Genomic BCR-ABL1 breakpoints in pediatric chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a rare disease in children and adolescents and various aspects—from molecular genesis to therapy regimen—have been taken over from studies on the more prevalent adult CML. However, differences have been observed between malignancies with identical underlying chromosomal translocations, but occurring at different age groups, suggesting some diversity in the mechanisms of formation and leukemogenesis. A multiplex long‐range PCR‐based assay was developed to allow fast and reliable amplification of patient‐specific BCR‐ABL1 fusion sequences from genomic DNA. The localization of breakpoints was analyzed with respect to distribution within the breakpoint cluster regions, sequence features, and association to repetitive elements or motifs associated with DNA recombination. The genomic fusion sites of 59 pediatric CML patients showed a bimodal breakpoint distribution in BCR that was different from the distribution in adult CML cases, but with similarities to BCR‐ABL1‐positive, acute lymphoblastic leukemia in adults. BCR breakpoints were found more frequently positioned within, or close to, Alu repeats than would be expected based on their overall sequence proportion. Technical aspects of the highly sensitive DNA‐based quantification of residual CML cells by specific fusion sequence during tyrosine kinase inhibitor therapy are exemplified in a subcohort of pediatric CML patients. © 2012 Wiley Periodicals, Inc.     

Bone marrow transplantation for patients with chronic myeloid leukemia

Between February 1981 and December 1984 we treated 52 patients with chronic myeloid leukemia in the chronic phase and 18 patients with more advanced disease by high-dose chemoradiotherapy followed by allogeneic bone marrow transplantation using marrow cells from HLA-identical sibling donors. In addition, the 40 patients who had not previously undergone splenectomy received radiotherapy to the spleen. To prevent graft versus host disease, cyclosporine was given either alone or in conjunction with donor marrow depleted of T cells. Of the 52 patients treated in the chronic phase, 38 are alive after a median follow-up of 25 months (range, 7 to 50); the actuarial survival at two years was 72 percent, and the actuarial risk of relapse was 7 percent. Of the 18 patients with more advanced disease, 4 have survived; the actuarial two-year survival was 18 percent, and the actuarial risk of relapse was 42 percent. We conclude that the probability of cure is highest if transplantation is performed while the patient remains in the chronic phase of chronic myeloid leukemia. T-cell depletion may have reduced the incidence and severity of graft versus host disease. The value of irradiation to the spleen before transplantation has not been established.     

Cytogenetic follow-up after bone marrow transplantation for Philadelphia-positive chronic myeloid leukemia

Forty-eight consecutive patients affected by chronic myelogenous leukemia (CML) Philadelphia (Ph) chromosome positive during the chronic phase of the disease underwent allogeneic bone marrow transplantation (BMT). Thirty-five patients had a follow-up over 12 months and were included in a cytogenetic study in order to evaluate the Ph clone eradication. In 25 cases, the Ph chromosome disappeared in all cytogenetic studies, and their hematologic picture is at present apparently normal. Ten patients showed cytogenetic relapse. In one case, the cytogenetic relapse was transitory without any clinical sign of the disease; in three cases, after a transitory cytogenetic relapse, a persistent relapse with clinical picture of progression of the disease occurred; in six cases cytogenetic and a clinical relapse were coincident. Structural chromosomal abnormalities other than Ph were temporarily seen in three cases. The so-called "nonrandom" chromosomal changes typical of the blastic phase were never detected. The reappearing Ph-positive clone spontaneously disappeared in three patients, and their hematologic picture reverted to complete chimerism. The present study confirms that the eradication of the Ph clone is often defective with BMT, and cytogenetic analysis can detect the competition between donor and residual host marrow. Furthermore, the karyotype evolution is different from that found in CML patients treated with conventional chemotherapy.     

p190 BCR-ABL rearrangement in chronic myeloid leukemia and acute lymphoblastic leukemia.

