Chromosome-positive Chronic Myelogenous Leukemia Research Articles

Imatinib mesylate (STI571) for myeloid malignancies other than CML

Imatinib mesylate, a small molecule tyrosine kinase inhibitor, has had a major impact on the treatment of Philadelphia chromosome positive chronic myelogenous leukemia. This review will explore its potential in the treatment of other myeloid neoplasms, based on its ability to inhibit Kit and PDGFR kinases in addition to Bcr-Abl. Imatinib’s potential role in the treatment of Philadelphia chromosome negative chronic myelogenous leukemia, systemic mastocytosis with associated hematologic neoplasms, chronic myelomonocytic leukemia, specific subtypes of acute myelogenous leukemia, myelofibrosis/myeloid metaplasia, and polycythemia vera is discussed.

Author reply

We appreciate the thoughtful comments of Drs. Drummond and Holyoake, which have highlighted several important issues in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (CML) with imatinib mesylate. The first issue is when to advise patients receiving imatinib that the treatment is unlikely to benefit them, and that they should consider allogeneic stem cell transplantation (SCT). Our concern is that such patients currently are often advised to consider allogeneic SCT if they have not achieved a major cytogenetic response after only 6–12 months of imatinib therapy or if they demonstrate persistence of residual disease by polymerase chain reaction (PCR). To our knowledge, there is little evidence to support such opinions. Our analysis in patients with chronic phase CML after interferon failure (with a median duration of 33 months) is, to our knowledge, the first to indicate that even if a minor cytogenetic response is obtained after 3–12 months of treatment with imatinib, 35–54% of patients may later achieve a complete cytogenetic response with continuation of imatinib.1 This information pertains not only to considerations of allogeneic SCT but also to the interpretation of data from investigational trials (e.g., vaccines, decitabine). In a study using a vaccine strategy, patients were categorized as having “resistant CML” based on the lack of a major cytogenetic response after only 6 months of imatinib therapy.2 The information in our analysis was derived from patients in chronic phase CML after interferon failure. In our experience in 167 patients with early chronic phase CML who were treated with imatinib, 131 patients (78%) had achieved a complete cytogenetic response at last follow-up. The prediction of later response based on initial response is preliminary but suggests similar trends (Table 1). Current data suggest that a complete cytogenetic response with different levels of quantitative PCR cannot be considered to be an indication for a change in imatinib therapy.3 The second issue is the significance of persistent PCR positivity during imatinib therapy. Only 10% of patients with newly diagnosed CML have been reported to achieve PCR negativity while receiving standard-dose imatinib, and this number increased to 30–40% with high-dose imatinib.4, 5 These low rates often are used to argue against the curative potential of imatinib. However, cures have been observed in other malignancies despite persistent minimal residual molecular disease (e.g., acute lymphocytic leukemia, acute promyelocytic leukemia, and lymphoma). Thus, long-term event-free survival perhaps depends not on the absolute presence or absence of molecular residual disease, but on reaching a critical threshold. This threshold in CML may be below a quantitative PCR value of 0.045% based on the interferon experience.6 Such levels reportedly are achievable in > 50% of patients with newly diagnosed CML who are receiving imatinib therapy.4, 5 Historically, the advice to consider upfront allogeneic SCT in patients with CML was based in part on an expected median survival of 3–7 years. The preliminary experience with imatinib suggests a reduction of the annual mortality in patients with chronic phase disease after interferon failure from approximately 15–20% to < 5% (estimated 2-year mortality rate of 8%).7 In newly diagnosed patients treated with imatinib, the estimated annual mortality rate has been reported to be < 2% in the first 2 years.8, 9 If this encouraging experience persists with longer follow-up, the expected 10-year survival rate in CML patients may exceed 50%.10 Finally, we agree that ultimately, the choice of therapy is the patient's, after considering the potential risks and benefits of each approach based on timely objective presentations of the updated results of different therapies.

