Dieter Lutz

Cell lineage heterogeneity in blast crisis of chronic myeloid leukaemia

Summary Blast cells from 45 patients with chronic myeloid leukaemia in blast crisis (CML‐BC) were immunologically phenotyped with a panel of 26 monoclonal antibodies and studied for terminal deoxynucleotidyl transferase (TdT) content. Out of 45 blast‐populations, 28 showed a myeloid, 14 a lymphoid, two a mixed and one an unclassifiable marker profile.

Prognostic impact of karyotype and immunologic phenotype in 125 adult patients with de novo AML

One hundred-twenty-five adult patients with de novo acute myeloid leukemia (AML) were treated according to a standard 7 + 3 induction regimen. Karyotype and immunological phenotype of blasts examined prior to treatment were correlated with each other, with response to treatment and duration of survival. The following monoclonal antibodies (mAbs) were used for immunological phenotyping: VIM-D5 (CD15), MY7 (CD13), MY9 (CD33), VIM-2 (CDw65), VIM-13 (CD14), 63D3 (CD14), VID-1 (anti HLA-DR), WT1 (CD7), CLB-Ery3 (antiblood group H antigen), C17-27 (CD61), and an antiserum against TdT. Despite a considerable overlap between the individual groups, patients with specific aberrations as defined by the MIC classification (n = 39) showed distinct, characteristic, myeloid or myelomonocytic immunophenotypes. In M2/t(8;21) there was a significant association with negativity to CD13, in M3/t(15;17) with negativity to CD15 and HLA-DR, whereas in M4/inv(16) expression of blood group H antigen was unexpectedly found. The response to therapy, as well as rate of complete remission as duration of survival, was better in patients with M2/t(8;21), M3/t(15;17), and M4Eo/inv(16) as compared to all other patients and significantly worse in patients with M5a/t/del(11)(q23). In 35 patients with normal karyotype and 16 patients with cytogenetic anomalies not presently associated with FAB subtypes the expected correlations of rather immature myeloid immunologic phenotypes with M1 and M2 morphology and CD14 expression in monoblastic leukemias was found. Remission rate and survival were significantly worse in 19 patients with complex nonrandom aberrations, where blast cell expression of blood group H antigen and of TdT were significantly increased.

Tumor cell detection in peripheral blood and bone marrow

Purpose of review Whether the occurrence of tumor cells in peripheral blood or bone marrow from patients with solid tumors is predictive for disease recurrence or of any other prognostic relevance remains unknown. This article reviews recently published results focusing on the various methods used, their correlations with clinical or biological parameters and their potential prognostic value. Recent findings An increasing number of marker genes and different techniques, alone or in combinations, have been used for the detection of tumor cells in peripheral blood and bone marrow. Various results obtained are hardly comparable, most often due to the different methods in use. The frequency of circulating tumor cells in peripheral blood varied within a broad range and their clinical relevance appeared to be contradictory, at least in part. Disseminated tumor cells in bone marrow reached an independent prognostic value in breast cancer patients, but several investigations led to inconsistent correlations with clinical or prognostic criteria. Summary Still many questions remain unanswered; hence, the detection of tumor cells in peripheral blood or bone marrow cannot yet be taken into account for therapeutic decisions.

Factors affecting long-term outcome after allogeneic haematopoietic stem cell transplantation for acute myelogenous leukaemia: a retrospective study of 172 adult patients reported to the Austrian Stem Cell Transplantation Registry.

Summary. Between 1982 and 2000, 172 patients with acute myelogenous leukaemia (AML) received haematopoietic stem cell transplants (SCT) from related (n = 132) or unrelated (n = 40) donors at four Austrian transplant centres and their results were reported to the Austrian Stem Cell Transplantation Registry. Conditioning for SCT consisted of cyclophosphamide and total body irradiation in 156 (91%) patients. Graft‐versus‐host disease (GVHD) prophylaxis was with standard cyclosporine and methotrexate in 95 (55%) patients. Median post‐transplant follow‐up was 5·6 years (range, 0·2‐‐16·7). Multivariate analysis of transplant‐related mortality (TRM) identified four variables associated with a lower risk: disease status of first complete remission (CR) at SCT, patient age of 45 years and younger, transplant performed during or after 1995, and lack of acute GVHD. Variables associated with significantly improved leukaemia‐free survival were: bone marrow as the stem cell source, disease status of first CR at SCT, and occurrence of chronic GVHD. In multivariate analysis, transplantation performed during or after 1995, first CR at SCT, occurrence of limited chronic GVHD and lack of acute GVHD grades III to IV were associated with increased overall survival. Based on these analyses, options for the improvement of results obtained with allogeneic SCT in patients with AML could be defined.

