Barbara Heinze

Detection of karyotypic aberrations in acute myeloblastic leukaemia: a prospective comparison between PCR/FISH and standard cytogenetics in 140 patients with de novo AML

In 140 patients with de novo acute myeloid leukaemia (AML) standard cytogenetics were compared with RT‐PCR for the detection of t(8;21), t(15;17) and inv(16) and fluorescence in situ hybridization (FISH) for numerical aberrations of chromosomes 7, 8, X and Y. RT‐PCR detected 18 cases with t(8;21), 12 with t(15;17) and seven with inv(16). In two cases with t(8;21), two with t(15;17) and four with inv(16) these aberrations had not been detected by standard cytogenetics. There were no false negative PCR results. In 12 patients with these chromosomal changes, standard cytogenetics revealed additional chromosomal aberrations. In 16 patients sole numerical aberrations of the chromosomes 7, 8, X or Y were found by FISH. In these patients the sensitivity of FISH and standard cytogenetics was comparable. In 87 patients no aberrations could be found by PCR and FISH whereas in 24 of these patients standard cytogenetics revealed an abnormal karyotype. These data recommend the combination of standard cytogenetics and molecular techniques to improve the sensitivity for the detection of genetic lesions in AML. Once chromosomal markers have been identified by combined analysis these markers could be used to monitor residual disease during/after chemotherapy, by RT‐PCR and/or FISH.

Marrow fibrosis, indicator of therapy failure in chronic myeloid leukemia – prospective long-term results from a randomized-controlled trial

Marrow fibrosis (MF) has rarely been considered in therapy studies on chronic myeloid leukemia (CML), and there is a lack of long-term observations on the basis of sequential bone marrow biopsies (BMBs) taken prospectively during the course of disease. A total of 848 BMBs from 400 patients with Ph+ CML recruited in the German randomized CML study I were examined for MF before and during therapy. In total, 110 patients had been randomized to receive interferon (IFN)-α, and 290 to receive chemotherapy (hydroxyurea (HU): 154, busulfan: 136). During IFN-α and HU medication, MF was reduced or did not increase for about 2 years. Evolving or progressive MF was an independent and early predictor of therapy failure about 2 years earlier than indicated by changes in the peripheral blood, spleen size, marrow blast count and cytogenetics (P<0.00005), resulting in a significant shortening of the survival times of patients independent of the type of therapy applied including allografting (multivariate analyses; P<0.00005). The analyzed long-term observations strongly indicate that MF is an independent poor prognostic complication of CML, allowing an early prediction of therapy failure. Consideration of the fiber content in marrow may therefore significantly improve the prediction of therapy efficacy and outcome of disease.

Chronic myelogenous leukemia in blast crisis: retrospective analysis of prognostic factors in 90 patients

Abstract Ninety patients with Philadelphia chromosome-positive chronic myelogenous leukemia in blast crisis were reviewed to identify significant prognostic associations. At diagnosis of blast crisis the main clinical, laboratory, and cytogenetic data were recorded and evaluated for prognostic significance. At the time of the analysis 89 patients had died, with a median survival of 11 weeks from diagnosis of blast crisis. Patient characteristics demonstrated in the univariate analysis to have significant association with shorter survival were: thrombocythemia, leukocyte count above 20×109, Karnofsky index <50%, nonlymphoid blast cell morphology, cytogenetic clonal evolution, the presence of a double Philadelphia chromosome or trisomy 8, and no response to therapy. In 17 of 59 patients (29%) evaluable for response to therapy a complete or partial remission was achieved. These responders had a significantly longer median survival (25 weeks) as compared with nonresponders (9 weeks). Response to therapy was significantly better in lymphoid blast crisis and in patients without clonal evolution. In a multivariate analysis containing all significant variables of the univariate analysis two parameters retained their prognostic significance: response to therapy and trisomy 8. In spite of the short overall survival in blast crisis, the determination of prognostic factors may be a useful tool for the clinician planning therapy, especially new therapeutic approaches.

Karyotype abnormalities and their clinical significance in blast crisis of chronic myeloid leukemia

Abstract We examined karyotypes and their prognostic significance in a series of 122 patients with chronic myeloid leukemia in blast crisis. Of 73 patients cytogenetically examined at the onset of blast crisis 63% had developed secondary cytogenetic abnormalities in addition to the Philadelphia chromosome. These newly emerging abnormalities included a double Philadelphia chromosome in 20 patients, a trisomy 8 in 17, and an isochromosome 17q in 11 patients. Development of such additional karyotypic abnormalities was significantly associated with a shorter median survival and less response to cytoreductive treatment and was significantly more common in nonlymphoid blast crisis than in the lymphoid-type blast crisis. Thus, assessment of karyotypes at the onset of chronic myeloid leukemia blast crisis appears to be of prognostic significance for both remission duration and survival.

