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

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Abstract

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.

Introduction

Thirty years ago, the prognosis of children suffering from acute lymphoblastic leukemia (ALL) was poor and most of them survived only 3 or 4 months after diagnosis. Several years later, in the mid-1970s, the 5-year event-free survival (EFS) for older children approached nearly 50% while the cure rate for infants with ALL (diagnosed within the first 12 months of life) still remained extremely poor. At that time, the few survivors of infant ALL usually had substantial neurologic damage from cranial irradiation. Although the EFS of infants who were treated with current chemotherapy protocols has dramatically improved over the last years, their EFS is still much lower as compared to older children. Aside from children with ALL and BCR/ABL rearrangement, infants have the worst prognosis of all pediatric ALL patients with an EFS of about 35–50%.1,2

Infant ALL accounts for about 5% of all leukemic cases in childhood and is characterized by an initial high white blood cell count, bulky extramedullary disease, an immature immunophenotype and translocations involving the MLL gene at chromosome 11q23. The spectrum of recombination partners of 11q23 comprise more than 35 loci, with a predominance of a translocation t(4;11) and t(11;19).3 The detection of these 11q23 aberrations is clinically most important as their occurrence has frequently been associated with poor treatment outcome.4,5,6 The careful evaluation of all infants with ALL for the presence of a rearrangement within the 11q23/MLL gene is therefore highly desirable. Previous studies have shown that the expression of the human homologue of the rat 220 kDa chondroitin sulfate proteoglycan (NG2) is highly correlated with 11q23/MLL abnormality.7,8 As compared to the cytogenetic or molecular methods for detection of 11q23/MLL abnormalities, the determination of the chondroitin sulfate expression by flow cytometry may be an attractive alternative approach with regard to cost and speed. The latter analysis is facilitated by the monoclonal antibody (moab) 7.1 which does not react with normal hematopoietic precursor cells but specifically reacts with 11q23/MLL-rearranged leukemic cells.7 In a previous series, we evaluated the reactivity of moab 7.1 in a large series of adults and older children suffering from various leukemias.9 We extended our former analysis and report herein the immunophenotypic, cytogenetic and molecular data of 77 infants with ALL. Apart from determination of the overall frequency of MLL/11q23 aberrations using karyotyping, FISH, Southern blotting and RT-PCR, we addressed the question whether flow cytometry of infant ALL with moab 7.1 and a panel of additional antibodies may substitute for molecular testing of 11q23/MLL rearrangements. The infants were enrolled within three German multicenter therapy trials ALL-BFM90, ALL-BFM95 and CoALL-05–92. The prognostic impact of 11q23/MLL abnormalities and the immunophenoytpe for patients with an adequate follow-up of 4 years is also presented.

Materials and methods

Patient samples

Between January 1994 and April 2000, 77 bone marrow and/or peripheral blood samples from infants were sent in by mail to the Oncogenetic Laboratory of the Children's University Hospital, Giessen, Germany. Informed consent from the guardians for each patient was obtained. The diagnosis of a previously untreated ALL was based on standard morphological studies, and cytochemical staining of leukemic cells.10

Cytogenetic analysis

Unstimulated isolated bone marrow and peripheral blood cells were cultured for 24–48 h. Chromosomes were prepared and G-banded according to standard procedures. Karyotyping was done according to the ISCN-guidelines.11

Fluorescence in situ hybridization (FISH) probes

The MLL-containing YAC-clone, 13 HH4, was kindly provided by Brian Young, St Bartholomew's Hospital (London, UK);12 the PAC clones with the 3′-and 5′-parts of the MLL gene by Ed Schuuring (Department of Pathology, University of Leiden, Leiden, The Netherlands).13

Southern blotting and RT-PCR

The protocols used for Southern blotting, and RT-PCR as well as the primer sequences for PCR amplification of the MLL/AF4, MLL/AF9, or MLL/ENL fusion transcripts were previously reported.14,15,16

