Infant acute lymphoblastic leukemia - combined cytogenetic, immunophenotypical and molecular analysis of 77 cases.

We used karyotyping, fluorescence in situ hybridization (FISH), Southern blotting, and RT-PCR in order to analyze prospectively 77 infants (less than 1 year of age) with acute lymphoblastic leukemia for the occurrence of 11q23/MLL rearrangements and/or other cytogenetic abnormalities. Out of the 69 informative samples we found an 11q23/MLL rearrangement in 42 cases (61%). Regarding only pro-B ALL cases, the incidence of 11q23/MLL rearranged cases, however, reached more than 90% The infants were treated within the therapy studies ALL-BFM90, ALL-BFM95 and CoALL-05-92. For patients with an adequate follow-up of 4 years the event-free survival of the 11q23/MLL-positive and 11q23/MLL-negative group was 0.2 or 0.64, respectively (P = 0.024). The monoclonal antibody 7.1. (moab 7.1) does not react with normal hematopoetic precursors or mature blood cells but was shown to specifically react with leukemic cells bearing a rearrangement of chromosome 11q23 or the MLL gene, respectively. We, therefore, specifically addressed the question whether the reactivity of moab 7.1, as determined by flow cytometry, may substitute for molecular testing of an 11q23/MLL rearrangement in this cohort of infant ALLs. Reactivity of moab 7.1 indicated a 11q23/MLL rearrangement with a specificity of 100%. However, five of the 11q23/MLL-positive cases did not react with moab 7.1 indicating a sensitivity of 84% only. Three of these five moab 7.1-negative but 11q23/MLL-positive cases could be identified by their unique expression pattern of CD65s and/or CD15. Thus, 95% of all 11q23/MLL-positive ALL cases in infancy may be identified by flow cytometry based on their expression of CD15, CD65s and/or moab 7.1.     

Acute myeloid leukemia with MLL rearrangements: clinicobiological features, prognostic impact and value of flow cytometry in the detection of residual leukemic cells.

The MLL gene, located at 11q23 band, is frequently disrupted by different chromosomal rearrangements that occur in a variety of hematological malignancies. MLL rearrangements are associated with distinct clinical features and a poor prognosis. The aim of this study was to analyze the incidence and the prognostic significance of MLL rearrangements in a consecutive series of adult AML patients and to determine the immunophenotypic features of these cases. The identification of abnormal immunophenotypes could be used for the detection of minimal residual disease (MRD). Ninety-three adult patients with de novo acute myeloid leukemia (AML) were analyzed by Southern blot in order to detect MLL rearrangements (MLL+). RT-PCR and genomic long-range PCR were performed to further characterize MLL partial tandem duplication (PTD) in those patients in whom conventional karyotype did not show 11q23 chromosomal translocations. All the patients were homogeneously immunophenotyped at diagnosis. MLL rearrangements were detected in 13 (14%) patients. Four patients (5%) showed 11q23 translocations by karyotypic conventional analysis. Nine patients (10%) revealed PTD of MLL and one patient showed a MLL cleavage pattern. The MLL+ patients usually expressed myeloid and monocytic antigens CD33 (12/13 cases), CD13 (9/13), CD117 (9/13), CD64 (11/13) and in some cases CD14 (4/11). HLA-DR was also positive in (12/13). Eight out of 13 cases expressed the stem cell marker CD34. Only one patient revealed lymphoid marker reactivity (CD7) and CD56 was expressed in 5/13 cases. All the MLL+ patients showed at least one aberrant phenotype at diagnosis, which allowed us to set out a simple panel for the MRD studies. Twenty-seven samples from eight patients in morphologic complete remission (CR) were analyzed using the aberrant immunologic combinations detected at diagnosis. Phenotypically abnormal cells were detected in all the patients who subsequently relapsed, whereas only one patient with MRD+ remained in CR. Owing to the high level of residual leukemic cells, the MLL+ patients showed a short CR duration and a poor survival. In conclusion, immunophenotyping may be a suitable approach to investigating MRD status in AML patients with PTD of the MLL gene.     

