Quantification of the breakpoint cluster region rearrangement for clinical monitoring in Philadelphia chromosome-positive chronic myeloid leukemia

The purpose of this report was to evaluate scintigraphy analysis of Southern blot hybridization as a method to quantify the breakpoint cluster region (BCR) rearrangement of Philadelphia chromosome (Ph)+ chronic myelogenous leukemia (CML). Cytogenetic and molecular studies performed simultaneously on 474 bone marrow and/or blood samples from 300 patients treated with alpha-interferon-based therapy were compared. Molecular results were expressed as the percentage of rearranged BCR bands versus the total scintigraphic signal. The percentage of Ph+ metaphases was calculated on 25 metaphases. The results of molecular studies obtained on both peripheral blood and bone marrow samples were identical. The rank correlation between the BCR quantification and the percentage of Ph positivity in 465 samples was excellent (r = .78). However, of 99 samples with a normal karyotype, 24% had a BCR rearrangement. Of 86 samples with no BCR rearrangement, 13% showed a Ph chromosome. Of 49 samples with partial cytogenetic remission (Ph+ metaphases, 1% to 34%), 23% had no BCR rearrangement. In samples with a minor or no cytogenetic response (Ph+ metaphases, > 34%), BCR analysis overestimated the degree of response in 73 of 326 samples (22%). Nevertheless, survival analysis by BCR quantification level showed statistically better outcome for patients in complete or partial molecular response (P < .01). Molecular quantification of BCR was useful in monitoring the course of Ph+ CML. This method, which can be used on peripheral blood, detected residual disease not shown by cytogenetic analysis and was prognostically relevant as a measure of disease suppression.     

Cytogenetic response to autografting in chronic myelogenous leukemia correlates with the amount of BCR-ABL positive cells in the graft

OBJECTIVE: An objective of autologous transplant in chronic myelogenous leukemia is to reinfuse the patient stem cells with the lowest amount of leukemic progenitors. We analyzed if the cytogenetic response after autografting was correlated with the amount of BCR ABL-positive cells present in the graft. MATERIALS AND METHODS: By BCR-ABL mRNA quantification, we studied the serial pheresis product from 40 Philadelphia (Ph)-positive patients who received ICE/mini-ICE mobilization therapy and underwent autologous stem cell transplant in our unit. Therefore, we correlated the residual disease in the graft reinfused to the patients with the cytogenetic response after autografting, taking into consideration only those responses that lasted 12 months or more. RESULTS: Thirty-two patients had a graft with 0-35% Ph-metaphases and 19 had a graft with BCR-ABL/ABL ratio < or =0.01. After a median of 27 months (range, 12-50) from transplant, 18 patients achieved complete or major cytogenetic response lasting at least 12 months and 14 of them (78%) had a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.0003-0.01). Twenty-two patients had short-lived response or showed to be >35% Ph-positive in the marrow after transplant, but only 5 of them (23%) had a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.001-0.01). Therefore, we found a strong association between BCR-ABL/ABL ratio < or =0.01 in the reinfused progenitor collections and the achievement of complete or major cytogenetic remission after autografting (chi2 test, p = 0.0001). Patients reinfused with low contaminated grafts also showed a longer duration of the response (logrank test, P = 0.0009). Eleven patients were reinfused with the lowest-contaminated stem cell collections, according to the BCR-ABL/ABL ratio. None of these patients had prolonged neutropenia or thrombocytopenia following stem cell reinfusion and 9 of them had long-lasting complete or major cytogenetic response after transplant. CONCLUSION: This study demonstrates that the amount of BCR-ABL positive cells present in the graft is an important predictive factor for the achievement and the duration of cytogenetic response after autografting.     