Immunophenotypical analysis

Leukemic cells from heparinized fresh bone marrow or peripheral blood samples were isolated by Ficoll–Hypaque (Pharmacia, Freiburg, Germany) density gradient centrifugation, and leukemia-associated antigens were detected by a panel of moabs either by a direct or an indirect immunofluorescence assay as previously described.17,18. Precursor B cell ALL cases were subclassified into pro-B-ALL (CD19+, CD10, CD20, cytoplasmatic IgM), common-ALL (CD19+, CD10+, cytoplasmatic IgM), and pre-B-ALL (CD19+, CD10+/−, cytoplasmatic IgM+) according to the EGIL guidelines.19 Cell samples were analyzed by flow cytometry (FACScan; Becton Dickinson, San Diego, CA, USA) using the Cell Quest software program (Becton Dickinson). Cell samples were considered positive for a specific antigen, if the antigen was expressed in at least 20% of the leukemic cells (‘20-cut-off-level’). Positivity for a specific intracytoplasmic antigen was assumed, if the antigen was expressed in at least 10% of the leukemic cells (‘10-cut-off-level’).19 Reactivity of monoclonal antibody (moab) 7.1, which was previously described to be a highly sensitive and specific marker for leukemic cells with MLL rearrangements (MLL-ra)20,21 was analyzed by indirect immunofluorescence using moab 7.1 (IgG1; Beckman Coulter, Marseille, France) as mentioned before.9 In selected experiments we also used the phycoerythrin-conjugated moab 7.1 (Beckman Coulter). Non-specific binding of Fcγ receptors was blocked by preincubation of the cells with a polyclonal rabbit serum (Gibco BRL, Paisley, UK). Non-viable cells were excluded from analysis by propidium iodide staining (Sigma, Deisenhofen, Germany). At least 10 000 cells per sample were acquired and analyzed by flow cytometry as described above.

Statistical analysis

Differences in the immunophenotypic expression patterns between cell samples with or without MLL-ra were analyzed using chi-square test (Pearson coefficient). P values <0.05 were considered significant. The sensitivity of distinct immunophenotypic markers (IM) in the detection of leukemia cells with MLL-ra was calculated as follows: {IM pos. MLL-ra pos. cases/(IM pos. MLL-ra pos. cases + IM neg. MLL-ra pos. cases)} × 100%. The specificity of distinct IM in the detection of leukemia cells with MLL-ra was calculated as follows: {IM neg. MLL-ra neg. cases/(IM neg. MLL-ra neg. cases + IM pos. MLL-ra neg. cases)} × 100%. The positive predictive value of distinct IM in the detection of leukemia cells with MLL-ra was calculated as follows: {IM pos. MLL-ra pos. cases/(IM pos. MLL-ra pos. cases + IM pos. MLL-ra neg. cases)} × 100%. EFS was estimated according to Kaplan–Meier.22 The starting point was the date of diagnosis and the end point was death during induction, relapse or death in continuous complete remission (CCR). Time was censored at last follow-up, if no failure was observed. Follow-up was updated in December 2000. The univariate comparison of the EFS of different groups of patients was performed by means of the two-sided log-rank test. All statistical analyses were done with the SPSS software program.

Results

The samples were primarily subjected to conventional cytogenetic analysis, immunophenotyping and RT-PCR for the presence of a MLL/AF4, MLL/AF9 or MLL/ENL fusion transcript. Fifty-seven and 32 samples were additionally tested by FISH and Southern blotting, respectively.

Frequency of 11q23/MLL abnormalities

In 47 out of the 77 samples (61%) a complete leukemic karyotype could be established. Among those, 31 (66%) had an abnormality of chromosome 11q23. The remaining 16 samples revealed hyperdiploidy (n = 3), an abnormality at chromosome 14q11 (n = 1), trisomy 8 (n = 1), random structural aberrations (n = 4) or a normal karyotype (n = 7). Table 1 summarizes the cytogenetic abnormalities among the successfully karyotyped samples.

