[Molecular biology in acute lymphoblastic leukemia].
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Adult↗
Biomedical subjects
P Hokland
Publications and source records attributed to P Hokland.
Pretreatment leukaemia cell drug resistance is correlated to clinical outcome in acute myeloid leukaemia.
In 85 adult patients diagnosed with acute myeloid leukaemia (AML) and treated at the same institution during a 5-yr period, the clinical significance of in vitro cellular drug resistance to the anthracyclines aclarubicin (Acla) and daunorubicin (Dau) as well as the nucleoside analogue cytarabine (Ara-C) was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. In 59 patients of whom 40 were treated by the combination of Acla and Ara-C we found that leukaemia cell drug resistance towards Acla was higher (by a factor 2.80) in patients who failed to enter complete remission (CR) after the first cycle of induction chemotherapy as compared to patients who entered complete remission. The relationship was significant in univariate as well as multivariate analysis (p=0.02 and 0.03, respectively). By contrast, no in vitro single drug resistance values were consistently correlated to other parameters of clinical outcome (overall CR rate, overall survival (OS), or continuous complete remission (CCR)), whereas the combined Acla and Ara-C drug resistance profile (Acla/Ara-C DRP) was of prognostic significance to overall survival of all 85 patients (p=0.004) as well as to the CCR of 39 complete responders (p=0.04). These findings remained statistically significant in multivariate analyses correcting for other variables influencing clinical outcome including patient age, leukocyte count, karyotype, FAB-subtype, and presence/absence of secondary AML. We conclude that the in vitro drug resistance of leukaemia cells at time of disease presentation appears to be independent of prognostic significance to short- and long-term clinical outcome in AML.
Aclarubicin↗
FAB M4 and high CD14 surface expression is associated with high cellular resistance to Ara-C and daunorubicin: implications for clinical outcome in acute myeloid leukaemia.
In 145 adult patients diagnosed with non-M3 acute myeloid leukaemia (AML) the relevance of FAB-subtype and immunophenotype to in vitro cellular drug resistance towards the anthracyclines aclarubicin (Acla) and daunorubicin (Dau), and the nucleoside analogue cytarabine (Ara-C), as well as other antileukaemic drugs, was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. We demonstrate that high CD14 expression is highly significantly associated with high cellular Ara-C and Dau resistance in univariate as well as multivariate analyses. FAB subtypes with highest and lowest cellular Ara-C resistance were M4 and M5, respectively (P < 0.01, one-way anova), whereas FAB subtypes with highest and lowest cellular Dau resistance were M4 and M1, respectively (P < 0.01, one-way anova). By contrast, no significant differences in cellular drug resistance towards Acla could be demonstrated among FAB subtypes. Furthermore, in two cohorts of AML patients treated by two different regimens for remission induction over a period of 15 yr (1985-94, n = 159 and 1995-99, n = 76, respectively) we demonstrate in univariate analyses a significance of CD14 expression with respect to clinical outcome. With the exception of significance to probability of obtaining complete remission in the first cohort (P = 0.03, logistic regression), this significance was, however, lost in multivariate analyses. It was demonstrated that FAB-M4 patients were older than M5 patients and that high CD14 expression was associated with the presence of secondary AML and older age. We conclude that although cases with high blast cell CD14 expression (and FAB-M4 cases) were more resistant to Ara-C as well as Dau in vitro, the clinical and biological significance of this may be debatable because of interactions with major prognostic factors in AML.
Aclarubicin↗
Characterization of t(12;21) breakpoint junctions in acute lymphoblastic leukemia.
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Base Sequence↗
Long-term culture of hematopoietic stem cells - validating the stromal component of the CAFC assay.
BACKGROUND: The stroma-based long-term culture is the assay of choice when a functional detection of primitive hematopoietic cells in vitro is sought. However, different stromal cell lines varying in supporting capacity have been raised and applied in different laboratories, resulting in a wide range in published frequencies of LTCIC alternative CAFC. METHODS: In order to identify the most suitable stromal source in terms of supportive capacity, reproducibility, and ease of handling, we have compared some of the most commonly employed murine cell lines to human bone marrow stroma in secondary long-term culture set-ups. RESULTS: Seeking an approximation to the supportive capacity of human BM stroma we found the FBMD-1 cell line supplemented with G-CSF and IL-3 superior to FBMD-1 cells alone, and to M2-10B4 and Sl/Sl cells. Moreover, in co-cultures of CD34(+) cells and the FBMD-1 line, we found week 5 CAFC content highly reproducible (50.5 +/- 6.66 - 54.6 +/- 7.07/10(4) plated cells, p value > 0.95) and the assay was suitable for inter-individual comparison in a clinical setting. In fact, the week 5 CAFC results were even more reproducible than those of the CFU assays (CV 0.03 for the CAFC assay versus 0.13-0.33 for the CFU assays). On the other hand, when extending the culture period to 8 weeks, the cobblestone area formation was best maintained by human BM stroma and the high reproducibility in CAFC enumeration in cultures supported by the FBMD-1 was lost. DISCUSSION: Among the stromal cell sources tested, the FBMD-1 line was found to be superior in terms of ease of handling and week 5 CAFC reproducibility. However, this robustness could not be extended to week 8 CAFC.
