TEL/AML1 gene fusion is related to in vitro drug sensitivity for L-asparaginase in childhood acute lymphoblastic leukemia

The t(12;21) translocation resulting in TEL/AML1 gene fusion is present in approximately 25% of patients with precursor B-lineage pediatric acute lymphoblastic leukemia (ALL). Studies suggest an association with a good prognosis; however, relapse can occur. We studied the relation between t(12;21), determined by fluorescence in situ hybridization or polymerase chain reaction, and in vitro drug resistance, measured by the MTT assay, in childhood B-lineage ALL at diagnosis. A total of 180 ALL samples were tested, 51 (28%) of which were positive for t(12;21). The median LC(50) values did not differ significantly between TEL/AML1-positive and -negative samples for prednisolone, dexamethasone, daunorubicin, thiopurines, epipodophyllotoxins, and 4-HOO-ifosfamide. However, the TEL/AML1-positive patients were relatively more sensitive to L-asparaginase (ASP; 5.9-fold; P =.029) and slightly but significantly more resistant to vincristine (1.5-fold; P =.011) and cytarabine (1.5-fold; P =.014). After matching for unevenly distributed patient characteristics-that is, excluding patients younger than 12 months, patients with CD10-negative immature B-lineage ALL, patients with Philadelphia chromosome, and patients who were hyperdiploid (more than 50 chromosomes) from the TEL/AML1 negative group-the only remaining difference was a relative sensitivity for ASP in the TEL/AML1-positive samples (10.8-fold; P =. 012). In conclusion, the presence of TEL/AML1 gene fusion in childhood precursor B-lineage ALL does not seem to be associated with a high in vitro drug sensitivity, except for ASP, indicating that these patients could benefit from treatment schedules with significant use of this drug.     

The histone deacetylase inhibitor AN-9 has selective toxicity to acute leukemia and drug-resistant primary leukemia and cancer cell lines

The novel prodrug of butyric acid, pivaloyloxymethyl butyrate (AN-9), a histone deacetylase inhibitor, shows great promise as an effective and relatively nontoxic anticancer agent for solid malignancies. However, little is known about its effects on hematopoietic malignancies. In this study, we show that 21 primary samples of acute leukemia were sensitive to the antiproliferative effects of AN-9, with a 50% inhibitory concentration (IC(50)) of 45.8 +/- 4.1 microM. In colony-forming assays, primary T-cell acute lymphoblastic leukemia (T-ALL) cells were 3-fold more sensitive to AN-9 than the normal hematopoietic progenitors, erythroid burst-forming units and granulocyte/monocyte colony-forming units. AN-9 induced apoptosis in the T-ALL cell line CEM. A common problem with cancer is chemoresistance, which is often typical of relapsed cancers. Remarkably, a T-ALL sample at diagnosis and an acute myeloid leukemia sample at relapse that were resistant to doxorubicin in vitro were sensitive to AN-9, with an IC(50) of 50 microM for both samples. More strikingly, samples from 2 infants with t(4;11) ALL obtained at diagnosis and relapse each were the most sensitive to AN-9, with IC(50) values of 25 microM and 17 microM, respectively. Furthermore, a doxorubicin-resistant clone of HL60, HL60/ADR, obtained by the transfection of the MDR-1 gene, was equally sensitive to AN-9 cytotoxicity as the parental cells. AN-9 induced the expression of p21 in an infant leukemia sample with 11q23 rearrangement, but not in T- or B-precursor ALL. Collectively, our results suggest that AN-9 is a selective agent for hematopoietic malignancies that can circumvent the mechanisms of chemoresistance limiting most conventional chemotherapy.     