Prolonged suppression of myeloid progenitor cell numbers after stopping interferon treatment for CML may necessitate delay in harvesting marrow cells for autografting

Fourteen patients with chronic myelogenous leukemia in chronic phase who had achieved hematological remission after alpha-interferon (IFN) therapy were evaluated for their ability to form hemopoietic colonies in vitro. Patients were studied both before and after discontinuation of IFN to assess the optimal timing for a further therapeutic approach consisting of stem cell collection followed by autologous bone marrow transplantation (ABMT). Before stopping IFN a profound inhibition of CFU-GM and BFU-E growth was observed. The numbers of CFU-GM and BFU-E in the bone marrow recovered to 50 per 2 x 10(5) cells plated at a median of 158 and 122 days, respectively, after stopping IFN. At that time, 12 patients were submitted to stem cell harvest and subsequently to ABMT. All patients showed complete engraftment and hematological reconstitution; cytogenetic improvement was observed in eight (67%) cases; two patients displayed complete disappearance of the Ph1 chromosome, confirmed at the molecular level. Eleven of the 12 patients are still in hematological remission at a median of 18 months from autografting and a median of 48 months from diagnosis.     

Use of alpha interferon for the treatment of relapse of chronic myelogenous leukemia in chronic phase after allogeneic bone marrow transplantation.

Eighteen patients with relapse of chronic myelogenous leukemia (CML) after allogeneic bone marrow transplantation (BMT) were treated with recombinant human alpha 2a interferon (IFN). Relapse was defined as greater than 90% metaphases containing the Philadelphia chromosome (Ph) and hematologic abnormalities consistent with chronic-phase (CP) CML. There were 11 males and seven females, with a median age of 38 years (range, 3 to 55). Three patients relapsed after second BMT. Only one patient had received T-cell-depleted marrow initially. The initial IFN dose of 3 x 10(6) U/m2/d was escalated to the maximum tolerated dose or to a maximum of 6 x 10(6) U/m2/d. IFN controlled the white blood cell (WBC) counts in 14 of 16 patients who had abnormal counts, and in all six patients with an elevated platelet count. Six patients (33%) have had a complete disappearance of the Ph and two have had a partial response (less than 35% Ph+ metaphases). One patient has a decrease in Ph+ metaphases after 9 months of IFN. Five patients had no significant cytogenetic response after 9 to 12 months, and four developed clinical accelerated phase or blast crisis after 3 to 6 months on therapy. Of four patients with a sex marker, the Ph- population was of donor origin in three and of host origin in one. Clonal cytogenetic abnormalities other than Ph were present in 13 patients and did not predict for lack of response to IFN. IFN is effective in suppressing the Ph clone in some patients who relapse with CML after allogeneic BMT and controls the blood counts in the majority.     

Hematological and cytogenetic response of interferon alpha 2b alone and combined interferon alpha plus cytarabine as a first-line treatment in chronic myeloid leukemia

Treatment with interferon-alpha (IFNalpha) prolongs survival in chronic myeloid leukemia (CML). Additionally, cytarabine (AraC) can reduce the number of Ph + metaphases. Fortythree previously untreated patients with CML in chronic phase were randomly assigned to receive either. IFNalpha 2b (5 MU sqm/daily) or IFNalpha 2b in the same dosages plus monthly courses of low-dose AraC. The aim were complete hematologic remission at 6 months and cytogenetic response at 12 months. A complete hematologic remission occurred in 60.4% patients with single IFNalpha 2b in 76.2% patients with combination therapy. A cytogenetic response was present in 13.9% (major in 2 patients) with IFN therapy and in 38.1% patients with combination therapy. Two of 21 patients treated with IFNalpha/AraC therapy achieved major (9.52%), 4 partial (19.04%) and 2 minor (9.52%) cytogenetic response. Major side effects were cytopenia (20.1%), flu-like syndromes (42.4%) and increase of hepatic transaminases (3.4%). The side effects were more significant in the group receiving combination therapy. Based on published data that show a survival advantage for patients who achieved any cytogenetic response, and high rate of cytogenetic response which we observed in our study we believe that IFN plus AraC regimen could be a front-line therapy for CML.     

