Deficiency of Bruton's tyrosine kinase in B cell precursor leukemia cells

Bruton's tyrosine kinase (BTK) deficiency results in a differentiation block at the pre-B cell stage. Likewise, acute lymphoblastic leukemia cells are typically arrested at early stages of B cell development. We therefore investigated BTK function in B cell precursor leukemia cells carrying a BCR-ABL1, E2A-PBX1, MLL-AF4, TEL-AML1, or TEL-PDGFRB gene rearrangement. Although somatic mutations of the BTK gene are rare in B cell precursor leukemia cells, we identified kinase-deficient splice variants of BTK throughout all leukemia subtypes. Unlike infant leukemia cells carrying an MLL-AF4 gene rearrangement, where expression of full-length BTK was detectable in only four of eight primary cases, in leukemia cells harboring other fusion genes full-length BTK was typically coexpressed with kinase-deficient variants. As shown by overexpression experiments, kinase-deficient splice variants can act as a dominant-negative BTK in that they suppress BTK-dependent differentiation and pre-B cell receptor responsiveness of the leukemia cells. On the other hand, induced expression of full-length BTK rendered the leukemia cells particularly sensitive to apoptosis. Comparing BTK expression in surviving or preapoptotic leukemia cells after 10-Gy gamma radiation, we observed selective survival of leukemia cells that exhibit expression of dominant-negative BTK forms. These findings indicate that lack of BTK expression or expression of dominant-negative splice variants in B cell precursor leukemia cells can (i) inhibit differentiation beyond the pre-B cell stage and (ii) protect from radiation-induced apoptosis.     

Molecular diagnostics in the treatment of leukemia.

The molecular characterization of childhood leukemias directly affects our treatment strategies. Acute lymphoblastic leukemia patients with the TEL-AML1 fusion have a favorable prognosis, whereas those with the E2A-PBX1 fusion require more intensive therapy to obtain a good outcome. Acute lymphoblastic leukemia patients whose leukemic lymphoblasts contain the MLL-AF4 or the BCR-ABL fusion are often candidates for allogeneic hematopoietic stem cell transplantation during first remission. Among acute myeloid leukemia patients, AML1-ETO and CBFbeta-MYH11 fusions are associated with a favorable response, especially when the chemotherapy regimen includes high-dose cytarabine. Patients with acute promyelocytic leukemia who carry the PML-RAR alpha fusion respond to all-trans retinoic acid and have an excellent outcome after treatment with all-trans retinoic acid in combination with anthracyclines. Several novel therapeutic agents targeted to molecular lesions of leukemic cells are under investigation.     

Clinical implications of recurring chromosomal and associated molecular abnormalities in acute lymphoblastic leukemia

Comprehensive study of the major chromosomal/molecular abnormalities in children and adults with acute lymphoblastic leukemia (ALL) has demonstrated prognostic utility for many of these anomalies, to the extent that cytogenetic and molecular genetic evaluations are now required for optimal clinical management of newly diagnosed cases. For example, the t(12;21)/TEL-AML1 (ETV6-CBFA2) or hyperdiploid karyotypes each identifies subgroups of children who can be cured with well-tolerated chemotherapy based primarily on drugs with few long-term toxicities, such as L-asparaginase and antimetabolites. By contrast, the t(1;19)/E2A-PBX1 identifies a subtype of ALL that responds much better to more intensive regimens that rely on genotoxic drugs. At the extreme end of the risk spectrum, the t(4;11)/MLL-AF4 and t(9;22)/BCR-ABL almost always confer a dire prognosis in both children and adults with ALL, who warrant high-dose chemotherapy and hematopoietic stem cell rescue to sustain or even induce first remission. Such chromosomal/molecular markers are being incorporated into risk classification schemes, as they convey prognostic information that cannot be gleaned from conventional risk factors such as immunophenotype, presenting age, and the initial circulating leukemic blast cell count. The most exciting prospect is the discovery of drugs that inhibit specific oncogenes, as illustrated by the BCR-ABL tyrosine kinase inhibitor STI-571.     