Flow cytometric analysis of de novo acute lymphoblastic leukemia in childhood: report from the Japanese Pediatric Leukemia/Lymphoma Study Group

Although the antigen expression patterns of childhood acute lymphoblastic leukemia (ALL) are well known, little attention has been given to standardizing the diagnostic and classification criteria. We retrospectively analyzed the flow cytometric data from a large study of antigen expression in 1,774 children with newly diagnosed ALL in JPLSG. T- and B-lineage ALL accounted for 13 and 87% of childhood ALL cases, respectively. Cytoplasmic CD3 and CD7 antigens were positive in all T-ALL cases. More than 80% of T-ALL cases expressed CD2, CD5 and TdT. In B-lineage ALL, the frequencies of early pre-B, pre-B, transitional pre-B and B-ALL were 81, 15.5, 0.6 and 2.9%, respectively. More than 90% of early pre-B ALL cases expressed CD19, CD79a, CD22, CD10 and TdT. CD34 was expressed in three-fourths of early pre-B ALL cases. The frequencies of TdT and CD34 expression were lower in pre-B ALL than in early pre-B ALL. B-ALL showed less frequent expression of CD22, CD10, CD34 and TdT than other B-lineage ALL cases. Expression of CD13 and CD33, aberrant myeloid antigens, was significantly more frequently associated with B-lineage ALL than with T-ALL. Based on this retrospective study of antigen expression in 1,774 de novo childhood ALL cases in JPLSG, we propose standardized clinical guidelines for the immunophenotypic criteria for diagnosis and classification of pediatric ALL.     

Cellular drug resistance profiles in childhood acute myeloid leukemia: differences between FAB types and comparison with acute lymphoblastic leukemia

Determining in vitro drug resistance may reveal clinically relevant information in childhood leukemia. Using the methyl-thiazol-tetrazolium assay, the resistance of untreated leukemic cells to 21 drugs was compared in 128 children with acute myeloid leukemia (AML) and 536 children with acute lymphoblastic leukemia (ALL). The differences between 3 French-American-British (FAB) types (M1/M2, M4, and M5) were also compared. AML was significantly more resistant than ALL to the following drugs, as noted by the median resistance: glucocorticoids (greater than 85-fold), vincristine (4.4-fold), L-asparaginase (6.9-fold), anthracyclines (1.8- to 3.4-fold), mitoxantrone (2.6-fold), etoposide (4.9-fold), platinum analogues (2.4- to 3.4-fold), ifosfamide (3.5-fold), and thiotepa (3.9-fold). For cytarabine and thiopurines, the median LC50 values (the drug concentration that kills 5% of the cells) were equal. Also, busulfan, amsacrine, teniposide, and vindesine showed no significant differences, but the numbers were smaller, and the median LC50 values were 1.3- to 5.2-fold higher in AML. None of the drugs demonstrated greater cytotoxicity in AML. FAB M5 was significantly more sensitive than FAB M4 to most drugs frequently used in AML, as indicated by the following ratios of median sensitivities: the anthracyclines (2.6- to 3.2-fold), mitoxantrone (12.5-fold), etoposide (8.7-fold), and cytarabine (2.9-fold). For etoposide and cytarabine (5.4- and 3.4-fold, respectively) FAB M5 was also significantly more sensitive than FAB M1/M2. FAB M5 was equally sensitive to L-asparaginase and vincristine as ALL. Only 15% of the AML samples were "intermediately" sensitive to glucocorticoids, mainly in FAB M1/M2. The poorer prognosis of childhood AML is related to resistance to a large number of drugs. Within AML, FAB M5 had a distinct resistance pattern. These resistance profiles may be helpful in the rational design of further treatment protocols. (Blood. 2000;96:2879-2886)     

CDKN2 gene deletion is not found in chronic lymphoid leukaemias of B- and T-cell origin but is frequent in acute lymphoblastic leukaemia