Intensive treatment and stem cell transplantation in chronic myelogenous leukemia: long-term follow-up

In the present study we combined interferon (IFN) and hydroxyurea (HU) treatment, intensive chemotherapy and autologous stem cell transplantation (SCT) in newly diagnosed chronic myelogenous leukemia patients aged below 56 years, not eligible for allogeneic SCT. Patients who had an HLA-identical sibling donor and no contraindication went for an allogeneic SCT (related donor, RD). After diagnosis, patients not allotransplanted received HU and IFN to keep WBC and platelet counts low. After 6 months patients with Ph-positive cells still present in the bone marrow received 1-3 courses of intensive chemotherapy. Those who became Ph-negative after IFN + HU or after 1-3 chemotherapy courses underwent autologous SCT. Some patients with poor cytogenetic response were allotransplanted with an unrelated donor (URD). IFN + HU reduced the percentage of Ph-positive metaphases in 56% of patients, and 1 patient became Ph-negative. After one or two intensive cytotherapies 86 and 88% had a Ph reduction, and 34 and 40% became Ph-negative, respectively. In patients receiving a third intensive chemotherapy 92% achieved a Ph reduction and 8% became Ph-negative. The median survival after auto-SCT (n = 46) was 7.5 years. The chance of remaining Ph-negative for up to 10 years after autologous SCT was around 20%. The overall survival for allo-SCT RD (n = 91) and URD (n = 28) was almost the same, i.e. approximately 60% at 10 years. The median survival for all 251 patients registered was 8 years (historical controls 3.5 years). The role of the treatment schedule presented in the imatinib era is discussed.     

Philadelphia chromosome-positive adult acute lymphocytic leukemia

The CML-specific Philadelphia (Ph1) chromosome is relatively common cytogenetic abnormality of ALL, which has been shown 20% of adult ALL and 5% of child ALL. We analysed here the 12 patients of Ph1-positive ALL, aged 35 to 69-years old, who were experienced in our hospital for latest eight years. In comparison with Ph1-negative ALL, these 12 patients were elder and showed high peripheral and bone marrow leukemic cell counts. Of these, seven patients had 100% Ph1 abnormality in the bone marrow and another five patients showed mosaic marrow patterns of Ph1 and normal chromosomes. Remissioned eight cases had no more Ph1 abnormalities in their bone marrows. Our Ph1-positive ALL revealed B-cell lineage leukemia, since their surface phenotype were Ia+ and CD10+ and they have rearranged immunoglobulin JH genes. Four out of these nine patients had such gene rearrangement in the 5.8kb bcr (major BCR: M-BCR) as CML's patient had. Eight out of twelve Ph1-positive ALL patients (66.7%) achieved complete remission, but the prognosis was so bad since they had shorter remission duration (median 6.7 mos) and survival months (median 11.9 mos) than those of Ph1-negatives.     

Tyrosine kinase inhibitor STI571 in the treatment of Philadelphia chromosome-positive leukaemia failing myeloablative stem cell transplantation

Eight patients with Philadelphia chromosome-positive (Ph(+)) leukaemia relapsing from stem cell transplantation (SCT) (one syngeneic and seven allogeneic) were treated with the tyrosine kinase inhibitor STI571. Five patients relapsing as chronic myeloid leukaemia (CML) in chronic phase achieved a complete haematological response, with complete and major cytogenetic responses occurring in four and one cases, respectively. One patient became negative for BCR/ABL in the bone marrow. Three patients relapsed as acute leukaemia (two CML in myeloblastic crisis and one Ph(+) acute lymphoblastic leukaemia), all of whom achieved haematological and cytogenetic responses. One patient also became BCR/ABL negative. However, pancytopenia and graft-versus-host disease led to cessation of treatment in the remaining two cases, which was followed by disease recurrence refractory to further STI treatment. Our results showed that Ph(+) leukaemic relapses after SCT might respond well to STI571 therapy.     