Table 1 Cytogenetic results of all successfully karyotyped infants (n = 47)

All infants with cytogenetic aberration at 11q23 showed a rearrangement within the MLL gene, 30 of them had a breakpoint within the 8.3 kb so-called breakpoint cluster region (BCR) of MLL which encompasses exon 5 to 11 of the gene.23 One exceptional patient had a MLL breakpoint outside the MLL-BCR (Viehmann et al, manuscript in preparation). In all infants with cytogenetic evidence of a translocation t(4;11), t(9;11), or t(11;19) the corresponding fusion transcript could be amplified by RT-PCR. In contrast, three infants with normal cytogenetic analysis were tested positive for a MLL-rearrangement by RT-PCR, FISH or Southern blotting, respectively. Two of these infants subsequently relapsed and succumbed to their disease (Table 2). Combining FISH with both RT-PCR and Southern blotting we found nine additional infants with MLL rearrangement, in whom the cytogenetic analysis failed because of the lack of metaphases.

Table 2 Data of infants with normal karyotype

In summary, in our cohort of infants we identified 42 samples with a MLL abnormality, whereas 27 samples remained MLL negative. In eight samples (10%) the status of the MLL gene could not undoubtedly be resolved. This was caused by the poor quality of RNA or the low amount of DNA which prevented PCR analysis or Southern blotting, respectively.

Immunophenotypic features of leukemic cells according to the status of MLL

Immunophenotypic data for correlation with MLL status were available in 65 patients examined. Most of the MLL rearrangement-positive cases (as detected with at least one molecular biological method) revealed a CD10-negative pro-B-ALL immunophenotype (Table 3).

Table 3 Immunophenotypes of infant leukemia with MLL rearrangements

Within precursor B cell ALL, MLL rearrangement positive cases were significantly more often positive for moab 7.1, CD34 and CD65s, and significantly more often negative for surface CD20, CD22 and CD54 expression compared with MLL-ra negative cases (Table 4).

Table 4 Immunophenotypic features of MLL rearrangement positive precursor B cell ALL cases in infant leukemia

Moab 7.1 emerged as the most valuable immunophenotypic marker for the detection of leukemic cells with MLL rearrangements in infants. In 32 out of the 42 MLL-positive cases flow cytometric data of moab7.1 were available. The specificity and positive predictive value (PPV) of moab 7.1 reached 100%. However, in five infants with MLL/11q23 aberration the moab 7.1 reactivity remained negative. This results in 84% sensitivity only (Table 5). Positivity of the myeloid markers CD15 and/or CD65s also reached a 100% specificity for the detection of leukemic cells with 11q23/MLL rearrangement in this series. In cases which were examined for both, CD65s and moab 7.1 or CD15 and moab 7.1, these markers detected three of five moab 7.1-negative cases with MLL rearrangements. Other markers, despite rather high sensitivity values, were less valuable for the detection of MLL rearrangement-positive leukemic cells, mostly due to rather low specificities (Table 5).

Table 5 Sensitivity, positive predictive value, and specificity of immunophenotypic markers for MLL rearrangements in infant leukemia

The expression of neither moab 7.1 nor CD15/CD65s was specific for a MLL/AF4 or MLL/ENL fusion transcript, respectively.

Treatment outcome

We determined the EFS for those infants for whom a follow-up of at least 4 years was available. Both highly interrelated parameters, immunophenotype, and 11q23/MLL status, showed a strong prognostic impact within the therapy trials ALL-BFM90, 95 and CoALL-05–95 (Figure 1a and b). As expected, infants without an 11q23/MLL rearrangement or a pre-B/common-ALL did significantly better than those with 11q23/MLL rearrangement or pro-B ALL. Thirty of the 31 infants with 11q23 aberrations and completely banded karyotype had an adequate follow-up. In this group, the outcome of patients with secondary chromosomal changes was compared with those showing the 11q23 abnormality as sole aberration. Even if the difference in EFS was not statistically significant, we surprisingly recognized that among infants with additional aberrations the number of relapses seems to be lower than in those with 11q23 aberration alone (Figure 1c).

Figure 1
figure1

(a) Prognostic impact of the 11q23/MLL rearrangement, (b) EFS according to the immunophenotype, (c) EFS of infants who had the aberration 11q23 as sole abnormality as compared to children with 11q23 aberration and additional chromosomal abnormalities.