Summary. Homozygous deletions of the cyclin-dependent kinase 4 (CDK4) inhibitor gene CDKN2 (pi6, MTS1) have been demonstrated to occur frequently in human cancer cell lines of different origin. However, in most primary tumours the frequencies of CDKN2 deletions are not well defined. We studied primary samples of 100 patients with lymphoid leukaemias [B-lineage acute lymphoblastic leukaemia (ALL), n = 23; T-ALL, n= 7; B-cell chronic lymphocytic (B-CLL) or prolymphocytic (B-PLL) leukaemia, =50; T-CLL/T-PLL, n = 20] using fluorescence in situ hybridization (FISH) with eight overlapping cosmid clones covering the region on chromosome band 9p21 containing CDKN2. We did not observe any CDKN2 deletions in the 70 patients with chronic lymphoid leukaemias of B- or T-cell origin. Of the 23 patients with B-lineage ALL, one (4%) exhibited a CDKN2 deletion: in this patient, two clones were detected, one exhibiting a hemizygous and the other a homozygous deletion. On chromosome banding analysis, four patients with B-lineage ALL had a 9p aberration, whereas all CDKN2 copies were retained. In contrast, six of the seven (86%) patients with T-ALL exhibited CDKN2 deletions (homozygous, n = 4; hemizygous, n = 2). We conclude that hemizygous or homozygous deletions of the CDKN2 gene occur at high frequency in T-ALL and at low frequency in B-lineage ALL, supporting the role of this gene as a tumour suppressor, especially in T-ALL. However, from our data there is no evidence that CDKN2 is involved in the pathogenesis of chronic lymphoid leukaemias of B- or T-cell origin.     

In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia

Cellular drug resistance is thought to be an important cause of the poor prognosis for children with relapsed or refractory acute lymphoblastic leukemia (ALL), but it is unknown when, to which drugs, and to what extent resistance is present. We determined in vitro resistance to 13 drugs with the MTT assay. Compared with 141 children with initial ALL, cells from 137 children with relapsed ALL were significantly more resistant to glucocorticoids, L-asparaginase, anthracyclines, and thiopurines, but not to vinca-alkaloids, cytarabine, ifosfamide, and epipodophyllotoxins. Relapsed ALL cells expressed the highest level of resistance to glucocorticoids, with a median level 357- and >24-fold more resistant to prednisolone and dexamethasone, respectively, than initial ALL cells, whereas the resistance ratios for the other drugs differed from 0.8- to 1.9-fold, intraindividual comparisons between initial and relapsed samples from 16 children with ALL showed that both de novo and acquired drug resistance were involved. Specific in vitro drug-resistance profiles were associated with high-risk relapsed ALL groups. In vitro drug resistance was also related to the clinical response to chemotherapy in relapsed/refractory childhood ALL. We conclude that drug resistance may explain the poor prognosis for children with relapsed/refractory ALL. These day may be helpful to design alternative treatment regimens for relapsed childhood ALL.     

Expression and production of aberrant PAX5 with deletion of exon 8 in B-lineage acute lymphoblastic leukaemia of children

Summary We investigated PAX5 expression in childhood B-lineage acute lymphoblastic leukaemia (ALL). Seven of 21 children with B-lineage ALL had multiple PAX5 variants, while 14 children and healthy controls showed full-length (FL) and one variant PAX5. By Western blotting, healthy controls displayed Pax5-FL, while one short Pax5, derived from the deletion of exon 8 (Pax5-DeltaE8) was produced in 90% of ALL samples, as well as in ALL cell lines. PAX5-DeltaE8 lacked more than 50% of the transactivation domain, indicating that aberrant Pax5 production might lead to the arrest of B-cell differentiation, contributing to the pathogenesis of B-lineage ALL.     

Circumvention of methotrexate resistance in childhood leukemia subtypes by rationally designed antifolates.