Autologous transplantation of Ph-negative peripheral blood stem cells for treatment of chronic myelogenous leukemia

A 21-year-old man, diagnosed in March 1997 as having chronic myelogenous leukemia (CML), received hydroxyurea followed by daily interferon (IFN) until December 1998, when the additional chromosome abnormality of +8 appeared. As no suitable matched donor was available, the patient received mobilization therapy consisting of mini-ICE (idarubicin, cytarabine, etoposide) followed by G-CSF subcutaneously. During hematopoietic recovery, a total of 12 x 10(6)/kg CD34-positive cells were harvested. Cytogenetic analysis of peripheral blood stem cell (PBSC) products using FISH revealed 1% BCR/ABL fusion signals. In March 1999, he received conditioning therapy consisting of busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) followed by infusion of 5 x 10(6)/kg CD34-positive cells. A neutrophil count of 500/microliter and a platelet count of 5 x 10(4)/microliter were attained by days 20 and 38, respectively. Bone marrow aspirates showed 2.6% BCR/ABL fusion signals on day 35 after autologous PBSC transplantation, and the patient remained in chronic phase until the sixth month, when a cytogenetic relapse (Ph, +8:4/20) occurred. These observations suggest that Ph-negative progenitor cells can be harvested using a mini-ICE regimen followed by G-CSF, and that autologous PBSC transplantation is feasible in patients with CML resistant to IFN.     

Apparent decrease and elimination of BCR/ABL mRNA-expressing residual cells in patients with chronic myelogenous leukemia after allogeneic bone marrow transplantation

Summary A modified two-step polymerase chain reaction (PCR) was used for the amplification of BCR/ABL mRNA in 16 patients with Philadelphia chromosomepositive (Ph+) chronic myelogenous leukemia (CML) following allogeneic bone marrow transplantation (BMT). At different intervals after BMT, patient cells were assessed for the presence of BCR/ABL mRNA by two subsequent rounds of PCR amplification; this procedure increased the sensitivity for the detection of one Ph+ cell in 10^4–5 to one cell in 10^5–6. Eight of 16 patients were negative by two-step PCR 1–39 months after BMT, suggesting an elimination of Ph-positive cells or a decrease below the threshold of detection. Although five patients showed negative results by the one-step PCR only, they were tested positive when nested primers were used, indicating a substantial decrease in the amount of BCR/ABL target mRNA compared with earlier pre- or post-transplant analyses. One patient who was still PCR positive 27 months after BMT became negative 12 months later. Persistence of BCR/ABL mRNA-expressing cells correlated with subsequent clinical relapse only when the transplantation was performed during blast crisis. All patients who underwent transplantation in chronic phase, including those with BCR rearrangement by PCR, are in clinical and hematological remission between 24 and 95 months after BMT. We conclude that aggressive chemotherapy combined with total body irradiation is unable to completely eradicate the malignant clone in all CML patients, and it might be speculated that other mechanisms (e.g., graft versus host reaction [GVHD] or graft versus leukemia effect [GVL]) may effectively eliminate residual leukemic cells.The studies were supported by grant Do 176/5-1 from theDeutsche Forschungsgemeinschaft     

甲磺酸伊马替尼治疗120例慢性髓性白血病的临床研究

目的观察甲磺酸伊马替尼（IM）治疗Ph染色体阳性和（或）BCR／ABL基因阳性的慢性髓性白血病（CML）的疗效和安全性。方法对90例Ph染色体阳性和（或）BCR／ABL基因阳性CML慢性期（CP）患者,持续口服IM,400mg／d;30例CML疾病进展期（加速期／急变期）患者,持续口服IM,600mg／d。服药期间定期复查血常规、骨髓细胞学、染色体和（或）BCR／ABL基因等指标,并随访观察。结果（1）CML—CP患者总的完全血液学缓解率（CHR）、完全细胞遗传学缓解率（CCyR）和完全分子遗传学疗效（CMR）分别为73．3％（66／90）、66．7％（60／90）、54．4％（49／90）;治疗前是否接受过干扰素治疗对CHR、CCyR和CMR均无明显影响;服药前病程≤6个月的CMR优于〉6个月者。初次达到CHR的时间与首次达CCyR的时间、首次达CCyR的时间与BCR／ABL首次转阴时间之间均存在相关性,而初次达CHR的时间与BCR／ABL首次转阴时间则无明显相关性。（2）进展期CML患者的CHR、CCyR、CMR分别为43．3％（13／30）、25．9％（7／27）、25．0％（7／28）,总病死率为30．0％（9／30）。（3）年龄≤25岁患者的病死率高于〉25岁者,差异有统计学意义（P〈0．05）。（4）白细胞减少达Ⅲ级者有19例（16．0％）,发生于治疗后5～20周。血小板减少达Ⅲ级者有21例（18．0％）,发生于治疗后3～16周。主要的非血液系统毒性为双下肢水肿、骨痛和皮疹等,但均程度轻微。结论IM对初治CML及经干扰素治疗失败的CML有较高的CHR及CCyR且起效迅速,对CML—CP疗效显著优于进展期;不良反应程度轻微,患者易于耐受。     