A phase I/II study of recombinant interferon alpha 2a and hydroxyurea for chronic myelocytic leukemia

SummaryNine previously untreated patients with Philadelphia chromosome-positive chronic myelocytic leukemia (CML) were treated with recombinant interferon alpha 2a (rIFN-alpha 2a) and hydroxyurea. Patients received 6×106 U rIFN-alpha 2a daily for the first week and 3×106 U rIFN-alpha 2a daily for the second week. As maintenance treatment starting on day 15, patients received 3×106 U rIFN-alpha 2 a 3 times a week. Simultaneously, hydroxyurea was given, starting at a dose of 40 mg/kg on day one. The maintenance dosage was adjusted to the white blood cell count. Two patients responded with complete hematological remissions but without cytogenetic and molecular-genetic improvements. Seven patients responded with partial hematological remissions. Response to therapy was rapid; normal white blood cell counts were reached after a median of 12 days. The doses of rIFN-alpha 2a and hydroxyurea needed to keep the leucocyte count in the normal range were low (3×106 U rIFN-alpha 2a 3 times per week, 0.5–1.5 g hydroxyurea/day). Acute toxicity of the combination therapy consisted of fever (9 of 9 patients), flulike symptoms (7 of 9 patients), pruritus and/or rash (3 of 9 patients) and evidence of a tumor cell lysis syndrome (1 of 9 patients). The side effects were not dose-limiting. Combination therapy with rIFN-alpha 2a and hydroxyurea for CML is well tolerated and allows quick and effective hematological control of the disease.

Chronic myeloid leukemia in the elderly: long-term results from randomized trials with interferon alpha

Chronic myeloid leukemia (CML) in older patients has not been studied well. To assess the long-term outcome of older patients with Philadelphia- and/or BCR-ABL-positive CML, 199 patients aged ⩾60 years representing 23% of 856 patients enrolled in the German randomized CML-studies I (interferon α (IFN) vs hydroxyurea (HU) vs busulfan (BU) and II (IFN+HU vs HU alone) were analyzed after a median observation time of 7 years. In all, 45 patients were treated with Bu, 63 with HU, and 91 with IFN. The 5-year survival was 38% in patients ⩾60 years and 47% in patients <60 years (P<0.001). Whereas 5-year survival in chemotherapy-treated older patients was inferior to that in younger patients (33 vs 46%, P=0.006 for HU and 29 vs 38%, P=0.042 for Bu), no significant survival difference could be verified in IFN-treated patients (46 vs 53%, P=0.077). Calculation of age-adjusted, relative survival confirmed these results. Adverse effects of IFN were similar in both age groups, but IFN dosage to achieve treatment goals was lower in older patients. We conclude that the course of CML is not different in the elderly. They require lower IFN doses, achieve the same hematologic and cytogenetic response rates and the same survival advantage at comparable toxicity.

Stable chromosomal aberrations in haemopoietic stem cells in the blood of radiation accident victims

PURPOSE The detection of long-term persistent chromosome aberrations in circulating haemopoietic stem cells after accidental radiation exposure. MATERIAL AND METHODS Peripheral blood samples from highly exposed persons were collected 7-25 years after the radiation accidents in Moscow (1971), Kazan (1975) and Chernobyl (1996). Haemopoietic blood stem cells were analysed when investigating individual colonies derived from haemopoietic progenitor cells: burst-forming units-erythroid (BFU-E), granulocyte-macrophage-colony-forming cells (GM-CFC) and multipotent granulocyte-erythrocyte-macrophage- megakaryocyte-colony-forming cells (GEMM-CFC). Colony formation was obtained in methylcellulose cultures. Chromosome preparations in single colonies were performed using a microtechnique. RESULTS Nine patients were investigated at 1 to 4 follow-up time points after radiation exposure. Three hundred and thirty-four single colonies were analyzed resulting in 1375 mitoses. It was found that colonies showed chromosome aberrations (ChA) up to 25 years after radiation exposure by classical cytogenetics and by fluorescence in situ hybridization (FISH). Stable aberrations were detected in 21% of colonies. They were clonal in 19% of colonies, i.e. the same abnormality was found in all cells derived from a single colony. In 2% of colonies ChA were stable but non-clonal; unstable ChA were not observed. CONCLUSIONS The results indicate that blood-derived haemopoietic stem cells may serve as a biological indicator to detect radiation-induced ChA. Since they are considered to be in dynamic and functional exchange with stem cells in the medullary sites of blood cell formation such as bone marrow, the use of blood stem cells as a marker of radiation effects should be explored to assess the repair status of the stem cell pool as such.