Imatinib in Philadelphia chromosome-positive chronic phase CML patients: molecular and cytogenetic response rates and prediction of clinical outcome.

Previous clinical trials with the tyrosine kinase inhibitor imatinib in chronic-phase Philadelphia chromosome-positive chronic myelogenous leukemia (CML) resulted in 95% of hematologic and 60% major cytogenetic remissions in patients who failed a previous interferon-alpha-containing regimen. In an identical clinical trial setting with 39 chronic-phase CML patients we achieved comparable cytogenetic response rates after a median follow up of 30.1 weeks, with an almost identical toxicity profile. In order to identify predictive markers for the therapeutical use of imatinib, we monitored apart from standard hematology parameters bcr/abl fusion transcripts by quantitative real-time fluorescence RT-PCR. As previous investigations demonstrated that the plasma protein alpha-1 acid glycoprotein might inactivate circulating levels of free imatinib by protein binding with high affinity, we assessed plasma alpha-1 acid glycoprotein concentrations in our study cohort as well. Median bcr/abl fusion transcripts declined gradually over the entire treatment period and became significantly lowered at month 3 after initiation of imatinib therapy. Further, we observed elevated pretreatment levels of alpha-1 acid glycoprotein in patients who relapsed with leukemia, whereas initial bcr/abl mRNA copy numbers were not of predictive value. In addition, we provide data showing molecular response to this therapy in the vast majority of patients. Finally, our results support the hypothesis, that initially elevated plasma levels of alpha-1 acid glycoprotein might serve as a predictive marker for the clinical outcome of treatment with imatinib.

Sudden onset of the blastic phase of chronic myelogenous leukemia: patterns and implications.

Therapeutic options in Philadelphia chromosome-positive chronic myelogenous leukemia (Ph-positive CML) in chronic phase include allogeneic stem cell transplantation (SCT) and treatments other than allogeneic SCT. The choice of therapy often depends on the mortality risk of allogeneic SCT. Delaying SCT until the disease demonstrates resistance to other strategies (e.g., imatinib mesylate or interferon-alpha) entails the risk of the development of a sudden blastic phase, which may not permit enough time to perform a SCT. The current study was performed to define the incidence and patterns of sudden-onset blastic-phase CML in relation to the risk inherent in delaying allogeneic SCT. Of a study group of 1286 patients who were referred in early chronic-phase CML between 1981-2001 and who received therapies other than allogeneic SCT, 1093 patients had regular follow-up at the study institution. A sudden onset of blastic phase was defined as having its onset within 3 months from a previously documented complete hematologic response. Among the 1093 patients, 183 patients developed blastic-phase disease, which was of sudden onset in 46 patients (25%; 4% of the total). The incidence of sudden blastic phase was 0.4% in the first year, 1.8% in the second year, and 2.6% in the third year. Patients with sudden blastic-phase disease were found to have more often low-risk features at the time of presentation, including low incidences of blasts and thrombocytosis. Sudden blastic-phase CML was found to be associated with a higher incidence of lymphoid blastic morphology (67% vs. 22%; P < 0.0001), which resulted in a higher response rate to blastic-phase therapy (70% vs. 29%; P < 0.0001) and significantly longer survival from the onset of blastic-phase disease (median of 12 months vs. 6 months; P < 0.01). Twenty-four of the 42 patients with sudden blastic-phase disease who were treated at the M. D. Anderson Cancer Center were able to undergo allogeneic SCT in second chronic phrase (n = 13) or transformed phase (n = 11); at the time of last follow-up, 6 patients were alive without evidence of disease at a median of 18 months. The low rates of sudden blastic transformation in the first 3 years of the course of disease for CML and the salvage rate of these patients with allogeneic SCT should be considered in relation to the transplantation mortality in patients with early chronic-phase disease and the early promising results with imatinib mesylate therapy.