Distinct lymphoblastic and myeloblastic populations in TdT positive acute myeloblastic leukemia: evidence by double-fluorescence staining.

Double-immunofluorescent staining for the enzyme terminal deoxynucleotidyl transferase (TdT) as a marker of primitive lymphoblasts, and for the VIM-D5 antigen as a differentiation antigen of the myeloid system gave direct evidence for distinct lymphoblastic and myeloblastic populations (mixed leukemic cell populations) in seven patients with acute leukemia. The percentage of malignant TdT positive cells contributing to a leukemic cell bulk with unequivocal signs of myeloid origin was between 10 and 80%. A defect at the level of a common progenitor cell giving rise to both the TdT and the VIM-D5 positive blast cell population is discussed.

OCT1 (SLC22A1) R61C polymorphism and response to imatinib treatment in chronic myeloid leukemia patients

Since 1998, patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) are treated successfully with abl-tyrosine kinase inhibitor imatinib. More than 95% of patients achieve a complete hematologic response and more than 80%, a complete cytogenetic response. However, a proportion of patients demonstrate resistance or suboptimal response to imatinib therapy; in many cases the mechanism is unknown [1]. The organic cation transporter 1 (OCT1, SLC22A1) has been shown to be responsible for the cellular uptake of imatinib. Differences in the activity of OCT1 are predictive for the response of CML patients to imatinib treatment [2]. Single nucleotide polymorphisms (SNPs) of the OCT1 transporter are involved in differences of transport activities and consequently might be associated with imatinib response in CML patients. The R61C (C286T) SNP in exon 1 (rs12208357) has been linked to a decreased transport activity of OCT1 [3]. Whether this polymorphism is involved in imatinib uptake into cells and imatinib response in CML patients is unknown. The DNA from 32 CML patients (18 females, 14 males, age 14–85 years, median 56 years) treated with imatinib was prepared out of peripheral blood using the QIAamp DNA Mini Kit (QIAGEN). The R61C genotype was assessed with a TaqMan SNP genotyping assay (Applied Biosystems). BCR-ABL transcripts in blood were quantified as described [4]. R61C genotype was related to imatinib response. Imatinib serum levels have not been determined in this study. All CML patients were in chronic phase when therapy was started. Heterozygote R61C genotype (C/T) was demonstrated in five of 32 (16%) CML patients, all others were wildtype (C/C). The frequency was higher than reported in European American with 7.2% [3]. Five patients (one heterozygote, four wildtype) were not included in the follow-up for several reasons: in one patient imatinib-therapy was immediately stopped due to severe toxicity, one young patient (14 years of age) was treated by allogeneic stem-cell transplantation and for three patients the period of follow-up was too short for evaluation. All patients achieved a complete hematologic response. Three of 17 patients (18%) with a complete cytogenetic response (0% Ph-positive metaphases within 12 months, CCyR) as well as three of 12 patients (25%) with a major molecular response (50.1% BCR-ABL transcripts within 18 months, MMolR) were R61C heterozygote. Similarly, one out of nine patients (11%) without MMolR had a heterozygote genotype (Table I). There is no statistically significant difference in the incidence of heterozygosity in patients ‘with’ or ‘without’ MMolR (Fisher Exact Test: p1⁄4 0.62). In this small cohort of patients there is no evidence that response to imatinib treatment is impaired in R61C heterozygote individuals, although none of

Factors influencing the timing of peripheral blood stem cell collection (PBSC)

High-dose conditioning regimens followed by autologous peripheral blood stem cell rescue are frequently used for the treatment of solid tumors and hematological malignancies. In 24 patients up to four peripheral stem cell collections (PBSC) were performed after priming with various chemotherapies and G-CSF (300 micrograms s.c. per day). In 16 patients (group A) more than 2 x 10(6) CD 34 positive cells per kg bodyweight could be collected; fewer were harvested in the remaining eight patients (group B). The amount of collected CD 34 positive cells correlated with the median number of these cells in the peripheral blood at the start of PBSC. The two groups differed both in recovery time after priming-induced cytopenia (4 vs 6 days from nadir) and in the number of WBC (21 x 10(6) mL-1 vs 6.1 x 10(6) mL-1) and platelets (133 x 10(6) mL-1 vs 58 x 10(6) mL-1) reached at first day of PBSC. No difference between the two groups was seen according to age, duration of disease or disease status. However, the intensity of prior treatment was significantly greater in group B than in group A. These observations indicate that the toxicity of previous chemotherapy is the most important factor for the mobilization of sufficient CD 34 positive cells into the peripheral blood.

