Gerlinde Mitterbauer

Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA.

Abstract Reverse transcription polymerase chain reaction (RTPCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol™, RNAzol™, FastTube™ reagent). RNA yield was slightly higher with RNAzol™ than with TRIzol™ as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube™ reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol™ and TRIzol™. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol™, and 1 BCR-ABL-positive (specific for translocation t [9; 22]) cell among 2×104 normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol™, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.

A comparative study on demographic, hematological, and cytogenetic findings and prognosis in acute myeloid leukemia with and without leukemia cutis

Abstract. We studied the incidence of leukemia cutis (LC) in 381 consecutive patients with acute myeloid leukemia (AML) in a single institution and compared the demographic, hematological, and cytogenetic findings in AML patients with and without LC. We also examined the response to intensive chemotherapy, overall survival, and duration of remission in this patient population with regard to the presence of LC. The prevalence of LC was 3.7% in clinically diagnosed patients and 2.9% in biopsy-proven cases, respectively. Patients with and without LC did not differ with regard to age, sex, white blood cell counts, hemoglobin, fibrinogen, and platelet counts at diagnosis, but lactate dehydrogenase (LDH) was significantly higher in patients with LC. Various karyotype abnormalities were found, but in patients with LC numerical abnormalities of chromosome 8 were significantly more common (P<0.0001). Patients with LC did not differ from patients without LC with regard to remission rate, but there was a trend towards shorter remission duration in patients with LC. We conclude that patients with LC have some features different from patients without this symptom. The increased frequency of numerical aberrations of chromosome 8 in patients with LC was the most interesting observation of our study. The pathophysiological significance of this finding remains to be determined.

Quantification of minimal residual disease in patients with BCR-ABL-positive acute lymphoblastic leukaemia using quantitative competitive polymerase chain reaction

We analysed 20 patients with BCR‐ABL‐positive acute lymphoblastic leukaemia (ALL) by quantitative competitive polymerase chain reaction (QC‐PCR) to study the kinetics of the leukaemic clone. Consecutive samples of 16 patients (minor‐bcr, n = 10; major‐bcr, n = 6) were analysed after conventional chemotherapy and/or bone marrow transplantation (BMT). DNA competitor templates co‐amplifying with either p210 or p190 BCR‐ABL cDNA were used for quantification of leukaemia‐specific BCR‐ABL mRNA. In all samples, total ABL transcripts were measured as internal control, and the percentage of BCR‐ABL/ABL molecules was calculated. Following induction chemotherapy the number of BCR‐ABL transcripts was reduced by a maximum of 2–3 logs. In most patients, additional chemotherapy did not lead to further reduction of BCR‐ABL mRNA. In two patients, conventional chemotherapy plus autologous BMT in complete haematological remission resulted in a total reduction of the transcript level of more than 3 logs. In two other patients, allogeneic BMT caused a transient reduction of the BCR‐ABL transcripts below the detection level of our method (<1 blast cell in 105 normal cells) for a period of 7 and 11 months, respectively. The achievement of PCR negativity did not guarantee sustained remission. Both patients relapsed and BCR‐ABL transcript levels rose by more than 1 log prior to frank relapse. Our data demonstrate that quantification of BCR‐ABL mRNA allows the evaluation of the dynamics of the leukaemic clone and thus is valuable for the evaluation of minimal residual leukaemia following various therapies and the early detection of increasing BCR‐ABL transcripts prior to relapse.

Competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction for quantitative assessment of minimal residual disease during postremission therapy in acute myeloid leukemia with inversion(16): a pilot study.

