scholarly journals BCR/ABL-negative primitive progenitors suitable for transplantation can be selected from the marrow of most early-chronic phase but not accelerated-phase chronic myelogenous leukemia patients

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4770-4779 ◽  
Author(s):  
CM Verfaillie ◽  
R Bhatia ◽  
W Miller ◽  
F Mortari ◽  
V Roy ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
High Speed ◽  
Large Scale ◽  
Autologous Transplantation ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Advanced Phase ◽  
Selection Of

We have previously reported that selection of marrow cells on the basis of the CD34+HLA-DR- phenotype (34+DR-) may result in the recovery of Philadelphia chromosome (Ph)- and BCR/ABL-negative long-term culture- initiating cells (LTC-IC) in selected patients with chronic myelogenous leukemia (CML). We now present data on 27 early chronic-phase ([ECP] studied within 1 year after diagnosis) and 23 advanced-phase ([AP] late chronic phase, ie, studied >1 year from diagnosis, or accelerated phase) CML patients. Fluorescence-activated call-sorting (FACS)- selected 34+DR- and 34+DR+ cells were subjected to reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization. These cells were also cultured in long-term bone marrow culture for 1 to 5 weeks to examine the number of LTC-IC and the presence or absence of the BCR/ABL gene rearrangement in progeny of primitive LTC-IC. The number of 34+DR- cells and LTC-IC present in ECP CML marrow was similar to that in normal (NL) marrow, whereas the numbers were reduced in AP CML. Furthermore, 34+DR- cells from more than 80% of ECP CML patients were BCR/ABL mRNA- and Ph-negative and contained only BCR/ABL mRNA- and Ph-negative LTC-IC, whereas 34+DR- cells and LTC-IC from less than 40% of AP CML patients were BCR/ABL mRNA- and Ph-negative. In contrast to NL marrow, 34+DR+ cells from CML marrow, irrespective of clinical stage, contained large numbers of LTC- IC. CML 34+DR+ cells and LTC-IC were BCR/ABL mRNA- and Ph-positive. Since these studies suggested that a population of primitive progenitors that are Ph-negative can be selected from steady-state marrow in some ECP CML patients, we determined if similar results could be obtained when large quantities of marrow sufficient for transplantation are processed. We demonstrate that 1 to 3 x 10(5) BCR/ABL mRNA-negative 34+DR- cells/kg recipient body weight, containing only BCR/ABL mRNA-negative LTC-IC, can be obtained from a 2- to 2.5-L marrow collection by sequential COBE Spectra apheresis (COBE BCT, Lakewood, CO), CD34+ enrichment using the CEPRATE SC Cell-Concentrator (CellPro, Bothell, WA), and high-speed FACS. Thus, large-scale selection of a BCR/ABL mRNA- and Ph-negative 34+DR- cell population is possible in a fraction of chronic-phase CML patients, in whom these cells could be used to reconstitute the hematopoietic compartment following autologous transplantation.

scholarly journals Percentage of Philadelphia chromosome (Ph)-negative and Ph-positive cells found after autologous transplantation for chronic myelogenous leukemia depends on percentage of diploid cells induced by conventional- dose chemotherapy before collection of autologous cells

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3257-3263 ◽  
Author(s):  
M Talpaz ◽  
H Kantarjian ◽  
J Liang ◽  
L Calvert ◽  
J Hamer ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Mononuclear Cells ◽  
Autologous Transplantation ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Conventional Dose ◽  
Diploid Cells ◽  
Conventional Dose Chemotherapy ◽  
Marrow Cells

We collected peripheral blood mononuclear cells and bone marrow cells soon after recovery from conventional-dose chemotherapy-induced myelosuppression and transplanted these cells into advanced chronic myelogenous leukemia (CML) patients after treating these patients with 120 mg/kg cyclophosphamide, 750 mg/m2 VP-16, and 1,020 cGy of total body irradiation (TBI). Of the 10 late chronic-phase patients and the eight accelerated-phase CML patients evaluable posttransplant, 90% and 87%, respectively, remain alive posttransplant, whereas none of the three blast crisis CML patients given this therapy remain alive posttransplant. We measured the percentage of Philadelphia chromosome (Ph)-negative cells in the autologous cells collected after conventional-dose chemotherapy-induced myelosuppression before autologous transplant and in the marrow of these same CML patients after autologous transplantation of these cells into recipients treated with the cyclophosphamide, VP-16, and TBI. A direct correlation (correlation coefficient = 0.91) was observed between the level of Ph+ cells in the transplanted cells and the percentage of Ph+ marrow cells after transplant in 21 patients so transplanted. The data show that the chance of generating cytogenetic remissions post-transplant depends on the percentage of diploid cells in the preparations of autologous cells used for transplant and the stage of disease of the patients at the time of collection of the autologous cells.