Comparison of cytogenetics, Southern blotting, and fluorescence in situ hybridization as methods for detecting MLL gene rearrangements in children with acute leukemia and with 11q23 abnormalities.

Bone marrow samples from 67 children with acute leukemia and with cytogenetic evidence of chromosome 11 band q23 (11q23) abnormalities were characterized by fluorescence in situ hybridization (FISH) and Southern blot analysis to determine whether FISH could reliably detect MLL gene rearrangements in this population. Among the 42 patients with acute lymphoblastic leukemia (ALL), MLL gene rearrangements were detected in cells from 23 patients (54.8%) by both FISH and Southern blot analysis. FISH identified allelic deletions of MLL gene in five of 12 patients (42%) with ALL and with deletion of 11q23. In 22 of 25 children (88%) with AML, FISH detected MLL gene rearrangements, whereas Southern blot analysis identified rearrangements in 24 of 25 patients (96%). For children with acute leukemia and with 11q23 abnormalities, we recommend that FISH be used for the rapid screening of MLL gene rearrangements and that Southern blot analysis be used for the definitive assessment of the MLL gene status.     

High frequency of pro-B acute lymphoblastic leukemia in adults with secondary leukemia with 11q23 abnormalities.

To evaluate the frequency and cytogenetic and immunophenotypic features of therapy-related, precursor B-cell acute lymphoblastic leukemia (ALL), 152 cases of immature B-cell ALL were reviewed. These were compared to the frequency of therapy-related acute myeloid leukemia (t-AML) during the same time period. Eight ALL cases with a prior diagnosis of malignancy were identified, including six (4.0%) with prior therapy considered to be therapy-related ALL (t-ALL). The t-ALL cases followed treatment for breast carcinoma (two cases), lung carcinoma (two cases), lymphocyte predominance Hodgkin's disease and follicular lymphoma with a latency period of 13 months to 8 years. All t-ALL cases had a pro-B (CD10-negative) immunophenotype with significantly higher expression of CD15 and CD65, compared to the de novo CD10-positive ALL cases. All six t-ALL cases had MLL abnormalities by fluorescence in situ hybridization, and four showed t(4;11)(q21;q23). These represented half of all 11q23-positive adult ALL cases. During the same time period, 4.9% of all AML cases were considered t-AML. There was a 16.7% frequency of 11q23 abnormalities in the t-AML group. Despite the similar frequency in therapy-related disease among ALL and AML cases, there were differences in the frequency of the diseases and t-ALL represented 12% of all therapy-related leukemias. However, t-ALL represented 46% of all 11q23-positive therapy-related leukemias. The immunogenetic features of t-ALL appear distinct and may aid in identifying more cases of this disease type in the future.     

Chromosome abnormalities and MLL rearrangements in acute myeloid leukemia of infants.

Of 29 infants with acute myeloid leukemia (AML), 14 (48%) had various 11q23 translocations. MLL rearrangements were examined in 21 of the 29 patients, and 11 (52%) showed the rearrangements. 11q23 translocations and/or MLL rearrangements were found in 17 (58%) of the 29 patients. While all but one of the 17 patients with 11q23/MLL rearrangements had M4 or M5 type of the FAB classification, the 12 patients without such rearrangements had various FAB types, including M2, M4, M4EO, M6 and M7. Of the 12 patients with other chromosome abnormalities or normal karyotypes, two had inv(16) ort(16;16), one had t(1;22)(p13;q13), and two had a novel translocation, t(7;12)(q32;p13). The breakpoint on 12p of the t(7;12) was assigned to intron 1 or the region just upstream of exon 1 of the TEL/ETV6 gene by fluorescence in situ hybridization. The event-free survival at 5 years for the 17 patients with 11q23/MLL rearrangements was 42.2%, and that for the 12 patients without such rearrangements was 31.3% (P = 0.5544). 11q231MLL rearrangements have been frequently reported and a poor prognosis in infant acute lymphoblastic leukemia implied. Our study showed that while 11q23/MLL rearrangements were also common in infant AML, AML infants with such rearrangements had a clinical outcome similar to that of AML infants without such rearrangements.     