Prognostic significance of cytogenetic clonal evolution in patients with chronic myelogenous leukemia on imatinib mesylate therapy

AbstractCytogenetic clonal evolution (CE) is a known poor prognostic factor in Philadelphia chromosome–positive chronic myelogenous leukemia (Ph-positive CML). However, its prognostic relevance in the era of imatinib therapy is unknown. We investigated the independent prognostic relevance of CE in 498 patients with Ph-positive CML treated with imatinib for chronic or accelerated phases. One hundred twenty-one patients had CE alone (n = 70) or with other accelerated phase criteria (n = 51). Patients were compared in 4 categories: chronic phase (n = 295), CE only (n = 70), accelerated phase without CE (n = 82), and accelerated phase with CE (n = 51). Statistical methods used established methodologies for univariate and multivariate analyses. In chronic and accelerated phases of CML, CE was not associated with significant differences in major or complete cytogenetic response rates, but it was an independent poor prognostic factor for survival by multivariate analyses in both chronic (P = .005) and accelerated phase (P = .03). Multivariate analyses conducted at the 3-month landmark (including the 3-month cytogenetic response) identified the lack of cytogenetic response at 3 months to be a stronger independent poor prognostic factor for survival than CE for both chronic (major cytogenetic response versus other) and accelerated phase (any cytogenetic response versus other). We conclude that cytogenetic CE is not an important factor for achieving major or complete cytogenetic response with imatinib mesylate therapy, but it is an independent poor prognostic factor for survival in both chronic and accelerated phases of CML. The 3-month cytogenetic response to imatinib mesylate refined the prognostic relevance of such studies in patients on imatinib mesylate therapy.

Prediction of initial cytogenetic response for subsequent major and complete cytogenetic response to imatinib mesylate therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia.

Obtaining a major (Philadelphia chromosome [Ph] of < 35%) or a complete cytogenetic response (Ph of 0%) has been associated with excellent long-term survival. Cytogenetic response may continue to improve with therapy. Because early allogeneic stem cell transplantation (SCT) may be associated with a better outcome, early parameters predicting for subsequent cytogenetic responses would optimize the treatment decision-making. The current study was performed to analyze whether early cytogenetic responses may be predictive of later major or complete cytogenetic responses to imatinib mesylate therapy in patients with Ph-positive chronic myelogenous leukemia (CML). Two hundred sixty-one patients with Ph-positive, chronic-phase CML after failure with interferon therapy who were treated with imatinib mesylate therapy were analyzed. A persistence of 100% Ph-positive cells after > or = 6 months of imatinib mesylate therapy was associated with a major cytogenetic response rate of 9-13% and a complete cytogenetic response rate of 0-4%. However, a minor cytogenetic response after 3-12 months of therapy still was associated with high rates of major (68-83%) or complete (35-54%) cytogenetic response rates. Patients with Ph-positive, chronic-phase CML who have persistent 100% Ph-positive disease after > or = 6 months of imatinib mesylate therapy may be offered allogeneic SCT or considered for alternative investigational therapies.

Quantitative real-time reverse-transcription polymerase chain reaction for diagnosis of BCR-ABL positive leukemias and molecular monitoring following allogeneic stem cell transplantation.