Cellular methotrexate (MTX) resistance may cause treatment failure in childhood common/preB-acute lymphoblastic leukemia (c/preB-ALL), T-lineage ALL (T-ALL), and acute myeloid leukemia (AML). The ex vivo potency of several antifolates (MTX, trimetrexate [TMQ], GW1843U89, multitargeted antifolate [MTA], Raltitrexed, and ZD9331) was studied via in situ inhibition of thymidylate synthase (TS). After short-term exposure, relapsed c/preB-ALL (rALL, n = 21), T-ALL (n = 22), and AML (n = 22) were 3-fold, 10-fold, and 6-fold less sensitive to MTX (P 相似文献   

New recurring cytogenetic abnormalities and association of blast cell karyotypes with prognosis in childhood T-cell acute lymphoblastic leukemia: a pediatric oncology group report of 343 cases

To further define the cytogenetic differences between B-cell lineage (B-lineage) acute lymphoblastic leukemia (ALL) and T-cell lineage ALL (T-ALL) and to determine the prognostic value of cytogenetics in childhood T-ALL, the blast cell karyotypes of 343 cases of pediatric T-ALL, the largest series reported to date, were evaluated. Cytogenetics were performed in a single central laboratory, and the children were treated using a single Pediatric Oncology Group protocol. Clear differences between the karyotypic characteristics of B-lineage ALL and T-ALL were confirmed. This study suggests that there may be survival differences associated with some T-ALL blast cell karyotypes. Better survival is associated with only normal karyotypes and with t(10;14) (translocation of chromosomes 10 and 14); worse survival is associated with the presence of any derivative chromosome. Two new recurring chromosome aberrations previously not reported in T-ALL were found: del(1)(p22) and t(8;12)(q13;p13). Ten aberrations found in this series, which were reported only once previously in T-ALL, can now be considered recurring abnormalities in T-ALL. All 12 of these new recurring aberrations are targets for discovery and characterization of new genes that are important in T-cell development and leukemogenesis.     

急性淋巴细胞白血病患者FLT3基因及FLT3/ITD基因突变的检测及临床意义

目的:研究急性淋巴细胞白血病(ALL)患者FLT3基因及其内部串联重复(ITD)突变情况。方法:采用多聚酶链反应(PCR)联合单链构象多态性(SSCP)方法检测76例不同免疫分型ALL患者FLT3基因及FLT3/ITD基因突变。结果:76例ALL患者经PCR扩增发现46例(60.5%)FLT3基因检测阳性,其中前前B细胞ALL、前B细胞ALL、成熟B细胞ALL及T细胞系ALL患者FLT3基因检测阳性率分别为88.2%(15/17),73.9%(17/23),40.0%(6/15)和23.5%(4/17);前前B细胞ALL和前B细胞患者ALL FLT3基因检测阳性率80.0%,显著高于成熟B细胞ALL(40.0%)(P<0.01);B细胞系ALL患者FLT3基因检测阳性率为69.1%,显著高于T细胞系ALL患者(23.5%)(P<0.01)。76例ALL患者中仅有2例(2.6%)出现FLT3/ITD基因突变,此2例均为伴有2种髓系抗原表达,免疫学检查诊断为急性混合细胞白血病患者,均伴有外周血高白细胞数、骨髓中高白血病细胞比例及预后较差。结论:B细胞系ALL和T细胞系ALL患者均可检测出FLT3基因,但B细胞系ALL患者FLT3基因检测阳性率显著高于T细胞系ALL;B细胞系ALL中细胞分化越成熟则FLT3基因检测率阳性越低。ALL患者一般不出现FLT3/ITD基因突变,FLT3/ITD基因突变检测可能有助于急性白血病基因分型及预后判断。     

Asparagine synthetase expression is linked with L-asparaginase resistance in TEL-AML1-negative but not TEL-AML1-positive pediatric acute lymphoblastic leukemia

Resistance to L-asparaginase in leukemic cells may be caused by an elevated cellular expression of asparagine synthetase (AS). Previously, we reported that high AS expression did not correlate to L-asparaginase resistance in TEL-AML1-positive B-lineage acute lymphoblastic leukemia (ALL). In the present study we confirmed this finding in TEL-AML1-positive patients (n = 28) using microarrays. In contrast, 35 L-asparaginase-resistant TEL-AML1-negative B-lineage ALL patients had a significant 3.5-fold higher AS expression than 43 sensitive patients (P < .001). Using real-time quantitative polymerase chain reaction (RTQ-PCR), this finding was confirmed in an independent group of 39 TEL-AML1-negative B-lineage ALL patients (P = .03). High expression of AS was associated with poor prognosis (4-year probability of disease-free survival [pDFS] 58% +/- 11%) compared with low expression (4-year pDFS 83% +/- 7%; P = .009). We conclude that resistance to l-asparaginase and relapse risk are associated with high expression of AS in TEL-AML1-negative but not TEL-AML1-positive B-lineage ALL.     