scholarly journals Autologous activated natural killer cells suppress primitive chronic myelogenous leukemia progenitors in long-term culture

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2476-2485 ◽  
Author(s):  
F Cervantes ◽  
BA Pierson ◽  
PB McGlave ◽  
CM Verfaillie ◽  
JS Miller
Keyword(s):  
Natural Killer Cells ◽  
Natural Killer ◽  
Chronic Myelogenous Leukemia ◽  
Marrow Transplantation ◽  
Autologous Transplantation ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Killer Cells ◽  
Long Term Culture

A graft-versus-leukemia effect has been well documented to prevent relapse in chronic myelogenous leukemia (CML) after allogeneic marrow transplantation. One type of lymphocytes that may contribute to this effect are natural killer cells (NK), which after activation with interleukin (IL)-2, exhibit a broad range of cytolytic activity against allogeneic and autologous cells. We have previously demonstrated that IL-2-activated NK (ANK) can be generated from blood of patients with CML and are benign in origin. Their proliferation and function, however, diminish with disease progression in CML, suggesting a role in tumor surveillance. We studied the effect of IL-2-activated NK (ANK) on normal and malignant primitive and committed progenitors in a novel long-term bone marrow culture (LTBMC) assay. Because ANK destroy marrow stromal layers, the use of classic stroma-dependent long-term cultures is not possible. Therefore, we used the stroma noncontact LTBMC system developed in our laboratory to analyze the effect of autologous ANK cells on primitive hematopoietic progenitors. Autologous ANK (CD56+/CD3- ) were generated from the peripheral blood of 10 patients with chronic phase CML and from six normal individuals by culturing CD5/CD8-depleted mononuclear cells for 14 days in 1,000 U/mL IL-2. At the same time ANK cultures were initiated, sorted normal (CD34+/DR+) marrow populations were plated in Transwell inserts of the stroma noncontact culture. On day 15, hydrocortisone, which rapidly inhibits ANK function, was removed, and autologous ANK were added to the Transwell inserts with fresh LTBMC medium without hydrocortisone but supplemented with 1,000 U/mL IL-2. After 48 hours, the number of colony-forming cells (CFC) was enumerated in methylcellulose culture. To determine the effect of ANK on more primitive long-term culture-initiating cells (LTCIC), the IL-2- supplemented LTBMC medium was replaced with fresh hydrocortisone containing LTBMC medium, and cultures were maintained for an additional 5 weeks. We demonstrate that autologous ANK did not suppress normal CFC or LTCIC. In contrast, ANK from eight patients with CML with potent cytotoxicity against NK-sensitive (K562) NK-resistant (Raji) tumor targets exhibited an ANK dose-dependent suppression of both CFC and LTCIC. Interestingly, ANK from two patients with CML who exhibited diminished cytotoxicity also did not suppress autologous CFC and LTCIC. These studies indicate that ANK with potent major histocompatibility complex unrestricted cytotoxic activity suppress malignant hematopoiesis. This effect was not mediated by soluble factors and was absolutely dependent on direct cell-to-cell contact. We further demonstrate that the beta2 integrin receptor is involved in ANK recognition of CML targets. These observations support the use of autologous ANK therapy to prevent relapse of CML after autologous marrow transplantation or use of ANK to purge CML marrow for autologous transplantation.

scholarly journals Association of a chromosomal 3;21 translocation with the blast phase of chronic myelogenous leukemia

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1338-1342 ◽  
Author(s):  
CM Rubin ◽  
RA Larson ◽  
MA Bitter ◽  
JJ Carrino ◽  
MM Le Beau ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Reciprocal Translocation ◽  
Clonal Evolution ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Cytogenetic Abnormality ◽  
Blast Phase ◽  
Male Patients

Abstract An identical reciprocal translocation between the long arms of chromosomes 3 and 21 with breakpoints in bands 3q26 and 21q22, t(3;21)(q26;q22), was found in three male patients with the blast phase of chronic myelogenous leukemia (CML). The abnormality was clonal in all three patients and was always accompanied by either a standard or variant 9;22 translocation resulting in a Philadelphia chromosome (Ph1). In two cases, the t(3;21) was the only abnormality other than a t(9;22) in the primary clone. Serial studies of one patient demonstrated that the t(3;21) occurred as a result of clonal evolution near the time of development of the blast phase. We have not observed the t(3;21) in greater than 500 patients with CML in the chronic phase. Thus, the t(3;21) is a new recurring cytogenetic abnormality associated with the blast phase of CML.