PURPOSE (1) Quantification of minimal residual disease (MRD) by competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction (RT-PCR) in patients with acute myeloid leukemia (AML) and inversion(16) [inv(16)] during postremission therapy, (2) comparison of this method with conventional two-step RT-PCR, and (3) evaluation of a potential prognostic value. PATIENTS AND METHODS MRD of six consecutive adult patients with AML and inv(16)(p13;q22) or t(16;16)(p13;q22) who entered complete remission (CR) was monitored by competitive CBFbeta/MYH11 RT-PCR in their bone marrow (BM) during postremission therapy with high-dose cytarabine (HiDAC) or after BM transplantation with a matched unrelated-donor marrow (MUD-BMT) during an observation period of 4.5 to 27 months after initiation of treatment. RESULTS Competitive PCR showed a gradual decline by at least 4 orders of magnitude after 7 to 9 months in patients in continuous CR (CCR), while one patient who relapsed after 13.5 months only achieved a reduction by 2 orders of magnitude at the end of consolidation therapy. A rapid decrease below the detection limit was observed within 1 month in two patients after MUD-BMT. A temporary reappearance of molecular MRD was observed in these patients during immunosuppression for graft-versus-host disease (GvHD). After reduction of immunosuppression, the level of MRD dropped again below the PCR detection limit. Molecular monitoring by conventional two-step RT-PCR yielded comparable results only when multiple assays per time point were performed, while single-assay RT-PCR gave misleading results. CONCLUSION Competitive RT-PCR is a valuable tool for molecular monitoring during postremission chemotherapy, as well as after BMT.

The immunophenotype of 325 adult acute leukemias: relationship to morphologic and molecular classification and proposal for a minimal screening program highly predictive for lineage discrimination.

Bone marrow cells of 325 adults with acute leukemia were immunophenotyped using a panel of monoclonal antibodies proposed by the European Group for the Immunological Characterization of Leukemias (EGIL). Of these, 97.2% could be assigned clearly to myeloid or lymphoid lineage (254 acute myeloid leukemias [AMLs], 48 B-cell lineage acute lymphoblastic leukemias [ALLs], 14 T-cell lineage ALLs), 1.8% as biphenotypic, and less than 1% as undifferentiated. Immunologic subtyping of ALLs revealed an association between early precursor phenotypes and coexpression of myeloid antigens, particularly CD15/CD65s coexpression and pre-pre-B cell-specific phenotypes and genotypes. The common ALL phenotype was associated with BCR-ABL translocation. Among AMLs, CD2 coexpression was almost exclusively restricted to French-American-British subtypes M3 variant and M4Eo and related molecular aberrations. The most valuable markers to differentiate between myeloperoxidase-negative AML subtypes M0 and ALLs were CD13, CD33, and CD117, typical of M0, and intracytoplasmic CD79a, intracytoplasmic CD3, CD10, and CD2, typical of B cell- or T cell-lineage ALL. Our results confirm excellent practicability of the EGIL proposalfor immunologic classification of acute leukemias. For myeloperoxidase-negative AMLs, we suggest a scoring system based on markers most valuable to distinguish between AML-M0 and ALLs.

FLAG (fludarabine, cytosine arabinoside, G-CSF) for refractory and relapsed acute myeloid leukemia.

Abstract Twenty-two patients with refractory or relapsed AML were treated with FLAG [25 mg/m2 fludarabine daily (days 1–5), 2 g/m2 daily Ara-C (days 1–5) and 400 μg/m2 daily G-CSF (day -1 till the absolute neutrophil count was >500/μl)]. Median age was 46 years (range 24–63). Eight patients had leukemia which was primarily refractory to conventional regimens, six were in first, seven were in second, and one was in third relapse.Overall, 11 of 22 (50%) patients achieved complete remission (CR), three had a partial response (PR), and seven did not respond (NR). One patient died of an early cerebral hemorrhage. The median remission duration from achievement of CR after FLAG was 9.9 months and median survival was 13.0 months. One patient is alive in CR at 31.9 months. Hematological toxicity of the regimen was severe. The median time to neutrophil recovery (ANC >500/μl) was 21 days (range 18–33). A median of seven red cell units (range 0–22) and of six platelet concentrate units (range 3–28) had to be given. Median duration of febrile neutropenia was 2 days (range 0–20 days) and patients were on i.v. antibiotics for a median of 16 days (range 0–51). There was no death from infection. Nonhematological toxicity was remarkably low, with almost no neurotoxicity and no major hepatotoxicity. In conclusion, FLAG seems to be an efficient and well tolerated regimen. It may be particularly useful for patients who have a sibling or unrelated donor for subsequent allogeneic bone marrow transplantation.