In vitro drug resistance profiles of adult versus childhood acute lymphoblastic leukaemia

The difference in the current cure rates between adult and childhood acute lymphoblastic leukaemia (ALL) may be caused by differences in drug resistance. Earlier studies showed that in vitro cellular drug resistance is a strong independent adverse risk factor in childhood ALL. Knowledge about cellular drug resistance in adult ALL is still limited. The present study compared the in vitro drug resistance profiles of 23 adult ALL patients with that of 395 childhood ALL patients. The lymphoblasts were tested by the MTT assay. The group of adult ALL samples was significantly more resistant to cytosine arabinoside, L-asparaginase, daunorubicin, dexamethasone and prednisolone. The resistance ratio (RR) was highest for prednisolone (31.7-fold) followed by dexamethasone (6.9-fold), L-asparaginase (6. 1-fold), cytosine arabinoside (2.9-fold), daunorubicin (2.5-fold) and vincristine (2.2-fold). Lymphoblasts from adult patients were not more resistant to mercaptopurine, thioguanine, 4-HOO-ifosfamide, mitoxantrone and teniposide. There were no significant differences in drug resistance between adult T-cell (T-) ALL (n = 11) and adult common/pre-B-cell (B-) ALL (n = 10). Additionally, adult T-ALL did not differ from childhood T-ALL (n = 69). There were significant differences between adult common/pre-B-ALL and childhood common/pre-B-ALL (n = 310) for prednisolone (RR = 302, P = 0.008), dexamethasone (RR = 20.9, P = 0.017) and daunorubicin (RR = 2.7, P = 0.009). Lymphoblasts from adults proved to be relatively resistant to drugs commonly used in therapy. This might contribute to the difference in outcome between children and adults with ALL.     

ZAP-70 expression in acute lymphoblastic leukemia: association with the E2A/PBX1 rearrangement and the pre-B stage of differentiation and prognostic implications

We evaluated the expression of 2 members of the Syk family, ZAP-70 and Syk, in acute lymphoblastic leukemia (ALL) samples, using data derived from a series of 33 T-ALL and 95 B-lineage adult ALL patients analyzed by oligonucleotide arrays. Of the B-lineage ALL cases, 37 were BCR/ABL+, 10 were ALL1/AF4+, 5 were E2A/PBX1+, and 43 carried no known molecular abnormality. ZAP-70 was highly expressed in T-ALL. A high ZAP-70 expression was also found in a proportion of B-lineage ALL, the highest levels being associated with the E2A/PBX1+ group and the lowest with ALL1/AF4+ cases (P < .001). A higher ZAP-70 expression was also observed in the pre-B group (P < .001). Remarkably, Syk expression was always preserved, suggesting that ZAP-70 expression is not substitutive of Syk. At the protein level, ZAP-70 was evaluated on 39 newly diagnosed ALL patients (25 adults, 14 children) and was detected in 23 cases (59%). ZAP-70 expression was consistently found in Ig mu+ cases. Evaluation of long-term outcome in cases without molecular abnormalities showed that the higher levels of ZAP-70 were coupled to a higher relapse rate. In ALL, ZAP-70 expression is associated with the E2A/PBX1 rearrangement and pre-B stage and may have a prognostic role and be a candidate molecule for targeted therapies.     

