Gerhard Heil

Real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients.

AML1/MTG8 was quantified relative to the expression of the GAPDH housekeeping gene by real‐time RT‐PCR in 22 patients with t(8;21)‐positive acute myeloblastic leukaemia (AML) at initial diagnosis and in seven of these patients also during/after chemotherapy and allogeneic bone marrow transplantation. Real‐time PCR was able to specifically detect and quantify AML1/MTG8 over a 5 log range. The detection limit for t(8;21)‐positive cells was a dilution of 1:105. The AML1/MTG8 expression varied considerably among the 22 AML patients at intial diagnosis with a ratio AML1/MTG8:GAPDH of 0.5135±0.536 (range 0.1–2.14, median 0.318). In six patients with t(8;21)‐positive AML a marked decline of AML1/MTG8 could be induced by chemotherapy. These patients are in ongoing complete haematological remission (CR) with a constant low‐level AML1/MTG8 expression. In another patient a rapid rise of AML1/MTG8 transcripts could be detected in CR after allogeneic bone marrow transplantation and the patient relapsed 10 weeks later. In conclusion, real‐time RT‐PCR is a suitable approach for the quantification of AML1/MTG8 transcripts in the monitoring of AML patients with t(8;21) during/after chemotherapy and can provide data of prognostic relevance.

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.

Intensive chemotherapy with idarubicin, cytarabine, etoposide, and G-CSF priming in patients with advanced myelodysplastic syndrome and high-risk acute myeloid leukemia

In an attempt to improve the complete remission (CR) rates and to prolong the remission duration especially in elderly patients >50xa0years of age, we have used a combination chemotherapy of idarubicin (10xa0mg/m2 IV×3xa0days), cytarabine (AraC, 100xa0mg/m2 CIVI×7d), and etoposide (100xa0mg/m2×5xa0days) in combination with granulocyte colony-stimulating factor (G-CSF) priming [5xa0mg/kg SQ day 1 until absolute neutrophil count (ANC) recovery] for remission induction. Responding patients received two consolidation courses of idarubicin, AraC, and etoposide, followed by a late consolidation course of intermediate-dose AraC (600xa0mg/m2 IV every 12xa0h×5xa0days) and amsacrine (60xa0mg/m2 IV×5xa0days). A total of 112xa0patients (57 male/55 female) with a median age of 58xa0years (range: 22–75) have been entered and are evaluable for response: 19 refractory anemia with excess of blast cells in transformation (RAEB-T), 84 acute myeloid leukemia (AML) evolving from myelodysplastic syndrome (MDS), and 9 secondary AML after chemotherapy/radiotherapy. The overall CR rate was 62%, partial remission (PR) rate 10%, treatment failure 16%, and early death rate 12%. The CR rate was higher in patients ≤60xa0years (68 vs 55%), mainly due to a lower early death rate (5 vs 21%, p<0.001). After a median follow-up of 58 months, the median overall survival is 14.5% and median duration of relapse-free survival 8 months. After 60 months, the probability of CR patients to still be in CR and alive is 16% (20% in patients ≤60xa0years and 13% in patients >60xa0years), while the probability of overall survival is 12% (15% in patients ≤60xa0years and 9% in patients >60xa0years). Compared to our previous trial (AML-MDS Study 01-92) which was done with identical chemotherapy but no G-CSF priming in 110xa0patients with RAEB-T, AML after MDS, or secondary AML (identical median age, age range, and distribution of subtypes), the CR rate in all patients, as well as CR rate, overall survival, and relapse-free survival in patients >60xa0years have significantly been improved. Thus, intensive chemotherapy with G-CSF priming is both well tolerated and highly effective for remission induction in these high-risk patients.

The oncogenic fusion protein RUNX1-CBFA2T1 supports proliferation and inhibits senescence in t(8;21)-positive leukaemic cells

BackgroundThe fusion protein RUNX1-CBFA2T1 associated with t(8;21)-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t(8;21)-positive cell lines.MethodsThe t(8;21)-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence.ResultsElectroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity.ConclusionsRUNX1-CBFA2T1 supports the proliferation and expansion of t(8;21)-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches.