T-cell receptor gamma and delta gene rearrangements and junctional region characteristics in south Indian patients with T-cell acute lymphoblastic leukemia

Clonal T-cell receptor (TCR) gamma and delta gene rearrangements were studied in 40 T-ALL cases (pediatrics, 29; adults, 11) using PCR with homo-heteroduplex analysis. At least one clonal TCRG or TCRD rearrangement was detected in 34 (85%) cases. TCR gamma (TCRG) rearrangement was detected in 25 (62.5%) cases that included 16 (55%) pediatrics and 9 (81.8%) adults. TCR delta (TCRD) rearrangement was detected in 14/40 (35%) cases, which included 12 (41%) pediatrics and 2 (18%) adults. The frequency of VgammaI-Jgamma1.3/2.3 was significantly more in adults than pediatrics (81.8% vs. 41.3%, P=0.02). In TCRD, Vdelta1-Jdelta1 was rearranged in 10 (25%) cases. The surface membrane CD3 positive cases are significantly associated with absence of TCRD rearrangements (surface membrane CD3+ TCRdelta- 84% vs. surface membrane CD3- TCRdelta- 48%, P value=0.03). Junctional region sequence analyzed with 10 cases each, of TCRG and TCRD, revealed an average junctional region of 7.4 nucleotides (range 2-18 nucleotides) in TCRG and 27 nucleotides (range 14-42 nucleotides) in TCRD-complete rearrangements. In TCRG, trimming at the ends of Vgamma and Jgamma germline nucleotides resulted in deletion, on an average of 9.2 nucleotides. In TCRD, deletion of nucleotides of the Vdelta and Jdelta gene segments on an average was 3.5 nucleotides. The junctional region of TCRD is more diverse than TCRG; nevertheless, the frequency of TCRG was more than that of TCRD and hence we rely more on TCRG clonal markers to quantitate the minimal residual disease in T-ALL.     

Unusual T-cell receptor-delta gene rearrangement patterns revealed by screening of a large series of childhood acute lymphoblastic leukaemia by multiplex polymerase chain reaction

Rearrangements of the T-cell receptor (TCR) and immunoglobulin genes are considered as useful clonal markers in lymphoproliferative disorders of B- and T-cell lineage, and are frequently used for the detection of minimal residual disease (MRD). In this paper, we report on the unexpected results of an extensive analysis of TCR-delta chain gene rearrangement frequencies and patterns in leukaemic bone marrow DNA samples collected from 438 children with initial (n = 112) or relapsed (n = 326) acute lymphoblastic leukaemia (ALL). By applying a previously described multiplex polymerase chain reaction, the overall incidence of non-deleted TCR-delta gene rearrangements in ALL was 47% (206/438), 52% in initial ALL (58/112) and 45% in relapsed ALL (148/326). As expected, the majority of B-cell precursor (BCP) ALL had incomplete Vdelta2-Ddelta3 or Ddelta2-Ddelta3 TCR-delta gene rearrangements, whereas most T-ALL showed complete rearrangements of the TCR-delta gene locus (Vdelta1-Jdelta1, Vdelta2-Jdelta1, Vdelta3-Jdelta1). However, unexpectedly, 5/206 rearranged TCR-delta alleles in BCP-ALL showed a complete Vdelta-(Ddelta)-Jdelta gene rearrangement pattern, and 3/31 T-ALL had an incomplete recombination. Theoretically, complete TCR-delta gene rearrangements should not occur in cells other than T-lymphocytes and have only been reported once previously in BCP-ALL. The data contribute to the discussion about the reliable screening for clonal markers in ALL.     

Immunoglobulin and T-cell receptor gene rearrangement and expression in human lymphoid leukemia cells at different stages of maturation.