Computer aided analysis of additional chromosome aberrations in Philadelphia chromosome positive acute lymphoblastic leukaemia using a simplified computer readable cytogenetic notation

BackgroundThe analysis of complex cytogenetic databases of distinct leukaemia entities may help to detect rare recurring chromosome aberrations, minimal common regions of gains and losses, and also hot spots of genomic rearrangements. The patterns of the karyotype alterations may provide insights into the genetic pathways of disease progression.ResultsWe developed a simplified computer readable cytogenetic notation (SCCN) by which chromosome findings are normalised at a resolution of 400 bands. Lost or gained chromosomes or chromosome segments are specified in detail, and ranges of chromosome breakpoint assignments are recorded. Software modules were written to summarise the recorded chromosome changes with regard to the respective chromosome involvement. To assess the degree of karyotype alterations the ploidy levels and numbers of numerical and structural changes were recorded separately, and summarised in a complex karyotype aberration score (CKAS). The SCCN and CKAS were used to analyse the extend and the spectrum of additional chromosome aberrations in 94 patients with Philadelphia chromosome positive (Ph-positive) acute lymphoblastic leukemia (ALL) and secondary chromosome anomalies. Dosage changes of chromosomal material represented 92.1% of all additional events. Recurring regions of chromosome losses were identified. Structural rearrangements affecting (peri)centromeric chromosome regions were recorded in 24.6% of the cases.ConclusionsSCCN and CKAS provide unifying elements between karyotypes and computer processable data formats. They proved to be useful in the investigation of additional chromosome aberrations in Ph-positive ALL, and may represent a step towards full automation of the analysis of large and complex karyotype databases.

German multicenter study group for adult ALL (GMALL): recruitment in comparison to ALL incidence and its impact on study results.

Abstract.Due to eligibility criteria not all patients with the disease under investigation can be recruited for therapeutic studies. Thus, the external validity of study results cannot per se be taken for granted. The representativity of the admitted patients is the most relevant determinant for external validity and has to be assessed. As an example we examined the representativity of the patients recruited for the German multicenter study group for adult acute lymphoblastic leukemia (ALL) (GMALL). Lacking nationwide ALL incidence figures available in Germany, a methodology was developed to estimate incidence figures, too. All relevant study groups, hospitals, and diagnostic labs were asked to provide data about patients with ALL newly diagnosed between 1997 and 1998. A matching procedure was developed, as heterogeneous databases had to be pooled and checked for duplicates. Age- and sex-specific incidences of ALL were estimated and compared with the number of patients recruited for the GMALL in the same time period. The purpose was to develop a methodology for estimating incidence figures and evaluating the representativity of patients of the GMALL. The combination of various data sources allowed estimation of reliable incidence data for ALL in Germany. Comparisons with the incidence figures for ALL in other countries and crosschecks within Germany confirm our results. Sixty-two percent of all ALL patients in Germany were admitted to the GMALL study. The recruitment rate of more than 60% of the annual incidence of ALL to the GMALL suggests a high external validity as well as an impact of the study on the patterns of treatment and referral of ALL in adults in Germany. There is no selection bias of patients admitted to the GMALL compared to those patients not included in the study.

Chronic myeloid leukemia in accelerated phase: treatment results with conventional chemotherapy and allogeneic bone marrow transplantation in 96 patients

Abstract: The treatment results of 96 patients with Philadelphia‐positive chronic myeloid leukemia (CML) in accelerated phase (AP) were reviewed retrospectively. Treatment of AP consisted of allogeneic bone marrow transplantation in 20 (14 related and 6 unrelated donors) or conventional chemotherapy (CC) in 76 patients. Three main treatment strategies were followed in the CC group: continuation (group A) or dose escalation (group B) of chronic phase therapy or change of chronic phase therapy to hydroxyurea (group C). Median survival was 7.0 months in group A (range 1.8–110), in group B 8.3 months (range 0.9–40) and in group C 9.6 months (range 1.5–47.6), p = 0.89. Survival in CC was dependent on response to therapy as the achievement of a second chronic phase was significantly associated (p < 0.001) with a longer median survival (21 months) compared with stable accelerated phase disease (11 months) or treatment failure (5 months). Median survival in the BMT group was 16.7 months (range 5–77), the 5‐yr probability of relapse was 25% and the 5‐yr disease‐free survial was 36%. For patients < 55 yr median survival after BMT was significantly prolonged compared with median survival after CC (n = 45, 8.3 months, p = 0.008). After developing criteria of AP, median survival in our analysis has been less than 1 yr. The results of conventional chemotherapy in the treatment of accelerated phase CML are disappointing. If a suitable donor is available allogeneic BMT should be performed without delay in patients with AP.