scholarly journals RAS mutations are rare events in Philadelphia chromosome-negative/bcr gene rearrangement-negative chronic myelogenous leukemia, but are prevalent in chronic myelomonocytic leukemia

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1214-1219 ◽  
Author(s):  
C Hirsch-Ginsberg ◽  
AC LeMaistre ◽  
H Kantarjian ◽  
M Talpaz ◽  
A Cork ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Philadelphia Chromosome ◽  
Point Mutations ◽  
Chronic Phase ◽  
Chronic Myelomonocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Ras Oncogene ◽  
Ras Mutations ◽  
Ras Oncogenes ◽  
Myelomonocytic Leukemia

Abstract Previous reports have indicated that mutations of the RAS oncogenes are not associated with the chronic phase of Philadelphia chromosome- positive chronic myelogenous leukemia (Ph1+ CML). However, further studies were needed to determine their association with Ph1- CML and chronic myelomonocytic leukemia (CMML). Therefore, 6 patients with Ph1- CML who were also negative for BCR rearrangements (Ph1-/BCR- CML) and 30 patients with CMML were analyzed for the presence of RAS oncogene point mutations to determine the similarities of these diseases at the molecular level. The assay used the polymerase chain reaction for amplification of the target RAS sequences and panels of specific synthetic oligonucleotide probes for hybridization to wild type and/or mutated sequences. None of the six Ph1-/BCR- CML patients had mutations in the RAS oncogenes, while 17 of 30 (57%) of the CMML patients had RAS oncogene mutations. Eighty percent of the mutations involved substitution of aspartic acid for glycine (G----A) in the 12th or 13th codons of N-ras or K-ras. Furthermore, although not statistically significant, survival studies raise the possibility of shortened survival in patients with RAS oncogene point mutations, with the average survival being 33 months for Ph1-/BCR- CML, 35 months for CMML without point mutations, and 11 months for CMML with RAS mutations. Thus, RAS mutations appear to be associated with CMML and not Ph1-/BCR- chronic phase CML, there is a high propensity for the K-ras or N-ras mutations to involve an G----A substitution in the 12th or 13th codons, and RAS mutations in CMML may relate to prognosis and require further studies.

Early treatment decisions with interferon-alfa therapy in early chronic-phase chronic myelogenous leukemia.

1998 ◽  
Vol 16 (3) ◽  
pp. 882-889 ◽  
Author(s):  
S Sacchi ◽  
H M Kantarjian ◽  
T L Smith ◽  
S O'Brien ◽  
S Pierce ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Univariate Analysis ◽  
Group Analysis ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Interferon Alfa ◽  
Myelogenous Leukemia ◽  
Costal Margin ◽  
Hematologic Response

PURPOSE To determine, in patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) on interferon alfa (IFNalpha), whether combining pretreatment characteristics and early response profiles would distinguish patients with differential benefits that would allow better decisions on subsequent therapy. PATIENTS AND METHODS A total of 274 patients treated from 1982 through 1990 with IFNalpha regimens were analyzed. A second group of 137 patients treated with IFNalpha and low-dose cytarabine (ara-C) between 1990 and 1994 was later used to confirm the guidelines derived from the original study group analysis. Patients' pretreatment factors and response to IFNalpha therapy at 3, 6, and 12 months were analyzed in relation to subsequent achievement of major cytogenetic response. After univariate analysis of prognostic factors, a multivariate analysis selected, at 6 months, independent pretreatment factors that added to the response status in predicting subsequent outcome. The results were then applied at the 3- and 12-month periods and confirmed in the subsequent population. RESULTS Response to IFNalpha therapy at 3, 6, and 12 months was a significant predictor of later major cytogenetic response. The presence of splenomegaly > or = 5 cm below the costal margin (BCM) or thrombocytosis > or = 700 x 10(9)/L pretreatment added significant independent prediction to response. At 6 months, patients with a partial hematologic response (PHR) or resistant disease had a less than 10% chance of achieving a later major cytogenetic response, as were those in complete hematologic response (CHR) and who had pretreatment splenomegaly and thrombocytosis. Applying the model at 3 months showed that only patients with < or = PHR and pretreatment splenomegaly or thrombocytosis at 3 months had such a low major cytogenetic response rate. Finally, at 12 months, patients with CHR still had a 15% to 25% chance of having a major cytogenetic response later if they did not have pretreatment splenomegaly and thrombocytosis. CONCLUSION This analysis allows better selection of patients with Ph-positive CML on IFNalpha therapy for continuation of IFNalpha versus changing therapy early in the course of CML. For treatment programs that choose to change patients to other investigational therapies (eg, intensive chemotherapy and/or autologous stem-cell transplantation [SCT]), baseline outcome expectations are provided for patients continued on IFNalpha therapy, against which the results of new approaches can be compared.