Adult↗
Expression of TEL-AML1 fusion transcripts and response to induction therapy in standard risk acute lymphoblastic leukemia.
We prospectively examined the frequency of the t(12;21)TEL-AML1 fusion in 504 children with newly diagnosed standard risk ALL using RT-PCR assays. Cells from 95 patients (18.8%) were TEL-AML1+. There was a significantly higher frequency of pseudodiploidy among the TEL-AML1+ cases (39.4% versus 14.1%, P = 0.001), primarily because structural abnormalities involving 12p and del(6q) occurred more frequently in the TEL-AML1+ group. TEL-AML1+ ALL was more sensitive to the induction chemotherapy than TEL-AML1- ALL. The percentage of "rapid early responders", i.e., patients who achieved an M1 (< 5% blasts) or M2 (5-25% blasts) marrow status on day 7 of induction chemotherapy, was significantly higher among TEL-AML1+ cases. The quality of remission of RT-PCR positive cases was excellent, as evidenced by the very low to absent MRD burden of their end-of-induction bone marrow specimens. TEL-AML1+ patients also had an excellent early EFS outcome. The probability of EFS at 30 months from study entry were 98.9 +/- 1.0% for the TEL-AML1+ group and 92.1 +/- 1.5% for the TEL-AML1- group (P = 0.0001). This prospective study significantly expands the knowledge gained from previous studies regarding the prognostic significance of t(12;21)TEL-AML1 fusion in pediatric ALL.
Bone Marrow↗
DAP-kinase CpG island methylation in acute myeloid leukemia: methodology versus biology?
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Acute Disease↗
[Hematopoietic stem cells I. Basal aspects].
The knowledge on basic aspects of hematopoietic stem cells (HSC) has markedly increased during the last decade to the extent that such cells are now routinely employed therapeutically in order to restore hematopoiesis following myeloablative chemotherapy and irradiation in patients with malignant disorders. Different methods can be applied to characterize HSC. Thus, by immunophenotyping and flow cytometry it is possible to delineate subpopulations of cells enriched for HSC. However, since phenotypic characteristics associated with HSC do not necessarily correlate to their developmental potential, measurements of HSC also rely heavily upon functional in vitro and in vivo assays, which demonstrate the presence of HSC through their ability to proliferate and differentiate in a clonogenic fashion. In this review we describe the assays developed to characterize and quantitate immature HSC, which form the basis for their clinical application.
Animals↗
[Hematopoietic stem cells II. Diagnostic and therapeutic aspects].
Characterization and isolation of haematopoietic stem cells (HSC) have resulted in their clinical application in patients with malignant disorders and--through gene therapeutic initiatives--also in the treatment of inherited diseases. Autologous stem cell transplantation (ASCT), which was introduced because of the high number of relapses in cancer patients in remission, involves dose-intensification (conditioning), which induces myeloablation. In this setting, reinfusion of HSC is performed to restore haematopoiesis. Flow cytometric determination of CD34+ cells and clonogenic assays for committed myeloid HSC (CFU-GM) are vehicles for quality control of the harvested HSC material and are integrated into the ASCT programs. Moreover, harvest of HSC and purification of CD34+ cells enables new treatment options such as removal of cancer cells from grafts, optimization of gene transduction as well as ex vivo expansion of HSC before reinfusion. In conclusion, the expanding insights into HSC in the 1990's have already been translated into valuable diagnostic and therapeutic modalities.
Antigens, CD34↗
Integration of molecular methods for detection of balanced translocations in the diagnosis and follow-up of patients with leukemia.
Molecular methods are emerging as important tools for the diagnosis and stratification of patients with leukemia. Together with rearrangements in immunoglobulin and T-cell receptor genes, balanced translocations are the most important genetic lesions amenable to molecular diagnosis. Moreover, many publications have identified significant differences in the prognosis of acute leukemia patients with such balanced translocations. Because not all balanced translocations can be diagnosed by cytogenetic techniques, reverse-transcriptase polymerase chain reaction (RT-PCR)-based methods are increasingly employed. This method has the added advantage that it can also be used to monitor for minimal residual disease (MRD). A disadvantage is that the multitude of balanced translocations in leukemia would make efforts to detect all lesions at diagnosis by such standard techniques extremely labor-intensive. Furthermore, difficulties in optimizing semiquantitative PCR assays have limited the utility of these methods for MRD. Recent advances in the design of multiplex PCR, which detects a number of genetic aberrations simultaneously, may improve the diagnostic process. Accurate quantitation of the fusion transcript for balanced translocations has become possible by use of fluorogenic probes and real-time PCR. Together, such methodologies may constitute a novel platform for the integration of molecular methods in clinical decision-making.