Discussion

The strong prognostic impact of the immunophenoytpe and 11q23/MLL rearrangement in infant ALL is already well known and was also confirmed in our series.24,25 If all immunophenotypes were included, 11q23/MLL rearrangements were found in 61% (n = 42) of them. With regard to the pro-B/CD10-negative ALL cases only, more than 90% showed an 11q23/MLL rearrangement. Because the data of CD10 expression are available in more than 95% of all cases, even in multicenter therapy trials, one might suggest that primarily the immunophenotype should pave the way for additional cyto- and molecular genetic assays of infant ALL cases. However, we detected three 11q23/MLL-positive cases which had either a common- or pre-B ALL. Thus, cytogenetic or molecular 11q23/MLL screening of the pro-B ALLs only would miss some rare rearranged cases. On the other hand, as routine cytogenetic and molecular screening procedures are expensive and time-consuming, especially in the context of multicenter or even multinational therapy trials, it would be helpful to have a flow cytometric marker which detects leukemic cells with MLL rearrangements with a high sensitivity and specificity. Moab 7.1, detecting the aberrant NG2 molecule expression, was reported to be a highly sensitive and specific marker for leukemic cells with MLL-ra in childhood ALL.8 Investigators from the St Jude Children's Research Hospital screened 104 consecutive children with ALL and found an NG2 expression in nine cases. All of them had a 11q23/MLL aberration whereas none of the 95 NG2-negative cases had such an abnormality. In their series, four of the 9 NG2-positive cases were infants. In our series of infant leukemias only, the specificity and positive predictive value of moab 7.1 was 100%. In spite of this high specificity it should be noted that the number of patients in the MLL-negative group was relatively small. The sensitivity was only 84%. However, three out of the five moab7.1-negative but MLL/11q23-positive cases could be identified by their co-expression of CD15 and CD65s. Thus, combining moAb 7.1 with the detection of these antigens emerged as a valuable additional immunophenotypic tool to detect leukemic cells with MLL rearrangements in nearly 95% of all infants. We, therefore, recommend that in infancy all moab 7.1-negative precursor B cell leukemias should be additionally investigated for these antigens. Future studies should closely investigate, whether the combination of several additional stem cell-related and myeloid-associated markers with moab 7.1 will lead to a characteristic immunophenotype with a 100% sensitivity for MLL rearrangement- positive precursor B cell ALL cases, in infant leukemia as well as in children and adults with ALL.

Using cytogenetics, we neither detected any of the rare translocation partners of chromosome 11q23 that mainly occur in acute myelogeneous leukemia (for an up-to date information see http://infobiogen.fr/services/chromcancer and3,26) nor patients with a deletion of 11q23. The latter subgroup do not have the same poor prognosis as patients with translocations involving this locus.27,28 This facilitates rapid RT-PCR screening procedures preferably by multiplexing MLL primers and various primers homologous to the respective MLL partner genes.14,15,29 Our intriguing observation that infants with 11q23 aberration and additional secondary cytogenetic abnormalities may have better treatment outcome as compared with infants who display the 11q23 aberration as sole abnormality, needs to be evaluated in larger studies.

In conclusion, all infants with ALL and aberrant expression of NG2 have a rearrangement of MLL/11q23. However, approximately 5% of 11q23/MLL-rearranged cases will be overlooked even if in addition to the expression of NG2, CD15 and CD65s are analyzed by flow cytometry. It remains enigmatic why in few 11q23/MLL-positive cases neither NG2 nor CD15, CD65s is expressed.

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Acknowledgements

This work was supported by grants from the ‘Mildred-Scheel-Stiftung for Cancer Research’ (10–1658-Bo 2), the ‘Monika Kutzner-Foundation’, and the ‘Forschungshilfe Station Peiper’.

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Correspondence to A Borkhardt.

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Keywords

  • infant ALL
  • 11q23/MLL rearrangement
  • prognosis
  • immunophenotype
  • moAb 7.1
  • flow cytometry

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