Detection and quantification of CBFB/MYH11 fusion transcripts in patients with inv(16)-positive acute myeloblastic leukemia by real-time RT-PCR

We used a newly established real‐time RT‐PCR assay for the quantification of the leukemia‐specific CBFB/MYH11 transcripts in inv(16)‐positive acute myeloblastic leukemia. CBFB/MYH11 could be quantified over a five log range, with a detection limit of 10 molecules of a CBFB/MYH11 plasmid and a 1:105 dilution of RNA of the inv(16)‐positive ME‐1 cell line, respectively. The fusion transcripts were also quantified in 19 patients with acute myeloblastic leukemia and an inv(16) at initial diagnosis. The expression of CBFB/MYH11 varied over a two log range without correlation to clinical response or relapse rate. In nine patients, CBFB/MYH11 was also quantified during/after chemotherapy and autologous or allogeneic stem cell transplantation. All of these patients showed a similar decline of CBFB/MYH11 after intensive therapy. Six of these patients are in complete remission with a stable low‐level or absent CBFB/MYH11 expression. Three patients relapsed, and their CBFB/MYH11 transcripts rose again to pretreatment levels. In two patients, this increase in CBFB/MYH11 could be detected by real‐time PCR before hematological relapse. These data indicate that real‐time RT‐PCR can be used for the sensitive detection and quantification of CBFB/MYH11 transcripts in the follow‐up of patients with inv(16)‐positive AML.

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

Abstractu2002Ninety 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.

Donor cell-derived acute myeloid leukemia developing 14 months after matched unrelated bone marrow transplantation for chronic myeloid leukemia

We report a patient with Ph-positive CML who developed a Ph-negative AML in donor cells 14 months after BMT from an HLA-identical male unrelated donor. The Ph translocation could not be detected by either conventional cytogenetics, FISH or RT-PCR analysis excluding relapse of CML in myeloid blast crisis. Chimerism studies were performed by variable number of tandem repeats (VNTR) analysis. These revealed donor-type hematopoiesis in both unseparated mononuclear cells and CD34+ selected blasts proving the leukemia to be of donor origin. The patient received three cycles of polychemotherapy with mitoxantrone, topotecan and ara-c resulting in CR after the first treatment cycle and reconstitution with donor hematopoiesis. A second transplant from a female alternative matched unrelated donor was performed after conditioning with fludarabine and 200 cGy TBI and was well tolerated. Nine months after the second transplant the patient is at home and in CR. T cell chimerism was studied by sex chromosome analysis and revealed complete female donor chimerism. Bone Marrow Transplantation (2001) 28, 705–707.

Aggressive chemotherapy combined with G-CSF and maintenance therapy with interleukin-2 for patients with advanced myelodysplastic syndrome, subacute or secondary acute myeloid leukemia--initial results

SummaryAggressive chemotherapy of advanced myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) evolving from MDS, subacute AML and secondary AML has usually been associated with low complete remission (CR) rates, a high incidence of early death, and low disease-free survival. We therefore have initiated a phase-III trial of aggressive chemotherapy consisting of idarubicin, cytosine arabinoside, and VP-16 to improve the CR rate. Each chemotherapy cycle is followed by G-CSF to accelerate neutrophil recovery and to reduce the incidence of infections. Until now, 19 patients with high-risk AML have been entered. The CR rate is 47%, with only one death during induction. Patients achieving CR are randomized to receive either high-dose or low-dose interleukin-2 to eliminate residual leukemic cells and to prolong the duration of remission.

Intensive chemotherapy with idarubicin, ara-C, etoposide, and m-AMSA followed by immunotherapy with interleukin-2 for myelodysplastic syndromes and high-risk Acute Myeloid Leukemia (AML)

Abstractu2002Intensive chemotherapy followed by treatment with interleukin-2 (IL-2) was evaluated in a prospective, randomized, multicenter trial including 18 patients with refractory anemia with excess of blasts in transformation (RAEB-T), 86 patients with acute myeloid leukemia (AML) evolving from myelodysplastic syndromes, and six patients with secondary AML after previous chemotherapy. Median age was 58u2009years (range: 18–76u2009years). Forty-nine patients (45%) achieved a complete remission (CR) after two induction cycles with idarubicin, ara-C, and etoposide, 52% of them aged ≤60u2009years and 35% aged >60u2009years (p=0.06). After two consolidation courses, patients were randomized to four cycles of either high- or low-dose IL-2. Patients aged up to 55u2009years with an HLA-identical sibling donor were eligible for allogeneic bone marrow transplantation. The median relapse-free survival was 12.5u2009months, with a probability of ongoing CR at 6.5u2009years of 19%. Overall survival of all patients was 8u2009months, and 21u2009months for the CR patients. Median survival was significantly longer among patients aged ≤60u2009years than among the older patients (16 vs 6u2009months, p<0.001). Median duration of survival and relapse-free survival were not statistically different in the two IL-2 treatment arms.

Myelodysplastic syndrome with monosomy 7 after immunosuppressive therapy in Behçet's disease.

Only few cases of Behçets and hematological malignancies have been reported until now. We recently observed a 39-year-old female patient with Behçets disease developing a myelodysplastic syndrome (MDS) FAB subtype refractory anemia with excess of blasts in transformation [RAEB-t] with a monosomy 7 after being treated with cyclosporin A and chlorambucil for several years. This case is reported and the occurrence of hematological malignancies and Behçets disease is reviewed.