A minority of chronic myeloid leukemia (CML) cases have breakpoint in the minor cluster region (m-bcr) of the BCR-ABL fusion gene. We report a patient with Ph-positive acute lymphoblastic leukemia and m-bcr breakpoint at diagnosis. The patient was treated with chemotherapy followed by an autologous peripheral blood stem cell transplantation, achieving a clinical and hematological complete remission but with persistence of the Philadelphia chromosome. One year later, she developed leukocytosis with a blood picture consistent with CML. She was treated with hydroxyurea and interferon alpha with no response. This is the second case of m-bcr CML reported presenting with features of lymphoid blast crisis or acute lymphoblastic leukemia.     

Appearance of Ph negative recipient clones in chronic myeloid leukemia patients following bone marrow transplantation.

Seven patients were studied following bone marrow transplantation for chronic myeloid leukemia. Cytogenetic heteromorphisms were used to determine the origin of cells present post-BMT. Differences were found between results from blood and bone marrow samples, and between karyotype and interphase Y-body studies on the same samples. Philadelphia negative (Ph-) hematopoietic chimerism was found in 6 of 7 patients, all of whom had been Ph+ before BMT. One patient also demonstrated hematopoietic chimerism with Ph+ recipient cells following clinical evidence of relapse. In two patients who had received T-cell depleted grafts, cytogenetically rearranged Ph- clones of recipient cells were prominent in PHA-stimulated blood. In one case two clones had appeared only 1 month post BMT. The appearance of these clones so soon after transplant suggests very rapid clonal expansion, or that they were already present pre-BMT but at levels too low to have been detected. In the second patient, clones were not observed until more than 12 months post-BMT, after which four were found. These collectively expanded to occupy an increasing proportion of the total cells. These two patients with clones both remain in good health 44 and 51 months post-BMT. Further studies are needed to determine the true frequency and the significance of such findings.     

Role of splenic irradiation in patients with chronic myeloid leukemia undergoing allogeneic bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) has become the treatment of choice for patients of appropriate age with chronic myeloid leukemia (CML). In an attempt to enhance tumor cytoreduction, splenic radiation therapy (RT) has been done before the allogeneic transplant, but the role of splenic RT in this setting remains controversial. The purpose of this study is to evaluate the role of splenic RT before allogeneic BMT in patients with CML. Thirty-seven patients with chronic (n=33) or accelerated (n=4) phase CML underwent BMT from April 1990 to January 1998. All patients received splenic RT consisting of 500 cGy in five daily fractions (n=36) or 250 cGy in five daily fractions (n=1) completed within 10 days before BMT. The conditioning regimen included total-body irradiation and cyclophosphamide; etoposide was added to the regimen of patients in the accelerated phase. Continuous-infusion cyclosporine and pulse methotrexate were administered to all patients for prophylaxis of graft-vs.-host disease (GVHD). All patients achieved hematologic and cytogenetic remission. At a median follow-up of 37 months, the freedom from progression (FFP) and overall survival (OS) were 90 and 82%, respectively. None of the patients in accelerated phase have relapsed. Five patients have died of late transplant-related complications while in complete remission. Acute GVHD of grade > or = II was observed in 20% (14% grade II, 6% grade III). Fifty-one percent of patients developed limited chronic GVHD. The median posttransplant creatinine level was 1.2 mg/dL (range 0.6-4.2). Renal dysfunction, manifested as a persistent elevation in serum creatinine level (> 1.2 mg/dL), was observed in 40% of the patients. Only 8.5% had a creatinine level > 2.0 mg/dL, and no patient required dialysis as a result of renal dysfunction. Seven patients (18.9%) developed pulmonary complications, which included idiopathic interstitial pneumonitis (two), biopsy-proven interstitial fibrosis (four), and alveolar hemorrhage (one). The low relapse rate observed in this study may reflect the use of splenic RT as a part of the cytoreductive regimen before BMT. The fractionation schedule of 500 cGy in five daily fractions was well tolerated and did not appear to increase the toxicity of the preparative regimen. These favorable results indicate that splenic RT deserves further investigation and may be of benefit as a part of the conditioning regimen for patients receiving allogeneic BMT for CML.     