Adult acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) in adults is a relatively rare neoplasm with a curability rate around 30% at 5 years. This consideration makes it imperative to dissect further the biological mechanisms of disease, in order to selectively implement an hitherto unsatisfactory success rate. The recognition of discrete ALL subtypes (some of which deserve specific therapeutic approaches, like T-lineage ALL (T-ALL) and mature B-lineage ALL (B-ALL)) is possible through an accurate combination of cytomorphology, immunophenotytpe and cytogenetic assays and has been a major result of clinical research studies conducted over the past 20 years. Two–three major prognostic groups are now easily identifiable, with a survival probability ranging from <10 to 20% (Philadelphia-positive ALL) to about 50–60% (low-risk T-ALL and selected patients with B-lineage ALL). These issues are extensively reviewed and form the basis of current knowledge. The second major point relates to the emerging importance of studies that reveal a dysregulated gene activity and its clinical counterpart. It is now clear that prognostication is a complex matter ranging from patient-related issues to cytogenetics to molecular biology, including the evaluation of minimal residual disease (MRD) and possibly gene array tests. On these bases, the role of a correct, highly personalised therapeutic choice will soon become fundamental. Therapeutic progress may be obtainable through a careful integration of chemotherapy, stem cell transplantation, and the new targeted treatments with highly specific metabolic inhibitors and humanised monoclonal antibodies.     

Different drug sensitivity profiles of acute myeloid and lymphoblastic leukemia and normal peripheral blood mononuclear cells in children with and without Down syndrome.

Children with Down syndrome (DS) have an increased risk for leukemia. The prognosis for DS acute myeloid leukemia (AML) is better than for non-DS AML, but the clinical outcome of DS acute lymphoblastic leukemia (ALL) is equal to that of non-DS ALL. Differences in prognosis may reflect differences in cellular drug resistance. In vitro drug resistance profiles were successfully investigated on leukemic cells from 13 patients with DS AML and 9 patients with DS ALL and were compared with reference data from 151 non-DS AML and 430 non-DS B-cell precursor (BCP) ALL. DS AML cells were significantly more sensitive to cytarabine (median, 12-fold), the anthracyclines (2-7-fold), mitoxantrone (9-fold), amsacrine (16-fold), etoposide (20-fold), 6-thioguanine (3-fold), busulfan (5-fold), vincristine (23-fold), and prednisolone (more than 1.1-fold), than non-DS AML cells. Compared with DS ALL, DS AML cells were significantly more sensitive to cytarabine only (21-fold). After short-term exposure to methotrexate, DS AML cells were 21-fold more resistant than non-DS AML cells, but no difference was observed after continuous exposure. DS ALL cells and non-DS BCP-ALL cells were equally sensitive to all drugs, including methotrexate. Normal peripheral blood mononuclear cells from DS and non-DS children without leukemia showed highly resistant drug profiles. It was concluded that the better prognosis of DS AML might, at least partially, be explained by a specific, relatively sensitive drug-resistance profile, reflecting the unique biology of this disease. (Blood. 2002;99:245-251)     

High-dose methotrexate in the treatment of adult acute lymphoblastic leukemia

 The application of high-dose treatment elements has an increasing importance in the therapy of acute lymphoblastic leukemia (ALL). High-dose methotrexate (HDMTX) has been introduced in clinical trials more than 20 years ago, since it has several theoretical advantages compared to conventional dose methotrexate. These trials revealed that the efficacy and toxicity of HDMTX depends on features such as dose level, infusion time, combination regimen and schedule of leucovorin rescue. Particularly the application of controlled leucovorin rescue and improved supportive care enabled the application of increasing doses of HDMTX in the treatment of childhood and adult ALL within a variety of schedules and at dose levels mostly ranging between 0.5 g/m² and 5.0 g/m². In childhood ALL, first devised to replace CNS irradiation in the prophylaxis of CNS relapse HDMTX has contributed to the reduction of bone marrow relapses particularly in low risk B-lineage ALL. In addition it proved to be an effective therapy element for prophylaxis and treatment of CNS disease. At least in low risk ALL CNS irradiation could be safely replaced by repeated cycles of HDMTX with additional intrathecal therapy. In adult ALL only few of the succesful treatment approaches with HDMTX have been investigated up to now. The results indicate that HDMTX has a beneficial effect with regard to overall outcome in adult B-lineage ALL. It provides effective CNS prophylaxis in combination with intrathecal therapy. In mature B-ALL HDMTX proved to be one of the most effective treatment elements and contributed to an impressive improvement of outcome in this subgroup. For T-ALL however sufficient data do not exist either in childhood or in adult ALL. Since HDMTX is an effective treatment element with manageable acute and long-term toxicity, its role in the management of adult ALL should be explored more intensively. Received: 15 January 1996 / Accepted: 17 January 1996     