Follow-Up Studies↗
Dynamic cell cycle kinetics in vitro and in vivo in myelodysplastic syndromes with special reference to the influence of hematopoietic growth factors.
We have investigated the effect of HGF in vivo and in vitro in MDS using a recently developed FCM assay involving the simultaneous measurement of cell surface antigens, DNA content, and BrdUrd or IodUrd incorporation. This allows for the determination of the dynamic cell kinetic parameters: LI, T(s), and T(pot) and we observed that in vitro HGF stimulation resulted in a significant decrease in mean T(pot) values from 6.6 to 3.5 days. Importantly, we demonstrated that in vivo GM-CSF administration to patients with RAEB resulted in a shortening of T(pot) within the 2 first weeks of GM-CSF treatment.
Aged↗
Mutational analysis of the tumour suppressor gene MMAC1/PTEN in malignant myeloid disorders.
The candidate tumour suppressor gene MMAC1/PTEN located at chromosome 10q23.3 has been reported to be frequently mutated in a number of solid tumours. Less is known about its status in leukaemia. In the present study we first analysed 13 leukaemia cell lines for mutations and homozygous deletions in MMAC1/PTEN using PCR and denaturing gradient gel electrophoresis (DGGE). We identified an intragenic deletion including MMAC1/PTEN exons 2-5 in an acute myelocytic leukaemia cell line, HL-60 blast, and an insertion of four nucleotides in exon 5 in an acute monocytic leukaemia cell line, U937. Analysis of 59 patients with acute myeloid leukaemia (AML), 26 patients with myelodysplastic syndromes (MDS) and 10 patients with chronic myeloid leukaemia (CML) only revealed a polymorphic base substitution in codon 44 in one AML patient, suggesting that mutations in the MMAC1/PTEN gene are infrequent genetic aberrations in myeloid leukaemia.
Acute Disease↗
Roquinimex (Linomide) vs placebo in AML after autologous bone marrow transplantation.
Roquinimex, Linomide, a quinoline derivative with pleiotropic immunomodulatory activity, has previously been shown to enhance natural killer (NK) cell number and activity after ABMT in patients with AML. In this study 278 AML patients in remission were randomized to receive Roquinimex 0.2 mg/kg body weight or placebo twice weekly for 2 years following ABMT. Out of 139 patients in each group, 109 Roquinimex patients and 108 placebo patients were in their first CR. Median age at inclusion was 41 years for Roquinimex patients and 39 years for placebo patients. Twelve patients in each group had their marrow purged prior to reinfusion. Relapse and death were study endpoints. Surviving patients were followed for 2.6 to 6. 9 years. The total number of relapses was 60 in the Roquinimex group and 63 in the placebo group (not significant). Leukemia-free and overall survivals were similar in the two groups. Recovery of platelet counts was significantly delayed in the Roquinimex group as compared to placebo. No other significant differences regarding toxicity parameters were recorded. In conclusion, previous findings on NK cells could not be confirmed and the study showed no benefit for Roquinimex over placebo regarding relapse or survival following ABMT for AML in remission.
Adolescent↗
Reduced total number of cobblestone area forming cells and in vitro stromal-cell growth in autografts from acute myeloid leukemia patients.
BACKGROUND: It is well known that ABMT in acute myeloid leukemia (AML) often results in delayed hematopoietic engraftment, but the reason behind this has not been resolved. Previous studies have largely dealt with measurements of committed myeloid progenitors as surrogate markers for hematopoiesis. METHODS: Measurements of Week 5 cobblestone area forming cells (CAFC) and stromal-cell growth in BM autografts from 14 AML patients were compared with those from 10 NHL patients. RESULTS: Grafts achieved from the AML patients contained a significantly lower total number of CAFC than those from the NHL patients. The reason for this was a lower total amount of mononuclear cells (MNC) obtained during harvest procedure (mean 0.4 x 10(8)/kg for AML, versus 0.8 x 10(8)/kg for NHL). In contrast, the frequency of CAFC was comparable both between patient groups (mean 1.47, range 0.15-6.33 per 10(4) MNC for AML versus mean 1.47, range 0.53-3.57 per 10(4) MNC for NHL) and compared with that of eight normal donors (mean 1.12, range 0.73-1.73 per 10(4) MNC). An inverse relationship was observed between the total CAFC number in the grafts and the hematopoietic reconstitution of both granulocytes > or = 2.0 x 10(9)/L and thrombocytes > or = 50 x 10(9)/L, in which the level of 9.0 x 10(3) CAFC/kg implied a prompt engraftment for both patient groups. Whereas the stromal cell outgrowth in vitro from 8/10 NHL patients was similar to that of six normal donors, only a few stromal cells appeared in the majority of nine evaluable AML patients. DISCUSSION: A decreased total CAFC content, as well as an inferior stromal-cell function, may be critical elements for prolonged hematopoietic reconstitution in AML.
