BCR gene was disrupted. Further FISH indicated a breakpoint within fibroblast growth factor receptor 1 (FGFR1), the receptor tyrosine kinase that is known to be disrupted in a distinctive myeloproliferative disorder, most commonly by fusion to ZNF198. RT-PCR confirmed the presence in both cases of an in-frame messenger RNA fusion between BCR exon 4 and FGFR1 exon 9. Expression of BCR-FGFR1 in the factor-dependent cell line Ba/F3 resulted in interleukin 3-independent clones that grew at a comparable rate to cells transformed with ZNF198-FGFR1. The growth of transformed cells was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, the farnesyltransferase inhibitors L744832 and manumycin A, the p38 inhibitors SB202190 and SB203580 but not by the MEK inhibitor PD98059. The growth of BaF3/BCR-FGFR1 and BaF3/ZNF198-FGFR1 was not significantly inhibited by treatment with STI571, but was inhibited by SU5402, a compound with inhibitory activity against FGFR1. Inhibition with this compound was associated with decreased phosphorylation of ERK1/2 and BCR-FGFR1 or ZNF198-FGFR1, and was dose dependent with an inhibitory concentration of 50% of approximately 5 μM. As expected, growth of BaF3/BCR-ABL was inhibited by STI571 but not by SU5402. The study demonstrates that the BCR-FGFR1 fusion may occur in patients with apparently typical CML. Patients with constitutively active FGFR1 fusion genes may be amenable to treatment with specific FGFR1 inhibitors.

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Down Regulation of TOPK, PBX3, SRPK, DDX21 and CLC Genes in Newly Diagnosed, Early-Chronic Phase Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate.

Blood ◽

10.1182/blood.v104.11.4650.4650 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4650-4650

Author(s):

Angela Poerio ◽

Marilina Amabile ◽

Simona Soverini ◽

Matteo Renzulli ◽

Gianantonio Rosti ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Chromosome 9 ◽

Pcr Analysis ◽

Newly Diagnosed ◽

Rt Pcr

Abstract Background: Due to the BCR/ABL chimeric protein expression in chronic myeloid leukemia (CML), several genes and signaling pathways have been reported to be activated. These alterations in gene expression contribute to the pathophysiology of p210BCR/ABL-mediated transformation. Some of the overexpressed genes are implicated in cellular processes known to be disturbed in CML, including the mitogen-activated protein kinase or the ubiquitin phatway, whereas overexpression of other genes may implicate new cellular pathways involved in CML. The characterization of new genes which could be implicated in the pathophysiology of CML, may lead to the identification of potentially novel therapeutic targets for CML. In a previous study we adopted a microarray analysis to study the gene expression profiling of CD34+ cells taken from 5 de novo CML patients and treated in vitro with Imatinib (data not shown). Five genes had shown a greater down-regulation and a possible involvement in the in vitro effects of Imatinib. Aims: Aim of the present study was to confirm these data in vivo through a quantitative real time PCR analysis in 10 CML patients enrolled in a multicenter clinical trial of the GIMEMA CML working party (newly diagnosed, early-CP CML patients treated with Imatinib 400mg/die and peghilated interferon). Five patients were known to have chromosome 9 deletion. Methods: this study was performed on 10 bone marrow samples from 10 consecutive patients enrolled in the protocol. Bone marrow bone was collected prior to treatment (baseline) and after 3 and 6 months of Imatinib therapy. Five genes (TOPK, PBX3, SRPK, DDX21 and CLC) were analyzed at the RNA levels by means of a quantitative RT-PCR analysis (TaqMan). β2 microglobuline was used as control gene. Results were expressed as a median ΔCt value. Expression of these 5 genes before treatment with Imatinib and after 6 months of therapy was compared. Statistical significance was tested by the student’s T-Test. Results: Quantitative RT-PCR analysis confirmed that the 5 tested genes were downregulated after 6 months of treatment with Imatinib. About 1 log reduction was observed. The differences between the baseline median values and median values after 6 months of treatment were statistically significant (p>0.005). We were not able identify any differences between patients who showed chromosome 9 deletion and patients who did not. Conclusions: treatment of CML patients with the ABL-specific tyrosine kinase inhibitor Imatinib decreased expression at the mRNA level of all the genes tested. This suggests that increased gene expression could be in some case tyrosine-kinase dependent and it could be implicated in the pathophisiology of CML. Study supported by: COFIN 2003 (M. Baccarani), AIRC, AIL, Fondazione del Monte di Bologna e Ravenna, FIRB 2001.