Immunosuppressive properties of mesenchymal stem cells derived from bone marrow of patients with chronic myeloid leukemia

Mesenchymal stem cells (MSCs) have emerged as excellent candidates for clinical application because of their capabilities of differentiating into multiple mesenchymal lineages and supporting hematopoiesis. Recently, MSCs have gained further interests after the demonstration of an immunosuppressive role. However, it is still unclear whether the immunosuppressive capability of MSCs will be altered with disease state. In this study, we obtained and expanded MSCs from bone marrow of patients with chronic myeloid leukemia (CML). Our results showed that MSCs derived from CML do not express costimulatory molecules CD40, CD80, and CD86. When MSCs derived from CML were added back to T cells stimulated by mitogens, a significant inhibition of T-cell proliferation was evident. MSCs differentiated into various mesenchymal lineages did not alter their immunosuppressive effect on T-cell proliferation. A significant T-cell inhibition was found in a transwell system, in which cell-cell contact between MSCs and effector cells was prevented. Furthermore, we found that transforming growth factor beta1 (TGF beta1) and hepatocyte growth factor (HGF) were major mediators of T-cell suppression by MSCs derived from CML. These results demonstrated that autologous MSCs derived from CML could effectively suppress T-cell proliferation.     

Chromosome studies in patients with Philadelphia chromosome-positive chronic myeloid leukemia submitted to bone marrow transplantation--results of a European Cooperative Study

The karyotypes of 74 patients with Ph-positive chronic myeloid leukemia submitted to bone marrow transplantation collected from nine institutions were studied serially before and after transplantation. In 13 cases sporadic Ph-positive metaphases were detected without signs of relapsing disease at various intervals after transplantation. These data indicate that the leukemic clone may not be completely suppressed by the conditioning treatment and that other biological mechanisms may be involved in destroying this clone or controlling its expansion.     

Megakaryopoiesis and myelofibrosis in chronic myeloid leukemia after allogeneic bone marrow transplantation: an immunohistochemical study of 127 patients.

An immunohistochemical and morphometric study was performed on 363 trephine biopsies of the bone marrow derived from 127 patients with chronic myeloid leukemia at standardized end points before and after allogeneic bone marrow transplantation (BMT). The purpose of this investigation was to evaluate features of CD61+ megakaryopoiesis related to successful engraftment. Further, we tried to elucidate possible associations of this lineage, including precursor cells, with the platelet count and reticulin fibrosis during the pretransplant and, specifically, post-transplant periods. A significant correlation was recognizable between the quantity of CD61+ megakaryocytes and the platelet values before BMT and also after completed hematopoietic recovery. In the very early post-transplant period, which is associated with severe thrombocytopenia, patchy regeneration of disarranged hematopoiesis occurred, including dysplastic megakaryocytes. According to planimetric measurements after BMT, the atypical micromegakaryocytes characteristic for chronic myeloid leukemia disappeared, and the engrafted donor bone marrow revealed a prevalence of normal-size cells of this lineage. On the other hand, normalization of megakaryocyte size was absent in sequential examinations of the few patients with a leukemic relapse who had a predominance of atypical dwarf forms comparable with chronic myeloid leukemia. Before BMT occurred, reticulin fiber density was significantly correlated with the number of CD61+ megakaryocytes and its precursor cell population. In 34 patients with myelofibrosis that occurred after myelo-ablative therapy and BMT, an initial regression was followed by an insidious recurrence of fibers concentrated in the areas of regenerating hematopoiesis. This postgraft reappearance of reticulin fibrosis was significantly associated with the quantity of megakaryocytes. Regarding engraftment parameters, pretransplant presence of (reticulin) myelofibrosis exerted a distinctive impact because of a delayed hematopoietic reconstitution according to standard clinical criteria. In line with this finding, slowed engraftment was also significantly related with higher pretransplant megakaryocyte and platelet counts.     