Real-time reverse-transcription polymerase chain reaction (RT-PCR) (qPCR) of the BCR-ABL mRNA is a suitable technique to measure the amount of circulating leukemic cells in chronic myelogenous leukemia (CML). In this study, we evaluated a BCR-ABL-specific qPCR method using the LightCycler technology in 95 patients with Philadelphia chromosome positive acute leukemia (n = 7) or CML in different stages (n = 88). Primers and hybridization probes were chosen to detect the most prevalent variants of BCR-ABL (b2a2, b3a2, b2a3, b3a3, e19a2, e1a2) with a sensitivity of 10-5 for b2a2 and b3a2. With median BCR-ABL/G6PDH ratios of 10.7% in the untreated chronic phase, 43.2% in the newly diagnosed accelerated phase, and 131.4% in newly diagnosed blast crisis the BCR-ABL mRNA levels varied significantly between different stages of CML whereas no difference was found between blast crisis and untreated acute leukemias (136.9%). There was a strong relationship between qPCR results and cytogenetics in patients treated with imatinib, interferon-alpha, or following allografting. Thirteen patients with CML were sequentially examined by qPCR following myeloablative or non-myeloablative allogeneic peripheral blood stem cell transplantation. Five patients received donor lymphocytes and became BCR-ABL negative as confirmed by nested RT-PCR. The gradual disappearance of BCR-ABL positive cells could be monitored by qPCR following non-myeloablative transplantation. Comparison of BCR-ABL levels with the degree of donor chimerism showed that 91% of samples with complete donor chimerism were BCR-ABL negative. In 22% of BCR-ABL negative samples chimerism between 71% and 98% was observed, indicating the persistence of normal recipient's hematopoietic cells. In conclusion, the qPCR protocol used in this study is a reliable and fast method for monitoring molecular response in CML.

Imatinib mesylate therapy in newly diagnosed patients with Philadelphia chromosome–positive chronic myelogenous leukemia: high incidence of early complete and major cytogenetic responses

AbstractFifty patients with Philadelphia chromosome–positive (Ph+) chronic myelogenous leukemia (CML) in early chronic phase received imatinib mesylate, 400 mg orally daily. After a median follow-up of 9 months, 49 patients (98%) achieved a complete hematologic response and 45 patients (90%) achieved a major cytogenetic response, complete in 36 patients (72%). Compared with similar patients who received interferon-α with or without hydroxyurea or other interferon-α combination regimens, those receiving imatinib mesylate had higher incidences of complete and major (Ph < 35%) cytogenetic responses at 3 months (34% and 74% versus 1%-4% and 9%-24%, respectively), 6 months (52% and 80% versus 3%-7% and 11%-28%, respectively), and 9 months (60% and 77% versus 5%-11% and 14%-30%, respectively;P< .001). Competitive quantitative polymerase chain reaction (QPCR) studies at 9 months showed a median QPCR value (ratio of BCR-ABL/ABL transcripts × 100) of 0.59% overall and of 0.24% (range, 0.001%-29.5%) for complete cytogenetic response.

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Simultaneous homoharringtonine and interferon-alpha in the treatment of patients with chronic-phase chronic myelogenous leukemia.

Homoharringtonine (HHT) has antileukemic activity in patients with Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML). Combinations of HHT, interferon-alpha (IFN-alpha), and cytarabine (ara-C) have been studied in various CML phases. The objectives of this study were to evaluate the efficacy and toxicity profiles of a combination regimen of simultaneous HHT and IFN-alpha therapy in patients with chronic-phase CML who were not exposed previously to either agent. Forty-seven patients were treated: 37 patients with early chronic-phase CML (2 patients with clonal evolution) and 10 patients with late chronic-phase CML. Their median age was 62 years (range, 23-73 years). HHT was given at a dose of 2.5 mg/m(2) by continuous intravenous infusion over 24 hours daily for 5 days every month, and IFN-alpha was given daily at a target dose of 5 x 10(6) units/m(2) subcutaneously. Response, survival, and treatment toxicity were analyzed. Overall, the complete hematologic response (CHR) rate was 85%; the cytogenetic response rate was 66%, with major cytogenetic responses (Ph positive in < 35% of metaphases) in 49% of patients and complete cytogenetic responses in 21% of patients. The CHR rate, cytogenetic response rate, and major cytogenetic response rate were 84%, 69%, and 52%, respectively, in patients with early chronic-phase CML. Among the 10 patients with late chronic-phase CML, the CHR rate, cytogenetic response rate, and major cytogenetic response rate were 80%, 50%, and 40%, respectively. Response rates in patients age > 60 years were 84%, 62%, and 49% for CHR, cytogenetic response, and major cytogenetic response. Myelosuppression was frequent but manageable: Anemia with hemoglobin < 8.0 g/dL occurred in 36% of patients, requiring dose adjustments and erythropoietin therapy. Nonhematologic toxicities were mainly fatigue, aches, and gastrointestinal disturbances. Dose reductions with multiple courses were significant and were due to myelosuppression: After 6-24 courses, the median daily IFN-alpha dose was 1 MU/m(2), and the median number of days on HHT per month was 2 days. With a median follow-up of 26 months, the estimated 2-year survival rate was 90% (95% confidence interval, 79-100%). The simultaneous combination of HHT and IFN-alpha is safe and effective, but the dose schedules that actually were delivered were significantly lower than the planned dose schedules. With the availability of signal-transduction inhibitor 571 (imatinib mesylate), studies of combination of HHT and IFN-alpha chemotherapy in patients with CML who have disease that fails to respond to imatinib mesylate and of combinations with imatinib mesylate need to be explored.