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Comparison of Cytogenetics and Interphase Fluorescence In Situ Hybridization in Newly Diagnosed Ph+ Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate. A Study by the GIMEMA Working Party on CML. On Behalf of GWP on CML.

Blood ◽

10.1182/blood.v106.11.4857.4857 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4857-4857

Author(s):

Nicoletta Testoni ◽

Simona Luatti ◽

Chiara Nicci ◽

Elena Montanari ◽

Giulia Marzocchi ◽

...

Keyword(s):

In Situ Hybridization ◽

Chronic Myeloid Leukemia ◽

Fluorescence In Situ Hybridization ◽

Myeloid Leukemia ◽

Chronic Phase ◽

Working Party ◽

Chromosome 9 ◽

Fish Analysis ◽

Imatinib Treatment

Abstract To asses cytogenetic pattern of early diagnosed chronic phase chronic myeloid leukemia (CML) patients and to evaluate the role of either conventional (CC) and molecular cytogenetics in three multicentric studies, karyotype and interphase fluorescence in situ hybridization (FISH) analyses were performed in 372 enrolled patients between April 2004 and July 2005 by the GIMEMA CML Working Party (WP). Local investigator laboratories (25 labs) or WP reference labs (12 labs) performed both analyses. Cytogenetic examinations was performed at baseline; after 6 and 12 months of imatinib treatment; thereafter every 6 months and in case of failure or disease progression. At the baseline, 257 patients have been studied and 237 (92%) are valuable for both analyses (CC and FISH). Additional abnormalities in Ph+ clone have been observed in 12 patients (5%). Moreover, 18 (8%) cases showed variant Ph translocations and in 23 (10%) patients the derivative of chromosome 9 was deleted. As yet, cytogenetic response (CR) was evaluated in 188 samples and 156 cases were valuable (83%): 20 at 3 months, 101 at 6 months and 35 at 1 year of treatment. One hundred and eighteen (76%) patients achieved complete CR (CCR) established with more than 20 metaphases in 84 cases, meanwhile in 34 CCR cases the number of examined metaphases was lower. In the first group, 70/84 (83%) samples showed absence of bcr/abl rearrangement in FISH, meanwhile 13/84 (16%) carried a low rate of positive cells (1–5%) and the last one showed the rearrangement in 12% of cells. In the latter group, 23/34 (68%) didn’t show any rearrangement in FISH, in 8/34 (24%) the amount of Ph+ cells was low (1–5%), in 2 was higher (7% and 10%) and the last one carried an high rate (72%) of rearranged cells. In this latter case the RCC was evaluated on 10 metaphases. Twenty-three patients in major CR (MCR), but not in CCR, showed retention of persisting Ph+ cells ranging from 2 to 21% in CC study and from 2 to 16% in FISH analysis. Moreover we found a patient with 2% of Ph+ metaphases and 53% of Ph+ cells in FISH: in this case the CC evaluation was established with 10 metaphases. We can suggest there was a good correlation between cytogenetic and FISH tests in terms of the kinetics of disappearance of the bcr/abl rearrangement. FISH is a reliable method to reveal submicroscopic deletions and to monitor the size of the Ph + clone in treated CML patients. However, a good CC analysis remains an excellent approach to the evaluation of response to Imatinib. Moreover it can detect the emergence of other abnormalities in Ph positive or negative clone.

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Overexpression of MEL1 as a Novel Fusion Partner of AML1 in the Blastic Phase of Chronic Myeloid Leukemia with the Recurrent Cryptic Translocation t(1;21)(p36.3;q22).