Interferon alpha-2c therapy of patients with chronic myelogenous leukemia: long-term results of a multicenter phase-II study

 In a prospective multicenter phase-II trial 80 patients with Philadelphia (Ph)-positive chronic myelogenous leukemia (CML) were treated with recombinant interferon (IFN)α-2c, administered subcutaneously at an absolute dose of 3.5 megaunits (MU)/day. Complete hematological remission was achieved in 29 (39%) and partial hematological remission in 26 (35%) of the 74 patients evaluable for response. Major cytogenetic responses were observed in ten (13%) and minor cytogenetic responses in 11 patients (15%). Median duration of cytogenetic response was 33 months (range, 2–90); relapses were seen in all of the 11 patients with minor and in three of the ten patients with major cytogenetic responses. Median survival estimates for pretreated (n=19) and untreated (n=58) patients were 51 months (95% confidence interval [CI], 30–72) and 77 months (95% CI, 43–111), and the survival probabilities at 5 years were 45% and 54% for the two groups, respectively. Hematological response after 3 months of treatment demonstrated a clear-cut discriminative capacity with 5-year survival probabilities of 100%, 67% and 24% for patients achieving CHR (n=6), PHR (n=34), and less than PHR (n=35), respectively. Landmark analysis at 12, 18, and 24 months after start of IFN therapy and an analysis treating time to cytogenetic response as a time-dependent covariate showed that cytogenetic response was associated with longer survival. The impact of a low-dose IFN regimen on survival in CML patients is unclear and requires further clarification by randomized clinical trials. Early hematological and cytogenetic response to IFN-α treatment identifies patients with a favorable long-term prognosis. Received: 26 October 1995 / Accepted: 3 January 1996     

Impact of interferon alpha-induced cytogenetic improvement on survival in chronic myelogenous leukaemia

Summary. The objective of this study was to investigate the prognostic impact of the reduction of Philadelphia chromosome (Ph) positive metaphases by treatment of chronic myelogenous leukaemia (CML) with interferon (IFN) alpha. Therefore, we evaluated the outcome of patients with previously untreated chronic phase Ph-positive CML, enrolled from 1984 to 1990 into two consecutive IFN trials at our institution. Of a total of 71 patients, 62 (87%) were evaluable for cytogenetic response. No cytogenetic improvement was seen in 16 patients (23%), 28 patients (38%) had a decrease in Ph-positive bone marrow metaphases to levels ranging from 35% to 95%, and nine patients (13%) to levels between 5% and 34%. In nine patients (13%), Ph-positive metaphases were no longer detectable. After a median follow-up period of 33 months, the projected 5-year survival is 55% for the 62 patients evaluable for cytogenetic response. In this patient population there was no significant difference in the survival probability according to patients'risk status as defined by the Sokal score. Categorization according to the extent of Ph reduction, however, allowed three groups with significantly different prognoses to be identified. Patients achieving a Ph reduction to less than 35% were found to constitute a low risk group with a median survival not yet known and a projected 5-year survival of 90%. The 5-year survival rate was 55% for patients with a Ph reduction to levels between 35% and 95%, and less than 10% for those without any cytogenetic improvement. Thus, this study demonstrates that cytogenetic improvement on IFN treatment is an important prognostic factor for survival.