Coexistence of independent myelodysplastic and Philadelphia chromosome positive clones in a patient treated with hydroxyurea

We report a patient with Philadelphia chromosome positive (Ph +ve) chronic myelogenous leukemia (CML), treated with hydroxyurea alone, who upon disease progression developed an additional Ph −ve clone containing chromosomal abnormalities typical of myelodysplastic syndrome (MDS). Retrospective analysis of a cryopreserved stem cell specimen from diagnosis confirmed that this second clone developed during the course of treatment. The development of a clone with additional cytogenetic abnormalities in CML has only been reported after leukemogenic treatment, stem cell transplantation or interferon. We report a case of secondary Ph −ve MDS/AML during blast crisis in a patient treated with hydroxyurea for CML.

Structure of the Bcr-Abl oncoprotein oligomerization domain.

The Bcr-Abl oncoprotein is responsible for a wide range of human leukemias, including most cases of Philadelphia chromosome-positive chronic myelogenous leukemia. Oligomerization of Bcr-Abl is essential for oncogenicity. We determined the crystal structure of the N-terminal oligomerization domain of Bcr-Abl (residues 1-72 or Bcr1-72) and found a novel mode of oligomer formation. Two N-shaped monomers dimerize by swapping N-terminal helices and by forming an antiparallel coiled coil between C-terminal helices. Two dimers then stack onto each other to form a tetramer. The Bcr1-72 structure provides a basis for the design of inhibitors of Bcr-Abl transforming activity by disrupting Bcr-Abl oligomerization.

Simultaneous Absolute Quantification of Target and Control Templates by Real-Time Fluorescence Reverse Transcription-PCR Using 4-(4′-Dimethylaminophenylazo)benzoic Acid as a Dark Quencher Dye

Despite the many advantages of real-time fluorescence reverse transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template. For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrument. Fluorescent probes were labeled with either 6-carboxy-fluorescein or hexachloro-6-carboxy-fluorescein as reporter dye and 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sensitivity and specificity of this assay, serial dilutions of reference and target templates were analyzed in one PCR reaction. In the presence of 10 beta-actin molecules as control templates, 10(5) bcr/abl molecules were amplified, and 10(5) beta-actin molecules were amplified in the presence of 10 bcr/abl copies. We also performed single and duplex measurements on samples from five patients with documented Philadelphia chromosome-positive chronic myelogenous leukemia disease courses (72 samples) and three with minor bcr/abl+ acute myelogenous leukemias (26 samples). For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for starting template amounts and threshold cycle values was 0.99; for m-bcr/abl, r = 0.96, indicating a precise log-linear relation for 10-10(5) copies/100 ng of cDNA. In the same PCR reactions, r = 0.99 for beta-actin (coamplified with M-bcr/abl or m-bcr/abl) for 10(3)-10(7) copies/100 ng cDNA. The linear correlation coefficient for single and duplex measurements was 0.98 for M- and m-bcr/abl in patient samples. DABCYL can be used as dark quencher fluorophore in real-time fluorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and beta-actin transcripts allows monitoring of bcr/abl+ leukemias.