Blood ◽

10.1182/blood.v106.11.4332.4332 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4332-4332

Author(s):

Sawcene Hazourli ◽

Pierre Chagnon ◽

Raouf Fetni ◽

Lambert Busque ◽

Josee Hebert

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Zinc Finger Protein ◽

Fish Analysis ◽

Fusion Partner ◽

Rt Pcr ◽

Enhancer Element ◽

Intron 1 ◽

Cryptic Translocation ◽

Pr Domain

Abstract Located at 1p36.3, MEL1 is a member of the MDS1/EVI1 gene family and encodes a zinc finger protein with a N-terminal PR-domain. It has been proposed that the overexpression of a modified version of MEL1 lacking the PR domain is oncogenic, whereas MEL1 retaining the PR domain is anti-tumorigenic. MEL1 is known to be overexpressed in some myeloid malignancies with reciprocal translocation t(1;3)(p36.3;q21) characterized by trilineage dysplasia and poor prognosis. It is suggested that the translocation of RPN1 gene at 3q21 in the vicinity of MEL1 gene might activate MEL1 expression through an enhancer element. Here we characterized a recurrent cryptic translocation t(1;21)(p36.3;q22) that fuses MEL1 to AML1 gene in a blastic transformation of chronic myeloid leukemia (CML). Fluorescence in situ hybridization (FISH) analysis with BAC/PAC clones revealed that the breakpoints are in intron 1 of MEL1 and between intron 1 and exon 8 of AML1. RT-PCR analysis showed that AML1-MEL1, but not the reciprocal MEL1-AML1 was expressed. Many splicing variants are present, and all fusions splice the 5′ end of AML1 that contains the RUNT domain with almost the entire MEL1. Furthermore two fusion transcripts contained open reading frames making possible the translation of two forms of AML1-MEL1 fusion proteins. To investigate if AML1-MEL1 leads to an inappropriate expression of MEL1 we performed a quantitative RT-PCR with primers outside and within the fused MEL1 allowing the detection of the normal and the rearranged allele respectively. Interestingly, our data show that while no normal MEL1 transcript was detected, there was an overexpression of the fused MEL1. These results suggest that similarly to AML1-EVI1 gene, overexpression of MEL1 could be regulated by the AML1 promoter in leukemic cells with the AML1-MEL1 fusion gene and might also play an important role in the progression of CML. Moreover, in contrast to previous studies showing an antioncogenic role for the PR domain, our findings indicate that in some leukemias, the overexpression of MEL1 is not restricted to the MEL1 PR-lacking form. This suggests that the mechanism by which the PR domain has his effect, is more complex than previously thought.

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Interleukin (IL)-1 Beta and IL-6 Activated Acute and Chronic Myelogenous Leukemia-Derived Myofibroblasts to Express Leukemia-Specific Transcripts

Blood ◽

10.1182/blood-2018-99-117704 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5094-5094

Author(s):

Takuji Matsuo ◽

Haruko Tashiro ◽

Ritsu Sumiyoshi ◽

Tadashi Yamamoto ◽

Kensuke Matsumoto ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myelogenous Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Specific Antigen ◽