Minimal residual disease monitoring in adult T-cell acute lymphoblastic leukemia: a molecular based approach using T-cell receptor G and D gene rearrangements

BACKGROUND AND OBJECTIVES: Minimal residual disease (MRD) is important in the measurement of response to treatment in childhood B- and T-cell acute lymphoblastic leukemia (ALL) and in adult B-cell ALL. Little is known about MRD evaluation in adult T-cell ALL. This study aimed to determine the prognostic significance of MRD measurements in adult T-cell ALL. DESIGN AND METHODS: T-cell receptor (TCR) gamma (G) and TCR delta (D) gene analyses were carried out at presentation in 49 patients with de novo T-ALL using a polymerase chain reaction (PCR) approach. In 26 of the patients bone marrow (BM) samples were collected at sequential time points (0-2, 3-5, 6-9, 10-24 months) after diagnosis for MRD investigation. The relationship between MRD status and clinical outcome was investigated and correlated with age, gender and white blood cell count at presentation. RESULTS: TCRG clonal gene rearrangements were found in 40 patients (82%). Eleven patients showed TCRD rearrangements (22%), in one of them as the sole molecular marker. V(gamma)I family rearrangements predominated (45 of 65 alleles) together with V(delta)1-J(delta)1/2 (9 of 13 alleles). Continuous clinical remission (CCR) occurred in 17 patients while nine patients relapsed. MRD analysis showed that negative tests during the first 6 months post-induction, and persisting negative MRD after induction were the best predictors of CCR. A positive test after 5 months was better at predicting relapse. In only four of seven patients was relapse preceded by a positive test the 5 months preceding relapse. INTERPRETATION AND CONCLUSIONS: Overall the ability of positive and negative tests to predict relapse or CCR was weaker in this cohort of adult T-ALL patients than in T- and B-lineage childhood ALL and B-lineage adult ALL. TCRG and TCRD gene analysis provides a clonal marker in the majority of adult T-ALL. These results suggest that caution should be taken in using MRD data based on TCR gene rearrangements to predict prognosis in adult T-ALL. Biological reasons may underlie differences between the performance of MRD tests in B- and T-lineage ALL. Further studies in a larger cohort of patients are needed to determine the exact role that MRD determination has in the management of T-ALL in adults.     

In vitro drug resistance and prognostic impact of p16INK4A/P15INK4B deletions in childhood T-cell acute lymphoblastic leukaemia

p16 gene deletions are present in about 70% of primary paediatric T-cell acute lymphoblastic leukaemia (T-ALL) and 20% of common/precursor B-cell ALL cases. It is not clear what the impact of the frequent p16 deletions is within the subgroup of T-lineage ALL. We studied the relationship between p16/p19ARF deletions, using fluorescence in situ hybridization, and in vitro drug resistance and prognosis in childhood T-ALL at diagnosis. The cellular drug resistance was measured with the methyl thiazol tetrazoliumbromide assay using a panel of drugs and the thymidylate synthase inhibition assay for methotrexate. There was a complete overlap of individual LC50 values of p16 gene homozygously deleted and p16 germ-line cases for most of the nine classes of drugs tested. The only difference was for dexamethasone: the p16-deleted group was more sensitive than the germ-line p16 group (P = 0.030). The homozygously deleted p16 T-ALL patients (n = 34) treated with the modern multiagent chemotherapy schemes of the Dutch Childhood Leukaemia Study Group ALL-VII/-VIII or Co-operative ALL-92/-97 protocols have a significantly lower 5-year disease-free survival (DFS) than germ-line p16 T-ALL (n = 25) (65.1 +/- 9.1% vs. 95.5 +/- 4.4%, Plog rank = 0.021). Hence, this study identifies a subpopulation of primary childhood T-ALL that appears to have an extremely high DFS. However, the observed differences in outcome do not seem to be related to intrinsic resistance for the tested drugs.     