Results of Therapy with Interferon Alpha and Cyclic Combination Chemotherapy in Patients with Philadelphia Chromosome Positive Chronic Myelogenous Leukemia in Early Chronic Phase

The objective of the study was to investigate the toxicity and efficacy of cyclic combination therapy offered to patients with Ph-positive CML having a sub-optimal response to IFN-α. Patients in early chronic phase CML were treated with IFN-α at 5MU/m2 daily. Patients who did not achieve cytogenetic response after 6 months of IFN-α therapy, or Ph-suppression to less than 35% Ph-positive cells (partial cytogenetic response) after 12 months of therapy were offered cyclic intensive chemotherapy every 6 months, with IFN-α maintenance between cycles. The initial 3 cycles included daunorubicin, vincristine, cytosine arabinoside (ara-C) and prednisone (DOAP). Later cycles were given with cyclophosphamide replacing daunorubicin (COAP).Of 74 patients treated, 61 (82%) achieved complete hematologic response (CHR): 51 (69%) had a cytogenetic response, which was major (Ph < 35%) in 31 (42%), and complete in 23 (31%). Fifty-five patients (74%) achieved CHR by 6 months of therapy, 38 (69%; 51% of total) with a cytogenetic response - 13 (24%) had a major cytogenetic response. Seventeen patients received at least 1 course of DOAP therapy. Median survival of the overall cohort of patients was 120 months. With a median follow-up of 145 months (103+ to 155+ months), 40 patients (54%) have died. The median duration of cytogenetic response was 35 months (range 3 to 149+ months) and the estimated 10-year cytogenetic response rate was 37%. A durable complete cytogenetic response was observed in 16 patients (20%) with a median duration of 139+ months (range 12+ to 149+ months), 11 of them (15%) are now off IFN-a therapy for a median of 57+ months (range 12+ to 128+ months). The projected 10-year survival was 50% for the study group versus 35% for 208 patients who received other IFN-α based regimens at the MD Anderson Cancer Center (p<.01). In conclusion, the addition of intensive chemotherapy may improve survival in patients with CML who have not obtained an adequate cytogenetic response on an IFN-α-based regimen.

Therapeutic choices in younger patients with chronic myelogenous leukemia.

Allogeneic stem cell transplantation (SCT) and interferon (IFN)-alpha therapy have significantly improved the prognosis of patients with Philadelphia (Ph) chromosome positive chronic myelogenous leukemia (CML). Both therapies may be suitable for younger patients. The authors reviewed current data to assist in prioritizing these modalities in an individual patient. The authors reviewed and summarized current data on outcomes of SCT and IFN-alpha therapy in patients with early chronic phase CML. Several disease-, patient-, and physician-related factors affect outcomes with both modalities. Interferon-alpha does not induce myelofibrosis. The course of CML is predictable in most patients; sudden emergence of blastic phase; disease is unusual. There is no significant adverse impact of delaying SCT for the 12 months usually necessary to assess cytogenetic response to an IFN-alpha-based regimen. Interferon-alpha may be discontinued some months before SCT and is not associated with an adverse impact on post-SCT outcomes. An individualized risk assessment-based approach is of value in prioritizing SCT and IFN-alpha in younger patients with chronic phase CML. The authors recommend a risk-based therapy algorithm based on the expected SCT associated 1-year mortality for an individual patient.

Changing patterns of histological subgroups during therapy of Ph1+ chronic myelogenous leukaemia.