Myelogenous Leukemia ◽

Pcr Analysis ◽

Fish Analysis ◽

Rt Pcr ◽

Single Colony

Abstract Background and Aims: We previously reported that interleukin 1-beta (IL-1-b) stimulated bone-marrow stromal myofibroblasts from normal individuals to express CD34. And, when myofibroblasts were cultured with IL-1-b and IL-6, GATA-2 and CD45 expressed. We report here that bone marrow-derived myofibroblasts from acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) patients were also activated to express leukemia-specific molecules when IL-1-b and IL-6 were added to the cultures. Materials and Methods: Bone marrow samples were collected from informed 7 AML patients (2 cases of M2 with t(8;21), 3 of M0, M2, and M7 positive for FLT-ITD mutation, 2 of Philadelphia-positive bi-phenotypic acute leukemia (Ph-AL)), and 3 CML ones, whose mononuclear cells were separated, and were cultured, split, and re-cultured for one month to eliminate non-adherent cells, vascular cells, and monocytes/macrophages to prepare myofibroblasts. The obtained cells were further cultured for one more month to obtain myofibroblasts with stably growth, and then leukemia-specific molecules (FLT3-ITD, BCR-ABL1 fusion (major and minor), RUNX1-RUNX1T1 fusion) were analyzed at DNA and RNA levels. Cells were further cultured with IL-1-b and IL-6 for two weeks, and morphological changes and expressions of specific molecules were observed. Results: When bone marrow-derived myofibroblast obtained from myeloid leukemia patients were analyzed, leukemia-specific molecules were detected at DNA levels (FISH analysis detected 0.5-5% BCR-ABL1 fusion in CML- and Ph-AL-derived myofibroblast-cultures, 1-2% RUNX1- RUNX1T1 in AML with t(8;21)-cultures, and positive FLT3-ITD mutation at genomic PCR in AML-cultures); however, RT-PCR analyses revealed that leukemia-specific transcripts were not detected in all cells. When myofibroblasts were cultured with IL-1-b and IL-6, leukemia-specific transcripts were detected with RT-PCR analysis in all cases. Also, CD45, and GATA-2 expressed in these cultures. Discussion: We reported previously that a few populations of bone marrow-derived myofibroblasts obtained from AML and CML patients expressed leukemia-specific transcripts and proteins when cultured as a separated single colony; however, when myofibroblasts were cultured not as a fractionated single colony but a whole stromal cells for a long term, expression of leukemia-specific molecules weakened and no longer confirmed. Myofibroblasts with leukemic character seem to stop their proliferation and keep dormant. One interesting point is that normal stromal myofibroblasts derived from myeloid leukemia patients can inhibit the growth of myofibroblasts that have leukemic characters. Some mechanisms may work on this observation. Also, an important issue is that myofibroblasts reportedly have an ability of antigen presentation. If some myofibroblasts can express leukemia-specific molecules as well as HLA when cultured with IL-1-b and IL-6, they can act as a leukemia-specific antigen-presenting cell. We now attempt to research this theme to develop a new cell-mediated vaccine for myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

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Deregulated Bcl-2 Family Expression Drives Chronic Myeloid Leukemia Cancer Stem Cell Survival.

Blood ◽

10.1182/blood.v112.11.1074.1074 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1074-1074

Author(s):

Daniel Jacob Goff ◽

Annelie Abrahamsson ◽

Ifat Geron ◽

Catriona Jamieson

Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Rt Pcr ◽

Significant Difference ◽

Apoptotic Pathways ◽

Serial Transplantation

Abstract Introduction: A growing body of evidence suggests that a relatively rare subset of cells within a cancer subverts properties normally ascribed to stem cells in regenerating tissues, such as enhanced self-renewal and survival capacity. Recent studies suggest that these cancer stem cells (CSC) are resistant to treatments that target rapidly dividing cells. In blast crisis chronic myeloid leukemia (BC CML) and some forms of acute myelogenous leukemia (AML), research performed by ourselves and others indicates that CSC originate from the CD34+CD38+lineage- compartment of hematopoietic cells and can serially transplant blast crisis leukemia in immunodeficient mice. Despite abundant data indicating that Bcl-2 family proteins are involved in CML progression, the importance of these proteins in CSC survival remains to be elucidated. Clinical data have shown that CML stem cells become more resistant to therapies targeting BCR-ABL with progression to blast crisis. As BCR-ABL targeted therapy initiates apoptosis, these results suggest that CML CSC may become increasingly resistant to apoptosis with progression. Based on these findings and the results from our serial transplantation experiments, we hypothesized that CML CSC deregulate apoptosis pathways by differential expression of Bcl-2 family molecules and that these changes contribute to CSC ability to survive serial transplantation. Methods: Quantitative FACS Aria analysis of Bcl-2 protein levels was compared in blast crisis (n=5) and chronic phase CML (n=3) patient samples. Mean fluorescence intensity (MFI) of Bcl-2 staining was compared between different hematopoietic populations within the patient samples. For gene expression analysis, cDNA was made from RNA isolated from sorted progenitor populations (CD34+CD38+Lin −). Isoform specific RT-PCR was used to determine expression levels of Bcl-2, Bcl-X, and Mcl-1 isoforms. Mcl-1 expression was confirmed using qPCR. In addition, preliminary experiments were performed (n=2) to determine if CSC engraftment could be reduced in vivo by targeted inhibition of Bcl-2 family molecules using Apogossypol, a clinically tested Bcl-2-family inhibitor. Briefly, immunocompromised neonatal mice were transplanted intrahepatically with luciferase GFP transduced granulocytic sarcomas from mice transplanted with BC CSC using our previously published methodology (Geron et al, Cancer Cell 2008). Transplanted mice were treated for 15 days with Apogossypol by oral gavage and engraftment was monitored by weekly bioluminescent imaging. Engraftment levels were determined by FACS analysis of human CD45+ expression in mouse livers on week 11 post-transplant. Results: Comparing the MFI of Bcl-2 staining in the entire live mononuclear cell population, we detected no statistically significant difference in levels between the blast crisis and chronic phase samples. However, when we gated on separate cell populations, differences in the Bcl-2 MFI emerged. There was a statistically significant increase (P<0.03) in Bcl-2 MFI exclusively in the CD34+CD38+lineage- population of the blast crisis samples indicative of cell type and context specific deregulation of apoptosis in the CSC population. Further, we were interested in whether there were differences in Bcl-2 family expression at the transcriptional level. Notably, while we detected no difference in the levels of the isoforms of Bcl-2 and Bcl-X, splice isoform specific RT-PCR and qPCR revealed a decrease in the expression of the short isoform of Mcl-1, which encodes a pro-apoptotic protein, in serially transplanted BC CSC (CD34+CD38+). Together these results indicate that CML CSC may indeed deregulate the expression of several Bcl-2 family proteins. To test the therapeutic potential of inhibiting these deregulated apoptotic pathways in CML, we treated mice engrafted with CML CSC with Apogossypol, a broad-spectrum inhibitor of pro-survival Bcl-2 molecules. We saw a statistically significant decrease (P<0.05) in the number of CD45+ cells engrafted in the mouse liver after 3 weeks of Apogossypol treatment. Overall, our results suggest that the subversion of apoptosis plays an important role in allowing CML CSC to be serially transplanted and that apoptotic pathways may be a useful target for therapeutics aimed at inhibiting these cells.