Mixed lineage leukemia-rearranged childhood pro-B and CD10-negative pre-B acute lymphoblastic leukemia constitute a distinct clinical entity.

PURPOSE: Mixed lineage leukemia (MLL) abnormalities occur in approximately 50% of childhood pro-B acute lymphoblastic leukemia (ALL). However, the incidence and type of MLL rearrangements have not been determined in common ALL (cALL) and CD10+ or CD10- pre-B ALL. EXPERIMENTAL DESIGN: To address this question, we analyzed 29 patients with pro-B ALL, 11 patients with CD10- pre-B ALL, 23 pre-B, and 26 cALL patients with CD10 on 20% to 80%, as well as 136 pre-B and 143 cALL patients with CD10 > or = 80% of blasts. They were all enrolled in four Austrian ALL multicenter trials. Conventional cytogenetics were done to detect 11q23 abnormalities and in parallel the potential involvement of the MLL gene was evaluated with a split apart fluorescence in situ hybridization probe set. RESULTS: We found that 15 of 29 pro-B ALL, 7 of 11 CD10- pre-B ALL, and 1 of 2 French-American-British classification L1 mature B-cell leukemia cases had a MLL rearrangement. However, no 11q23/MLL translocation was identified among the CD10+ pre-B and cALL patients. MLL-rearranged pro-B and CD10- pre-B ALL cases had similar clinical and immunophenotypic (coexpression of CDw65 and CD15) features at initial diagnosis. CONCLUSIONS: The striking similarities between the two CD10- ALL subsets imply that CD10- pre-B ALL variants may represent pro-B ALL cases that maintained the propensity to rearrange and express their immunoglobulin heavy chain rather than actual pre-B ALL forms transformed at this later stage of B-cell differentiation. However, direct experimental data are needed to confirm this observation.     

FLJ10849, a septin family gene, fuses MLL in a novel leukemia cell line CNLBC1 derived from chronic neutrophilic leukemia in transformation with t(4;11)(q21;q23).

A t(4;11)(q21;q23) has been described in 50-70% of cases of infant acute lymphoblastic leukemia and, less frequently, in cases of pediatric and adult acute lymphoblastic leukemia and acute myeloid leukemia (AML). In t(4;11)(q21;q23) leukemias, the AF4 gene has been cloned as a fusion partner of the MLL gene. A human myeloid leukemia cell line, chronic neutrophilic leukemia (CNL)BC1, was established from a peripheral blood specimen of a patient with CNL in leukemic transformation. As with the original leukemia cells, the established line had a t(4;11)(q21;q23). We showed that the MLL gene on 11q23 was fused to the FLJ10849 gene on 4q21. The protein encoded by FLJ10849 belongs to the septin family, sharing highest homology with human SEPT6, which is one of the fusion partners of MLL in t(X;11)(q13;q23) AML. Our results suggest that FLJ10849 might define a new septin family particularly involved in the pathogenesis of 11q23-associated leukemia. The established cell line, CNLBC1, could provide a useful model for analyzing the pathogenesis of MLL-septin leukemias and chronic neutrophilic leukemia.     

Characterization of acute myeloid leukemia with MLL rearrangements--no increase in the incidence of coexpression of lymphoid-associated antigens on leukemic blasts.