Bone marrow histopathology reveals a striking heterogeneity at diagnosis of Philadelphia chromosome positive (Ph1+) chronic myelogenous leukaemia (CML). Based on semiquantitative evaluations of the number of megakaryocytes and the content of fibres, various histological subtypes have been postulated. However, little information exists on whether these groups represent stable categories of the different classification systems and whether therapeutic regimes exert any influence on the putative shift of histological patterns. A retrospective clinicopathological study was performed on 396 bone marrow biopsies derived from 173 patients. There were at least two representative trephines taken at diagnosis and at median intervals of 16 months. Processing of the specimens involved immunostaining with CD61 (megakaryopoiesis) and Ret40f (erythropoiesis) and Gomori's silver impregnation technique. Based on morphometric analysis and in accordance with the general appearance of bone marrow histology three different histological subtypes were distinguished. These consisted of a granulocytic (51 patients), a predominantly megakaryocytic (73 patients) and a myelofibrotic pattern (49 patients). Follow-up biopsies revealed that a significant transition of histological groups occurred and that, independently of treatment modalities, the myelofibrotic category was associated with an unfavourable prognosis. Of the 124 patients without myelofibrosis at onset, 42% later transformed into the myelofibrotic subtype. However, these patients showed no prevalence of either a pre-existing granulocytic or megakaryocytic growth. Myelofibrotic changes were significantly associated with interferon (IFN) and busulfan (BU) therapy. On the other hand, a transition of a myelofibrotic into a nonfibrotic subtype was detectable in 17 of the 49 patients under study and related to hydroxyurea (HU) treatment. Histological classification systems of bone marrow features in CML do not represent stable patterns, but may be significantly altered by therapy, in particular IFN and HU.

Therapy-related Changes of CD20+ and CD45RO+ Lymphocyte Subsets in Chronic Myeloid Leukemia (CML): An Immunohistochemical and Morphometric Study on Sequential Trephine Biopsies of the Bone Marrow

Little information exists about the amount of CD45RO+-T- and CD20+-B-lymphocytes in the bone marrow of patients with Philadelphia chromosome-positive chronic myelogenous leukemia (Ph1+-CML) at presentation or regarding corresponding changes during therapy. On the other hand, quantification of this cell compartment seems to be imperative for two reasons: first, the presumed association of immunocompetent lymphocyte subsets in the expansion of the leukemic cell clone; and second, a speculated relationship with the complex generation of myelofibrosis. Therefore, an immunohistological and morphometric study was performed on 219 representative trephine biopsies of the bone marrow derived from 70 patients with repeated examinations during the course of Ph1+-CML. For the identification of the different lymphocyte populations, the monoclonal antibodies UCHL-1 (CD45R0) and L26 (CD20) were applied on formaldehyde-fixed and decalcified specimens. In comparison to a control group and calculated per hematopoietic cells, the CML bone marrow showed about a 50% decrease in the total amount of lymphocytes. Determination of CD45RO+ and CD20+ subsets revealed a significant enhancement during treatment. Because of the different intervals (range, 10 to 25 mo) between first and last biopsy in the various therapeutic groups, results had to be modified by considering dynamic features. This calculation included changes of the lymphocyte subpopulations related to time. Contrasting the CD45RO+ lymphocytes, a relevant increase in the CD20+ subset could be observed after interferon-α treatment or corresponding combination regimens. No significant correlations were found between fiber density at onset (first biopsy) or development of fibrosis and lymphocyte proliferations in the course of CML. Our results are in keeping with the finding that a proper immune response consistent with an increased lymphocyte growth seems to be associated with a regression of the clonally-transformed cell population. Opposed to a repeatedly discussed pathomechanism, we failed to demonstrate any quantitative relationships between the extent of lymphocyte proliferations and occurrence or progression of myelofibrosis.

Effects of chemotherapy (busulfan-hydroxyurea) and interferon-alfa on bone marrow morphologic features in chronic myelogenous leukemia. Histochemical and morphometric study on sequential trephine biopsy specimens with special emphasis on dynamic features.