Adult↗
Biology of multiple drug resistance in acute leukemia.
Since the early 1970s, multiple drug resistance (MDR) has been known to exist in cancer cells and is thought to be attributable to a membrane-bound, energy-dependent pump protein (P-glycoprotein [P-gp]) capable of extruding various related and unrelated chemotherapeutic drugs. P-gp is coded for by the MDR1 gene, which in the human genome is located on the long arm of chromosome 7 (7q21-31). At the cellular level, the function of P-gp has been extensively investigated in human cancer. Although innumerable reports have been published in which P-gp has been shown to confer MDR to malignant (including leukemia) cells, so far, large-scale studies in the clinical setting have not convincingly proven that MDR1 plays a major role in clinical drug resistance when the influence of other known prognostic factors in human leukemia are taken into account. At present, results from phase 3 clinical trials evaluating the efficiency of inhibiting (or reversing) the function of P-gp in hematologic malignancies are eagerly awaited. Moreover, the horizon of cellular drug resistance in human cancer has during recent years widened dramatically. Thus, an array of different molecules and mechanisms by which resistant cells can escape the cytotoxic effect of anticancer drugs has now been identified. These molecules and mechanisms include apoptosis-related proteins. In this article, we review the different methods for determining MDR and, in particular, methods for determining P-gp/MDR1, with special reference to their potential importance for therapeutic strategies in human acute leukemia.
Acute Disease↗
[Scientific reporting--any future development?].
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Clinical Trials, Phase III as Topic↗
Extensive intra- and interindividual heterogeneity of p15INK4B methylation in acute myeloid leukemia.
Silencing of the cyclin-dependent kinase inhibitor gene p15INK4B by cytosine methylation of the promoter region has been associated with some types of hematological malignancy. To study in detail the patterns of p15INK4B methylation in patients with acute myeloid leukemia, we adopted a novel approach based on PCR amplification of bisulfite-treated DNA followed by resolution of differentially methylated sequences by denaturing gradient gel electrophoresis. This method visually displays the degree and heterogeneity of DNA methylation and enables the isolation and characterization of distinct clonotypic epigenotypes. A surprisingly high degree of intra- and interindividual heterogeneity of p15INK4B methylation was observed in the 65 acute myeloid leukemia patients examined. Methylation was detected in 46 (71%) of the patients and was observed more frequently in the French-American-British subtypes M1/M2 than in M4/M5 (P < 0.025). Examination of the same panel of samples using a highly sensitive methylation-specific PCR method showed methylated p15INK4B alleles in 61 (94%) of the samples. We present evidence that the higher frequency of p15INK4B methylation determined by methylation-specific PCR may, at least in part, be due to the presence of a small fraction of p15INK4B-methylated lymphocytes in normal blood.
Alleles↗
Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript.
Because previous PCR-based methodologies for detection of minimal residual disease (MRD) in leukemia patients have been too cumbersome to allow for widespread clinical usefulness, we have employed a real-time quantitative PCR (RQ-PCR) system to develop an MRD assay for t(12;21). We initially determined the expression of the different alternatively spliced TEL-AML1 mRNAs found in t(12;21) breakpoint variants I and II. We then optimized PCR primers for the RQ-PCR system and, using the t(12;21)+ REH cell line in spiking experiments, found a linear detection of TEL-AML1 over at least five logs. Moreover, 1 malignant cell in a background of 1,000,000 normal cells could be detected. The expression of the GAPDH, ABL, and beta(2)-microglobulin (beta2M) housekeeping genes were then compared in normal donors and in leukemic patients, and the very stably expressed beta2M was selected as an internal reference gene, allowing us to compensate for variation in RNA quality and day-to-day variation. In 12 samples from t(12;21)-positive patients at diagnosis, the levels of the TEL-AML1 fusion transcripts were found to vary up to 14-fold after normalization to beta2M. Interestingly, in samples obtained from seven patients at diagnosis, during induction chemotherapy, or relapse, the level of TEL-AML1 in peripheral blood (PB) and bone marrow (BM) was found to differ only by threefold, suggesting that MRD may be evaluated in PB samples in most patients. We conclude that this assay could set new standards for t(12;21) MRD detection with its accuracy, its high throughput, and its short turnover time for samples. Genes Chromosomes Cancer 26:355-365, 1999.
Bone Marrow↗