Mammaglobin as a Marker for the Detection of Tumor Cells in the Peripheral Blood of Breast Cancer Patients

The detection of circulating tumor cells in the peripheral blood (pB) of breast cancer (BC) patients might become an important factor for the prognosis of BC patients. Sensitive molecular techniques, primarily based upon the reverse-transcriptase polymerase chain reaction (RT-PCR), have been developed using the expression of tissueand/or tumor-specific genes as a marker for the presence of tumor cells. In 1996, the cDNA of a novel gene termed human mammaglobin (hMAM) was described.1 As far as known, the expression of hMAM is restricted to the adult mammary gland and to breast carcinoma cells; therefore, it might be a specific marker for BC cells. We developed a nested RT-PCR assay for the detection of hMAM mRNA molecules in the pB of BC patients as a marker for the presence of tumor cells.2 The assay is highly sensitive (1 tumor cell detectable in 106–107 white blood cells) and specific for breast carcinoma cells (no hMAM expression in pB of healthy volunteers). Peripheral blood samples from 286 BC patients (27–94 years, mean of 58 years) were classified into four defined clinical subgroups: before (pre) and after (post) surgery (without metastases and before any further adjuvant treatment), no evidence of disease (NED, stages I–III or relapsed, and after chemotherapy-induced remission), and metastatic disease (MD, stage IV and relapses of earlier stages). RNA was isolated out of 2× 5 mL pB per sample and patient, cDNA was synthesized, and nested PCR was performed with two PCR setups per cDNA, resulting in four PCR setups per sample.2 In all assays, freshly prepared cDNAs from dilutions of 10 cells of the mammary carcinoma cell line SKBR5 in 5 mL pB and from pB of healthy volunteers were used as positive and negative controls, respectively. Results of pB samples from BC patients tested for hMAM mRNA expression via a nested RT-PCR assay were as follows: 2/46 (4%) pre, 2/24 (8%) post, 4/135 (3%) NED, and 35/81 (43%) MD (TABLE 1). Significantly more MD patients were hMAM-positive than patients of the other subgroups (p < 0.0001). The detection of tumor cells in pB of BC patients is limited to tumors that do express hMAM. It has been shown that about 80% of mammary carcinomas are strongly immunopositive for hMAM protein.3 In addition, cells derived from breast

In vitro detection of methylated DNA via recombinant protein MBD2b

Members of the methyl binding domain (MBD) protein family are known for binding to methylated DNA by recognizing methylated cytosines. Their original function is to regulate protein biosynthesis by recruitment of transcriptional repression complexes to silence gene expression. The aim of the presented work was to detect methylated DNA spotted onto nitrocellulose membranes with recombinant proteins MBD2b, MBD2b-GFP and directly labeled protein MBD2b. Proteins were affinity purified and tested for functionality before application. We were able to show that these functional recombinant proteins bind to unilaterally and symmetrically methylated oligonucleotides and genomic DNA in vitro and thus can be used in various detection assays.

Hypo- and hypertetraploidy in a case of erythroleukemia analyzed by fluorochrome banding techniques

Chromosome studies of a case of erythroleukemia in a 57-year-old female patient were made from bone marrow aspirates using the fluorescent primary stain/counterstain methodology. The chromosome number ranged from 42 to 110. There was a high proportion of hypotetraploid cells and a few hypertetraploid and hypooctaploid ones. Structurally normal chromosomes varied in number from cell to cell, ranging from one to seven in the polyploid cells. A number of marker chromosomes were observed, some of which occurred repeatedly in two copies per hypotetraploid cell. The chromosomes involved in aberrations were tentatively identified as #3, #5, #7, #12, #13, #15, #16, #18, #19, and #21. In the abnormal chromosome #16, which was missing a normal short arm, a new kind of heterochromatin was demonstrated by sequential staining with DA-DAPI and DAPI-AMD, suggesting de novo amplification of an A-T-rich satellite DNA sequence.