MLL gene rearrangements are associated with coexpression of myeloid- and lymphoid-associated antigens on leukemic blasts and a dismal outcome in acute lymphoblastic leukemia (ALL). Whether the same conditions can apply to acute myeloid leukemia (AML) is not quite clear. Rearrangements of the MLL gene were analyzed on 113 patients with newly diagnosed de novo AML in a single institution. Sixteen (14%) of them showed rearranged bands by Southern blot analysis, including three (50%) of six infants, three (14%) of 21 children between 1 and 15 years and 10 (12%) of 86 adults. MLL rearrangements were not only detected in M5 (four of 12 patients, 33%) and M4 (six of 31, 19%) subtypes but also in other non-M4-M5 AML (six of 70, 9%), including M1, M2 and M7, but not M3 subtype. Seven patients had chromosomal abnormalities involving 11q23, but nine did not. The latter comprised three (6%) of 48 patients with normal karyotype, one with t(8;21), none with t(15;17), inv(16) or t(9;22), and four (15%) of 27 with cytogenetic aberrations other than those specific structural abnormalities. In contrast to ALL, AML patients with MLL rearrangements did not tend to coexpress lymphoid- and myeloid-associated antigens simultaneously on leukemic blasts and have similar outcome as those without the gene rearrangements.     

Recent advances in the treatment of infant acute myeloid leukemia

Infant acute myeloid leukemia (AML) of less than 12 months old is generally characterized by a high incidence of acute monoblastic or myelomonoblastic leukemia with hyperleukocytosis and extramedullary involvement. Most of the leukemic cells have 11q23 translocations, which lead to the MLL gene rearrangements. The MLL gene rearrangements occur at a high frequency in monoblastic subtype, hyperleukocytosis or young age in infant AML. Compared with acute lymphoblastic leukemia, however, it remains unknown whether prenatal origin exists in the pathogenesis of infant AML. Recently, the treatment outcome of infant AML has been clarified by two study groups, which confirmed the effect of intensive chemotherapy including repeated cycles of cytarabine and anthracyclines for infant AML. Presence of the MLL gene rearrangements, gender, age and white blood cell count showed no influence on the outcome of infant AML. The allogeneic hematopoietic stem cell transplantation (HSCT) remains the treatment of choice for infant AML when a matched related donor is available. Monitoring of minimal residual disease by real-time PCR is a useful technique to predict the outcome or efficacy of the treatment in infant AML. Although intensive chemotherapy and/or allogeneic HSCT have cured most AML infants, some still relapse and ultimately die. A need remains for future development by exploiting the unusual biologic properties of leukemic progenitor cells expressing the abnormal MLL gene product.     

Incidence of MLL rearrangement in acute myeloid leukemia, and a CALM-AF10 fusion in M4 type acute myeloblastic leukemia

To determine the incidence of the mixed lineage leukemia (MLL) gene rearrangements in acute myeloid leukemia (AML) without cytogenetically-detected 11q23 abnormalities, we screened 64 cases of AML at diagnosis for MLL rearrangement by FISH. Three cases (4.7%) had a MLL rearrangement detected; one was shown to have a cryptic t(11;22)(q23;q11) and another to have a t(9;11)(p21-22;q23) which had been missed by the conventional cytogenetic study. No 11q23 structural abnormality was visible in the third case. Twenty-six of the 64 cases were further studied by Southern blotting and DNA hybridization, and four of these cases (15%) were found to have MLL rearrangement: in three of these, FISH had not detected any abnormality. FISH was also used to confirm MLL involvement in eight cases of AML that had a cytogenetic abnormality at 11q23; in one of these, Southern blot did not show a rearrangement. The survival of patients with MLL abnormalities identified by cytogenetics, FISH and/or DNA analysis was significantly worse than that of patients without MLL abnormalities (event-free survival p = 0.016) although two patients with a t(9;11)(p21-22;q23) were long-term survivors, consistent with this particular translocation having a better prognosis. One further case with a cytogenetic abnormality close to 11q23 was studied; it was found to have a t(10;11)(p13;q21), and the breakpoints were shown by FISH to involve the Clathrin Assembly Lymphoid Myeloid (CALM) gene at 11q21 and the AF10 gene at 10p13. Our data confirm the value of combining cytogenetic, FISH and molecular analyses to define the incidence and precise nature of MLL and 11q23 abnormalities in AML.     