Expression of Biomarkers That Predict Progression in Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4241-4241
Author(s):  
Mariana Selena Gonzalez ◽  
Patricia Martha Gargallo ◽  
Beatriz Moiraghi ◽  
Irene Larripa
Keyword(s):  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Apoptotic Index ◽  
Myelogenous Leukemia ◽  
Control Group ◽  
Qrt Pcr ◽  
Advanced Phase ◽  
Bmi1 Expression ◽  
Healthy Donors

Abstract Chronic Myelogenous Leukemia (CML) is associated with a chromosomal translocation, t(9;22)(q34;q11.2), that produces the Philadelphia chromosome (Ph). The molecular consequence of this translocation is the generation of the BCR/ABL oncogene, which encodes a chimeric protein of 210 kDa (p210Bcr/Abl) with elevated tyrosine kinase activity. BCR/ABL exerts its oncogenic effect in CML cells essentially by stimulating cell proliferation, inhibiting apoptosis and altering cell adhesion to bone marrow stroma. Despite of this consistent molecular abnormality, a marked heterogeneity in prognosis and response to treatment has been reported. Different molecular markers have been studied, such as: BMI1, ELA2, PR3, E2F1 and apoptotic genes (BCL-2, BCL-XL, BAX, BAD, BAK) in order to predict progression and overall survival in myeloid leukemia. The polycomb group gene BMI1 plays an essential role in regulating the proliferative activity in leukemic stem cell. The expression of this gene is related to a higher degree of malignancy. On the other hand, BCL-2 family genes involved in the mitochondrial-apoptotic pathway are related with clinical response and treatment failure. Enhanced expression of the apoptotic inhibitor BCL-2 or its homolog BCL-XL lead to tumor cells having a decreased susceptibility to cell death. Other BCL-2 family members such as BAX are able to induced apoptosis, so that the ratio of expression of proapoptotic and anti-apoptotic members might determine the apoptotic potencial of cancer cells. In this study we evaluated the expression of BMI1 and BAX/BCL-XL ratio (apoptotic index) to determine whether these genes could behave as biomarkers to predict disease aggressiveness and progression from chronic phase to more advanced phases. Total RNA was extracted from leucocytes of peripheral blood. using Trizol method. cDNA was synthesized with random hexamer primers and reverse transcriptase. The expression was assessed by quantitative real time (QRT-PCR) using the LightCycler 2.0 instrument (Roche), based on the Syber-Green method. All QRT-PCR reactions were performed in 20ul volume. The β-actin expression was used as the endogenous cDNA quality control. Groups of patients were compared using the Mann-Whitney test. The study was performed in 31 patients: 16 in chronic phase (CP), 15 in advanced phases (accelerated and blast crisis) and 10 healthy donors (control group). BMI1 expression levels were significantly lower in CP (mean ± SEM: 0.54±0.15) than in more advanced stages of CML (mean ± SEM: 4.54±1.4) (P&lt;0.0005). In peripherical blood of healthy donors, the expression of this gene was similar to CML-CP patients (0.4±0.13). The relationship of BAX/BCL-XL values were higher in CP (mean ± SEM: 13.81± 1.85) and lower in advanced phase (mean ± SEM: 0.88±0.17) than in the control group (mean ± SEM: 4.82 ± 0.49) (P&lt;0.0044 and P&lt; 0.0002, respectively). The CP patients showed a low BMI1 expression level and a high apoptotic index, this inverse correlation is associated with a benign stage of the disease and good treatment response. On the contrary, cases in more advance stage displayed overexpression of BMI1 gene and low BAX/BCL-XL ratio suggesting an aggressive stage and poor response. The identification of a genetic hostile profile in CP phase could predict an impending disease progression. Our results show that the simultaneous use of two biomarkers: BMI1 and the ratio BAX/BCL-XL represent sensitive indicators of clinical outcome in CML-CP. Therefore, the prospective screening of these biomarkers would help to refine CML disease staging and would be useful prognostic indicators for optimizing therapeutic strategies.

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