The use of probes to genes (IG and TCRB) encoding immunoglobulins (IG) and the beta chain of the T-cell antigen receptor (TCRB), respectively, have become a sensitive means to assess clonality and lineage in lymphoid malignancies. It has become apparent that some individual cases show rearrangements of both IG and TCRB genes. In an attempt to more accurately define cell lineage we have analyzed cells from patients with B- or T-cell leukemia (n = 26) at various stages of maturation with probes to two additional TCR genes, TCRG and TCRA (encoding the TCR gamma and alpha chains, respectively), as well as the IG heavy chain joining region (IGHJ) and TCRB genes. On Southern blot analysis, the mature T-cell leukemia cells studied had rearranged TCRG and TCRB while IGHJ remained as in the germ line. The mature B-cell leukemia cells studied had rearranged IGHJ with germ-line TCRG and TCRB. These data suggest that, in the majority of more mature leukemias, cells have rearranged IG or TCR genes but not both. In contrast, cells from five of nine precursor B-cell leukemia patients and cell lines from one of four precursor T-cell leukemia patients had rearranged both IGHJ and TCR genes. TCRG and TCRB mRNAs were expressed in the cells of precursor T- but not B-cell leukemia patients studied. The spectrum of leukemia cells studied within the T-cell series permitted an assessment of the order of TCR gene rearrangements. Two of 13 patients had cells with germ-line TCRG and TCRB, 2 patients had cells with rearranged TCRG alone, and the remainder had cells with rearranged TCRG and TCRB. TCRG and TCRB mRNAs were expressed in precursor T-cell leukemia cells, whereas TCRB and TCRA were expressed in mature T-cell leukemia cells. These results parallel observations from mouse studies on gene expression and support the view of a hierarchy of TCR gene rearrangements in T-lymphocyte ontogeny. TCRG genes are rearranged first, subsequently TCRB genes are rearranged, followed by TCRA gene activation.     

A comprehensive genetic classification of adult acute lymphoblastic leukemia (ALL): analysis of the GIMEMA 0496 protocol

The Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) 0496 protocol, through the central handling of bone marrow samples at presentation, allowed us to combine cytogenetic and molecular information on a large series of adults with acute lymphoblastic leukemia (ALL) treated homogeneously, enabling us to define as broadly as possible their genetic profile and to determine the impact on outcome of the cytogenetic-molecular signature. Of 414 patients centrally processed, 325 were considered for the categorization into the following cytogenetic-molecular subgroups: normal, t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1;19)/E2A-PBX1, 9p/p15-p16 deletions, 6q deletions, miscellaneous structural abnormalities, and hyperdiploid. The inclusion into each subgroup was based on a hierarchical approach: molecular abnormalities with adverse prognosis had precedence over karyotypic changes with less-defined prognosis and the latter over ploidy. Patients without abnormalities and those with isolated 9p/p15-p16 deletions showed a relatively favorable outcome (median disease-free survival [DFS], > 3 years). The t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1; 19)/E2A-PBX1 defined a group with dismal prognosis (median DFS, 7 months), whereas 6q deletions, miscellaneous aberrations, and hyperdiploidy predicted an intermediate prognosis (median DFS, 19 months). This study highlights the importance of a combined cytogenetic-molecular profiling of adult ALL at presentation as a critical independent determinant of their outcome, providing further evidence of the necessity of a risk-adapted therapeutic algorithm for an optimal management of these patients.     

Prognostic significance of CD20 expression in childhood B-cell precursor acute lymphoblastic leukemia

CD20 expression is associated with inferior survival in adults with acute lymphoblastic leukemia (ALL). We analyzed the prognostic impact of CD20 expression in 353 children with B-cell precursor ALL treated in 3 consecutive St Jude Total Therapy studies. CD20 expression (> 20%) was found in 169 patients (48%) and was more frequent in patients between 1 and 10 years of age than in those younger than 1 or older than 10 years (P = .001). None of 14 patients with MLL-AF4 expressed CD20. There was no association between CD20 expression and E2A-PBX, TEL-AML1, ploidy, white blood cell count at diagnosis, or sex. In contrast to the experience in adult ALL, our patients with CD20 expression tended to have a better treatment outcome than those without the expression: 5-year event-free survival 84% +/- 2.9% versus 78% +/- 3.1% (P = .08). These data suggest that CD20 expression is not associated with inferior outcome in pediatric patients treated with contemporary regimens.     