Evaluating the volume ratio of bone marrow affected by fibrosis: a parameter crucial for the prognostic significance of marrow fibrosis in chronic myeloid leukemia

Marrow fibrosis (MF) is a complication of bone marrow neoplasms that usually impairs quality of life and shortens survival time. Proof and exact quantification of MF is not yet standardized, thus impeding the comparability of results and the evaluation of its prognostic impact. In this study on 360 bone marrow biopsy specimens from 135 patients with either chronic idiopathic myelofibrosis (CIMF) (evaluation group) or chronic myeloid leukemia (CML) (test group), marked differences were detected between six different approaches systematically compared with respect to proof and quantification of MF. A new volumetric approach quantifying the marrow volume affected by fibrosis turned out to be superior to all of the other morphometric methods considering practicability and specificity of results, and superior to a semiquantitative procedure considering sensitivity, precision, and reproducibility (P < 0.00005). The assessment of the marrow volume with fibrosis was the only feature of MF independently influencing the survival time of patients (test group with CML; multivariate analysis, P = 0.0008). We conclude that an approach estimating the marrow volume affected by fibrosis is the method of choice to exactly quantify and prove MF. The loss of marrow volume due to fibrosis appears to be crucial with respect to the prognostic significance of MF in CML. Hum Pathol 34:391-401.     

Elevated IL-35 in bone marrow of the patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common hematological malignancy in adults, but the etiology of it remains poorly understood. IL-35 is a recently described cytokine composed of an IL-12 subunit p35 and an IL-27 subunit Epstein–Barr virus induced gene 3 (EBI3), and has an immunosuppressive effect on inflammation through induction of regulatory T cells (Tregs) and suppression of Th1 and Th17. Recently, we have illustrated that concentrations of IL-35 in peripheral blood are up-regulated in newly diagnosed (ND) AML patients. However, whether IL-35 in bone marrow is increased in AML patients is not clear. In this study, we examined IL-35 in bone marrow by various methods including RT-PCR, ELISA, FCM and IHC, and found that IL-35 levels are also increased significantly in bone marrow of adult AML patients. Furthermore, we investigated that concentrations of bone marrow IL-35 in ND group were higher than that in complete remission (CR) group and control group, but there was no significant difference compared to that in relapse group. In conclusion, IL-35 was elevated in bone marrow of adult AML patients and this increase was correlated with the clinical stages of malignancy, suggesting that IL-35 is involved in pathogenesis of AML.     

Relapse in chronic myeloid leukemia after bone marrow transplantation: biomathematical modeling as a new approach to understanding pathogenesis

A biomathematical model was developed to simulate relapse development in patients with chronic myeloid leukemia (CML) following bone marrow transplantation (BMT). The purpose of this study was to better understand the pathophysiology of the time evolution of CML relapse and to provide means whereby the outcomes of patients with CML relapse can be projected and treatment modified accordingly. The model consists of three parallel series of catenated compartments representing granulopoiesis in normal (donor) cells from the marrow, in CML cells from the marrow, and in CML cells from extramedullary sites. It was assumed that CML stem cells were resistant to feedback control and that CML-derived neutrophils, as well as normal neutrophils, exercised feedback regulation of normal stem cells. The known longer generation times for CML neutrophil precursors compared with normal neutrophil precursors were used, and it was assumed that 10(7) pluripotential stem cells were infused with BMT. The model was evaluated for its ability to simulate the reappearance of CML (Philadelphia chromosome positive) metaphases in the marrow and the recovery pattern in the blood neutrophil count in six patients who had relapsed following BMT (allogeneic in three patients, allogeneic with T-cell depletion in two patients, and syngeneic in one patient). The variables tested included the site of origin of the CML stem cells responsible for relapse (marrow alone versus marrow and extramedullary sites), the minimum number of CML stem cells responsible for relapse, and the time delay between BMT and the onset of relapse. Model profiles based on the observed values were obtained in each case. The simulations pointed to the fact that relapse began from a small number of CML cells in medullary and extramedullary sites. The time delay between BMT and the onset of relapse varied from 15 to 240 days. We suggest that this biomathematical model should be further investigated as a possible means of predicting outcome and guiding the treatment for patients with CML relapsing after BMT.     