Candidate tumor-suppressor genes MTS1 (p16INK4A) and MTS2 (p15INK4B) display frequent homozygous deletions in primary cells from T- but not from B-cell lineage acute lymphoblastic leukemias [see comments]

Using a Southern blot approach, deletions of MTS1 (multiple tumor- suppressor gene 1) and MTS2 (multiple tumor-suppressor gene 2) candidate tumor-suppressor genes have been studied in primary neoplastic cells from 55 acute lymphoblastic leukemia (ALL) patients. Homozygous MTS1 deletions were found in 20 of 24 T-ALL cases and in only 2 of 31 B-lineage cases (P < .001). The deletions involved MTS1 and MTS2 in most cases. Homozygous MTS2 deletions were observed in 16 of 24 T-ALL cases and in 1 of 31 B-lineage ALLs (P < .001), all of them displaying homozygous MTS1 deletions. In 5 cases (4 T and 1 B), deletions involved MTS1 but spared the MTS2 gene, showing that one deletion breakpoint was located between the two genes within a 25-kb region. In 1 T-ALL case, an MTS1 gene rearrangement has occurred downstream to exon 2. Possible hemizygous deletions were found in 6 cases, 4 of them of the B-cell lineage. In 7 ALL cases, cells obtained at presentation and at first relapse were studied and identical results were observed in 6 cases. In 1 B-lineage case, a germline pattern was found at presentation and a possible monoallelic MTS1/MTS2 deletion was observed at relapse. The high frequency of MTS1 and MTS2 homozygous deletions in T-ALLs supports the view that inactivation of these genes plays an important role in the pathogenesis of this type of human leukemia.     

Resistance of bcr-abl-positive acute lymphoblastic leukemia to daunorubicin is not mediated by mdr1 gene expression

In vitro resistance to anthracyclines is thought to be a poor prognosis in achieving long-term remission in patients with acute lymphoblastic leukemia (ALL). Expression of a multidrug resistance gene (mdr1) that codes for 170 Kd transmembrane glycoprotein is responsible for conferring resistance to malignant cells to anthracyclines. The t(9:22) translocation, resulting in bcr-abl fusion gene, is commonly found in B-lineage ALL and is known to be a poor prognostic factor for long-term remission. To investigate whether resistance to anthracyclines contributes to poor prognosis in bcr-abl-positive ALL, we studied daunorubicin sensitivity by an in vitro colorimetric methyl tetrazolium (MTT) assay in B-lineage ALL patients who were bcr-abl-positive and compared them with the B-lineage, age-matched bcr-abl-negative group. We also looked for and compared the presence of mdr1 gene expression in these two groups of patients by RT-PCR. Of the 46 patients included in the study, 16 (34.7%) were positive for the bcr-abl fusion gene. mdr1 gene expression was seen in 14 of these 46 patients (30.4%). However, the expression of the mdr1 gene was relatively lower in the bcr-abl-positive group (3 out of 16, 18.7%) compared to the bcr-abl-negative group (11 out of 30, 36.6%). The median LD(50) of daunorubicin (concentration lethal to 50% of the leukemic blasts) differed significantly between bcr-abl-positive and -negative patients (P = 0.018). This in vitro study suggests that bcr-abl-positive ALL is relatively resistant to daunorubicin, but this resistance is not mediated through mdr1 gene expression.