Myelomastocytic Leukemia: Evidence for the Origin of Mast Cells from the Leukemic Clone and Eradication by Allogeneic Stem Cell Transplantation

Purpose: Myelomastocytic leukemia is a term used for patients with advanced myeloid neoplasms, in whom elevated numbers of immature atypical mast cells are found, but criteria for a primary mast cell disease are not met. The origin of mast cells in these patients is presently unknown. Patient and Methods: We have analyzed clonality of mast cells in an 18-year-old patient suffering from acute myeloid leukemia with a complex karyotype including a t(8;21) and mastocytic transformation with a huge increase in immature mast cells and elevated serum tryptase level, but no evidence for a primary mast cell disease/mastocytosis. Results: As assessed by in situ fluorescence hybridization combined with tryptase staining, both the tryptase-negative blast cells and the tryptase-positive mast cells were found to contain the t(8;21)-specific AML1/ETO fusion gene. Myeloablative stem cell transplantation resulted in complete remission with consecutive disappearance of AML1/ETO transcripts, decrease of serum tryptase to normal range, and disappearance of neoplastic mast cells. Conclusion: These data suggest that mast cells directly derive from the leukemic clone in patients with myelomastocytic leukemia.

PCR‐monitoring of minimal residual leukaemia after conventional chemotherapy and bone marrow transplantation in BCR‐ABL‐positive acute lymphoblastic leukaemia

We report the results of consecutive tests in nine BCR‐ABL‐positive ALL patients by one‐step and two‐step (nested primer) reverse transcriptase‐polymerase chain reaction (RT‐PCR). Six patients could be tested in complete haematological remission (CHR). One patient remained one‐step positive; four patients became one‐step negative, but remained two‐step positive; only one patient became two‐step negative. In five patients the haematological relapse was preceded by one‐step positivity by 1.5‐5 weeks. In two patients who received autologous BMT in CHR, BCR‐ABL was still detectable by two‐step PCR, whereas allogeneic BMT was able to transiently reduce BCR‐ABL below the two‐step detection level. Our results show that one‐step combined with two‐step RT‐PCR analysis gives valuable information about the efficacy of treatment and the dynamics of the leukaemic clone.

Low curative potential of bone marrow transplantation for highly aggressive acute myelogenous leukemia with inversion inv (3)(q21q26) or homologous translocation t(3;3) (q21;q26)

Abstract Structural rearrangements of the long arm of chromosome 3 involving bands 3q21 and 3q26 and leading either to a paracentric inversion inv (3)(q21q26) or a translocation between both homologous chromosomes – t (3;3)(q21q26) – have been reported in patients with acute myelogenous leukemia (AML), myelodysplastic syndromes, myeloproliferative disorders, and chronic myelogenous leukemia in blast crisis. We describe three patients with de novo AML with these structural abnormalities who received multiple courses of conventional chemotherapy followed by unrelated donor (n=2) and autologous (n=1) bone marrow transplantation (BMT). All three patients had early relapse: patients 1 and 2 had relapse 69 days and 306 days after BMT, respectively, and patient 3 immediately after autologous BMT. Despite further chemotherapy, they died without achieving another remission. These findings, together with other recorded similar cases, show that AML with structural abnormalities of the long arm of chromosome 3 as described has an extremely poor prognosis even with the most potent anti-leukemic treatment modalities.

Spontaneous remission of acute myeloid leukemia after infection and blood transfusion associated with hypergammaglobulinaemia

Abstract Spontaneous remissions of acute myeloid leukemia (AML) have been documented in association with infection as well as blood transfusions. Activation of the immune system including an increased number of NK cells and cytokine release have been implicated in the mechanism of this phenomenon. We have observed spontaneous remissions in two patients with AML (one with a t(8;21)-positive M2, one with M5b), both occurring after infection and blood transfusions. The bone marrow showed a reduction of blast cells from 65% to 2% or 40% to 1%, respectively. Remission was accompanied by a marked polyclonal hypergammaglobulinemia in both cases (IgG values of 6420 and 2160 mg/dl, IgA of 802 and 811 mg/dl, respectively). A concomitant increase in bone marrow plasma cells was observed in both patients. Reduction of AML1/ETO PCR positivity from one-step to two-step PCR (approximately 100-fold) was documented in the patient with a t(8;21), while a regression of lymph node and skin leukemic infiltrations occurred in the patient with M5b. One patient relapsed after 4 months, at a time when his serum immunoglobulin levels had markedly decreased. The other patient is in continuous remission after 14 months. These cases suggest a potential role for a humoral immune response in the mechanism of spontaneous remission.