We performed a retrospective clinicopathologic study on sequential biopsy specimens from 90 patients with Philadelphia chromosome-positive chronic myelogenous leukemia to study therapy-specific effects of busulfan (28 patients), hydroxyurea (32 patients), and interferon-alfa (IFN-alfa; 30 patients). Bone marrow specimens were evaluated by morphometry after silver impregnation and staining with monoclonal antibodies to identify reticulin fibers, nucleated erythroid precursors, megakaryocytes, and macrophages. To compute dynamics of histopathology implicating corresponding changes in time, relevant indices were calculated. Quantification of megakaryocytopoiesis and its precursor cell population showed a significant increase in the IFN-alfa and busulfan groups compared with the hydroxyurea group. These changes were associated with a development of myelofibrosis during therapy. Although a significant increase in fiber density was detectable in the busulfan group, the progression index proved to be twice as high after IFN-alfa therapy. In contrast, a considerable number of patients displayed a regression of myelofibrosis after hydroxyurea treatment. The general association of the megakaryocyte lineage with myelofibrosis was in line with experimental findings. The mature macrophage population and its activated subfraction revealed a marked proliferation (IFN-alfa group) during treatment. Growth and activation of macrophages may be compatible with their putative function during erythrocytopoietic regeneration and with stimulation of their phagocytic properties.

Expression of thrombopoietin and its receptor (c-mpl) in chronic myelogenous leukemia: correlation with disease progression and response to therapy.

Chronic myelogenous leukemia (CML) represents a paradigm of the stepwise increment in disease aggressiveness, resistance to therapy, and transformation. Thrombopoietin (TPO) and its receptor, c-mpl, support the proliferation of multiple types of immature hematopoietic progenitor cells, and induce clonal growth of leukemic cells. The authors investigated whether TPO or c-mpl overexpression might correlate with progression of CML, disease aggressiveness, or response to therapy. Expression of c-mpl and TPO was measured in bone marrow samples from 208 patients with CML by Western blot analysis and solid-phase plate radioimmunoassay (used for quantification). Samples obtained from individuals without evidence of hematologic abnormalities were used as controls. There were no significant differences in TPO or c-mpl expression among patients in different phases of CML or between patients with Philadelphia chromosome positive and negative CML. When TPO and c-mpl levels were analyzed in relation to prognostically important host and disease characteristics in early chronic phase CML, platelet and white blood cell counts demonstrated significant differences in both TPO and c-mpl expression, but age and spleen size demonstrated significant differences in TPO expression only. Responses to interferon (INF)-alpha-based therapy and survival were not influenced by TPO or c-mpl levels. TPO or c-mpl overexpression did not correlate with different CML phases, suggesting that they were not involved in CML progression from early to advanced phase. Neither TPO nor c-mpl overexpression was particularly evident in any risk group, suggesting lack of correlation between their expression and disease aggressiveness. This was supported by the finding of similar response to IFN-alpha-based therapy and survival regardless of the level of TPO or c-mpl expression.

Fluorescence In Situ Hybridization Analysis of Complex Translocations in Two Newly Diagnosed Philadelphia Chromosome-Positive Chronic Myelogenous Leukemia Patients

Two different complex translocations from newly diagnosed cases of Philadelphia chromosome-positive chronic myelogenous leukemia (CML) were characterized by G-banding and fluorescence in situ hybridization (FISH) analysis. In one case, a unique balanced t(9;22;9;11)(q34;q11;p22;q23) was identified by G-banding, and confirmed by FISH using MBCR/ABL and painting probes. In the second case, an apparently balanced t(19;22) was identified by G-banding analysis. FISH using MBCR/ABL probe detected the fusion gene on the derivative chromosome 22, indicating the involvement of chromosome 9. Further FISH analysis with selected painting probes showed that the t(19;22) was a result of a complex translocation involving chromosomes 9, 19, 21, and 22.