Childhood acute leukemia with t(11;19) (q23;p13).

From 583 cases of acute lymphoblastic leukemia (ALL) and 181 cases of acute myeloid leukemia (AML) in childhood, seven patients were identified to have t(11;19) (q23;p13) by sequential cytogenetic analyses. The t(11;19) was associated with B-precursor ALL at diagnosis in three patients and at relapse in one patient. All four tested patients with B-precursor failed to express the CD10 antigen when the t(11;19) was detected, and one of three patients tested expressed myeloid-associated markers. In three other patients the translocation was detected either at lineage conversion from ALL to M5 AML (n = 2) or from AML to CD10- B-precursor ALL (n = 1). Leukemic blasts of four patients had an entirely different karyotype at the time of lineage conversion or loss of CD10 expression, suggesting an induction of a second neoplasm. Thus the t(11;19) can be found in de novo or secondary acute leukemia with lymphoid (CD10-) or myeloid (monoblastic) phenotype. Further investigation of the gene(s) involved in the 11q23 chromosomal region and the breakpoints in the 19p13 region is needed to understand the leukemogenesis of this apparently heterogeneous group of disorders.     

Immunobiological diversity in infant acute lymphoblastic leukemia is related to the occurrence and type of MLL gene rearrangement.

The aim of this study was to identify immunobiological subgroups in 133 infant acute lymphoblastic leukemia (ALL) cases as assessed by their immunophenotype, immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement pattern, and the presence of mixed lineage leukemia (MLL) rearrangements. About 70% of cases showed the pro-B-ALL immunophenotype, whereas the remaining cases were common ALL and pre-B-ALL. MLL translocations were found in 79% of infants, involving MLL-AF4 (41%), MLL-ENL (18%), MLL-AF9 (11%) or another MLL partner gene (10%). Detailed analysis of Ig/TCR rearrangement patterns revealed IGH, IGK and IGL rearrangements in 91, 21 and 13% of infants, respectively. Cross-lineage TCRD, TCRG and TCRB rearrangements were found in 46, 17 and 10% of cases, respectively. As compared to childhood precursor-B-ALL, Ig/TCR rearrangements in infant ALL were less frequent and more oligoclonal. MLL-AF4 and MLL-ENL-positive infants demonstrated immature rearrangements, whereas in MLL-AF9-positive leukemias more mature rearrangements predominated. The immature Ig/TCR pattern in infant ALL correlated with young age at diagnosis, CD10 negativity and predominantly with the presence and the type of MLL translocation. The high frequency of immature and oligoclonal Ig/TCR rearrangements is probably caused by early (prenatal) oncogenic transformation in immature B-lineage progenitor cells with germline Ig/TCR genes combined with a short latency period.     

Enhanced myeloid specificity of CD117 compared with CD13 and CD33

The c-kit proto-oncogene encodes a 145 kd tyrosine kinase transmembrane receptor, which plays a key role in haemopoiesis. The c-kit has been classified as CD117 and is especially useful in the differential diagnosis of acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). We analysed 104 consecutive cases (55 AML, 23 B-cell lineage ALL, three T-cell ALL, 11 blast crisis of chronic myeloproliferative disorders and 12 cases of myelodysplastic syndromes with more than 10% of blasts) referred to our Hospital for immunophenotypic diagnosis and compared the expression pattern of CD13, CD33 and CD117 using the same fluorochrome (phycoerythrin-PE). The recommendations of the EGIL group were followed in order to establish lineage involvement of the blastic population. The threshold used to assign positivity for CD117 was 10%. Bcr/abl, TEL/AML-1 and MLL rearrangements were assessed by molecular methods. CD117 expression was detected in 91% of AML and MDS. All the negative cases corresponded to acute monocytic leukemias. The calculated specificity for myeloid involvement was 0.86 for CD117, 0.36 for CD13 and 0.44 for CD33 (P < 0.005). CD117 was also positive in four cases of ALL. None of these cases showed bcr/abl or MLL rearrangements. In the light of these findings, CD117 expression should yield a higher score, at least one point, in the system currently applied for the diagnosis of biphenotypic acute leukemias (BAL) as its myeloid specificity is greater than that of CD13 and CD33. Moreover, its absence in AML could identify two subgroups of M5b cases. The coexpression of CD117 with cytoplasmic CD79a is often associated with CD7 reactivity, suggesting a stem cell disorder. CD117 should be included on a routine basis for the immunophenotypic diagnosis of acute leukemias.     