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FLT3 Is Up-Regulated in Blast Crisis of Chronic Myeloid Leukemia and Combination of Small Interference RNA of FLT3 and Gleevec (STI571) Synergistically Induces the Apoptosis in K562 Cells.

Blood ◽

10.1182/blood.v110.11.4532.4532 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4532-4532

Author(s):

Young Y. Lee ◽

Kwang-Sung Ahn ◽

Sung-Soo Yoon ◽

Jung H. Choi ◽

Byoung B. Park ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

K562 Cells ◽

Pcr Analysis ◽

Wild Type ◽

Small Interference Rna ◽

Expression Levels ◽

Significant Difference

Abstract To identify a gene signature for prognostic markers at transition from chronic phase to blast crisis of chronic myeloid leukemia (CML), we have applied Affymetrix Genechips of 22,000 transcripts to analyze total RNA of CML cells from 12 patients with chronic phase and 12 patients with blast crisis. Data analysis using GeneSpring 6.0 generated a list of 143 differentially expressed genes. A total of 89 genes were up-regulated and 54 genes were down-regulated in blast crisis of CML, and vice versa in chronic phase of CML. Array data for 32 genes was validated using quantitative realtime PCR analysis. The expression levels of HSA6591, FLT3, NTE5, RSG1, LAF4, CPA3, ATF, FCGR3A, MYD88, IFIT1, TP73L, DTNA, MDA, and IL18R1 showed statistically significant difference (p < 0.05) between chronic phase and blast crisis. Since CML cells of blast crisis were generally unresponsive to STI571, we further analyzed roles of FLT3 which is known to be a poor prognositic marker in acute myeloid leukemia. For this experiment, K562 cells (CML blast cells) were transfected with small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human FLT3, resulting in the significant inhibition of FLT3 expression at mRNA and protein levels. MTT assay demonstrated that FLT3 knockdown K562 cells by shRNAs were more sensitive to STI571 compared to wild type of K562, although there was no difference at high concentration of STI571 (320 nM) between FLT3 knockdown K562 cells and wild type of K562 cells. The higher expression levels of apoptosis related genes (PARP, caspase-3, Bax) were observed in FLT3 knockdown K562 cells compared to wild type of K562 cells. Thus, RNA interference-directed targeting of FLT3 might be a novel treatment modality in STI571 refractory CML patients.

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