Bone marrow trephine biopsy findings in chronic myeloid leukemia

Sixty patients with chronic myeloid leukemia (CML) underwent bone marrow trephine biopsy at presentation. All the biopsies were decalcified, paraffin-embedded and stained with H&E, Gomori's reticulin and Masson Trichrome. A detailed study of the histology including the morphology and topographic distribution of megakaryocytes was done. 55 patients presented in chronic phase. Of these there were 37 cases (67%) of CML-granulocytic (CML-G) type and 18 cases (33%) of CML-granulocytic megakaryocytic (CML -GM) type. Five cases presented in blast crisis. 73% of CML-G had low-grade fibrosis while 83% of CML-GM had high-grade fibrosis. This was statistically significant. On follow-up 25% of CML-G went into blast crisis while all the CML-GM patients remained stable to date. Bone marrow biopsy is a useful investigation in patients of CML at diagnosis as it provides prognostic information. Evaluation of megakaryopoiesis, grading of fibrosis and localization of blasts are possible on a trephine biopsy.     

Consequences of BCR-ABL expression within the hematopoietic stem cell in chronic myeloid leukemia

Chronic myeloid leukemia (CML) was the first human malignancy shown to be associated with a specific cytogenetic lesion, the Philadelphia chromosomal translocation. Forty years on, many biological and biochemical properties have been ascribed to its molecular product, the BCR-ABL tyrosine kinase fusion protein. However, it has been difficult to establish their precise contribution to the deregulation of normal survival, proliferative and differentiative control in chronic phase CML and the degree to which the involvement of stem cells extends beyond their role as the aetiological target. This review will focus on our current understanding of the pathogenesis of CML from the perspective of stem cell involvement, and how the biological and biochemical properties ascribed to BCR-ABL from studies of in vitro transformation and in vivo leukemogenesis systems relate to the abnormalities manifest in the human disease.     

Infectious complications in patients undergoing unrelated donor bone marrow transplantation: experience from a single institution

Objective   To analyze the incidence and characteristics of documented infections in patients with hematologic malignancies undergoing unrelated donor bone marrow transplantation (UD-BMT).
Methods   We studied the occurrence of infections in 22 patients with hematologic malignancies or severe aplastic anemia who underwent UD-BMT from April 1990 to December 2000. The median age was 26 years (range 13–46). Acyclovir–ganciclovir, co-trimoxazole, fluconazole–nystatin and ciprofloxacin were administered for anti-infectious prophylaxis.
Results   We registered 61 infectious episodes. During the early post-transplant period, there were eight clinically documented infections (CDIs), four cases of fever of unknown origin (FUO), seven cases of bacteremia, two cases of cytomegalovirus (CMV) antigenemia, and one case of CMV disease. In the intermediate period (days 30–100 after BMT), there were nine cases of CMV antigenemia, three bacterial infections, two fungal infections, one case of disseminated toxoplasmosis, and one case of FUO. In the late period (day 100 and later), we documented 13 viral infections, eight bacterial infections, one CDI, and one case of invasive aspergillosis. Infections contributed to death in 10 of 17 patients. Citrobacter bacteremia and sepsis of unknown origin were the main causes of infectious mortality in the early period. Infection was the main cause of death in six of seven patients in the late period.
Conclusion   A high incidence of life-threatening infections and infection-related mortality was observed. A high rate of CMV infection in the early period, and death caused by multiresistant Gram-negative microorganisms in the late period, were the main findings in this series.     

Analysis of BCR-ABL mRNA in chronic myelogenous leukemia patients and identification of a new BCR-related sequence in human DNA

The Philadelphia chromosome is present in more than 95% of chronic myelogenous leukemia patients and in up to 25% of patients with acute lymphocytic leukemia. The major consequence of the aberration is the fusion of the ABL and BCR genes. The position of the breakpoint on chromosome 22 determines which species of the potential three fused mRNAs and proteins will be synthesized. We have used the polymerase chain reaction (PCR) to detect these mRNAs in 53 patients and cell lines and found that around 20% contain simultaneously two BCR-ABL mRNAs, presumably due to a process of alternative splicing. The results also indicate that most patients in lymphocytic blast crisis of CML contain the mRNA in which bcr exon 2 is linked to ABL exon II. Finally, we identified, cloned, and characterized a BCR-related sequence that originated from mRNA.