Characterization of Philadelphia-chromosome-positive acute leukemia by clinical, immunocytochemical, and gene analysis.

Philadelphia chromosome (Ph') was detected at presentation in 10 out of 110 patients with acute lymphoblastic leukemia (ALL) and five of 168 patients with acute myelogenous leukemia (AML). Two other ALL patients who had studies at relapse were also included in the analyses. One of the 12 Ph'-positive (Ph+) ALL patients had simultaneous expression of myeloid-associated antigen on the leukemic blasts, while all the five AML patients coexpressed markers of lymphoid cells. Double labeling of the cells with myeloperoxidase and CD10 on three Ph+ AML cases showed that most leukemic blasts expressed either myeloperoxidase activity or CD10 but not both. Cross-lineage gene rearrangements of T-cell receptor (TCR) beta-chain gene were detected in three of the eight Ph+ ALL patients tested. All the four Ph+ AML cases studied showed immunoglobulin heavy chain gene rearrangements, and three of them also had simultaneous rearrangements of TCR beta-chain gene. The results revealed that Ph+ acute leukemia in this study belonged either to ALL or mixed lineage leukemia, and none was pure AML. This finding is contrary to that of acute blast crisis of chronic myelogenous leukemia in which the majority of patients had myeloid transformation. Rearrangements of bcr were detected in four of the 10 Ph+ ALL and three of the four Ph+ AML patients tested. No significant difference was noted in the clinical or hematologic manifestations among Ph+ leukemia with or without bcr rearrangements.     

Maternal diet and infant leukemia: the DNA topoisomerase II inhibitor hypothesis: a report from the children's oncology group.

BACKGROUND: The MLL 11q23 translocation arises in utero and is present in 75% of infant leukemias. That MLL+ acute myeloid leukemia (AML) can arise following chemotherapy with DNA topoisomerase II (DNAt2) inhibitors suggests that these substances, which also occur naturally in foods, may contribute toward infant leukemia. We hypothesized that maternal consumption of dietary DNAt2 inhibitors during pregnancy would increase the risk of infant leukemia, particularly AML(MLL+). METHODS: This Children's Oncology Group case-control study consisted of 240 incident cases of infant acute leukemia [AML and acute lymphoblastic leukemia (ALL)] diagnosed during 1996 to 2002 and 255 random digit dialed controls. Maternal diet during pregnancy was determined through a food frequency questionnaire. An index of specific foods identified a priori to contain DNAt2 inhibitors as well as vegetables and fruits were created and analyzed using unconditional logistic regression. RESULTS: There was little evidence of an association between the specific DNAt2 index and leukemia overall and by subtype. An exception was AML(MLL+); odds ratios (95% confidence intervals) comparing the second to fourth quartiles to the first were 1.9 (0.5-7.0), 2.1 (0.6-7.7), and 3.2 (0.9-11.9), respectively (P for trend = 0.10). For the vegetable and fruit index, there were significant or near-significant inverse linear trends for all leukemias combined, ALL(MLL+), and AML(MLL-). CONCLUSION: Overall, maternal consumption of fresh vegetables and fruits during pregnancy was associated with a decreased risk of infant leukemia, particularly MLL+. However, for AML(MLL+) cases, maternal consumption of specific DNAt2 inhibitors seemed to increase risk. Although based on small numbers, these data provide some support for distinct etiologic pathways in infant leukemia.