Low frequency of clonotypic Ig and T-cell receptor gene rearrangements in t(4;11) infant acute lymphoblastic leukaemia and its implication for the detection of minimal residual disease

Infant t(4;11) acute lymphoblastic leukaemia (ALL) is a rare but cytogenetically well defined subgroup of immature B-cell precursor (BCP) ALL. To date, the configuration of their antigen receptor genes has not been studied in a large group of patients so far. In this study on 27 t(4;11) infant ALL, we have used standardized primer sets for the detection of all incomplete and complete immunoglobulin (Ig) heavy chain (IGH) rearrangements, as well as for the Ig light chain kappa (IGK), T-cell receptor delta (TCRD) and gamma (TCRG) rearrangements that are most common in childhood BCP ALL. Only 52% of cases displayed clonotypic antigen receptor gene rearrangements (IGH in 48%, IGK, TCRD and TCRG in 12%, 41% and 6% respectively). This low frequency suggests, together with the findings of predominantly incomplete DJh joins and monoallelic IGH rearrangements, that they are derived from an immature progenitor cell. As 48% of the t(4;11) infant ALL cases had no detectable antigen receptor gene rearrangements that could be used for minimal residual disease (MRD) analysis, we established an expression-independent, leukaemia-specific polymerase chain reaction (PCR) using the genomic sequence of the MLL-AF4 fusion genes. This method had high sensitivity and specificity and resulted in identical estimations of tumour loads when compared with IGH targets. Thus, genomic MLL-AF4 fusion genes are a good alternative target for the analysis of MRD in patients with t(4;11) leukaemias.     

A common Vδ2—Dδ2—Dδ3 T cell receptor gene rearrangement in precursor B acute lymphoblastic leukaemia

Despite their apparent commitment to the B lymphocytic lineage, human precursor B cell acute lymphoblastic leukaemias (ALL) frequently rearrange their T cell antigen receptor (TCR) alpha, beta and gamma chain genes. Since these three genes are active sites of rearrangement in precursor B cell neoplasms, it seemed that the recently discovered fourth TCR gene, delta, might be similarly rearranged. To investigate this possibility, a series of precursor B cell leukaemias was analysed for rearrangements at the delta chain gene locus, using probes of the variable, joining, and constant regions of the delta chain gene. The majority of precursor B cell ALLs in this series (25/32, 78%) showed rearrangement or deletion of one or more TCR delta genes. This contrasts sharply with a series of 16 mature B cell neoplasms (chronic lymphocytic leukaemia) in which no TCR delta gene rearrangements were detected. An unusual TCR delta rearrangement, rarely observed in normal or neoplastic T cells, was seen in the majority (14/18) of precursor B cell ALLs with TCR delta rearrangements. In contrast to the utilization ov V delta 1 in T cell ALL, detailed restriction mapping of precursor B ALL revealed an incomplete rearrangement without involvement of J delta segments. Direct genomic sequencing was performed on one example and demonstrated a nonproductive V delta 2-D delta 2-D delta 3 recombination in this precursor B ALL. We conclude that the TCR delta chain gene is an active locus in precursor B cell neoplasia, involves an unusual type of rearrangement and provides a clonal tumour marker for diagnosis of precursor B ALL.     

Classification of pediatric acute lymphoblastic leukemia by gene expression profiling

Contemporary treatment of pediatric acute lymphoblastic leukemia (ALL) requires the assignment of patients to specific risk groups. We have recently demonstrated that expression profiling of leukemic blasts can accurately identify the known prognostic subtypes of ALL, including T-cell lineage ALL (T-ALL), E2A-PBX1, TEL-AML1, MLL rearrangements, BCR-ABL, and hyperdiploid karyotypes with more than 50 chromosomes. As the next step toward developing this methodology into a frontline diagnostic tool, we have now analyzed leukemic blasts from 132 diagnostic samples using higher density oligonucleotide arrays that allow the interrogation of most of the identified genes in the human genome. Nearly 60% of the newly identified subtype discriminating genes are novel markers not identified in our previous study, and thus should provide new insights into the altered biology underlying these leukemias. Moreover, a proportion of the newly selected genes are highly ranked as class discriminators, and when incorporated into class-predicting algorithms resulted in an overall diagnostic accuracy of 97%. The performance of an array containing the identified discriminating genes should now be assessed in frontline clinical trials in order to determine the accuracy, practicality, and cost effectiveness of this methodology in the clinical setting.     

Gamma/delta lineage relationship within a consecutive series of human precursor T-cell neoplasms

We analyzed the gene rearrangements associated with the newly described delta T-cell receptor (TCR) gene from a series of 19 consecutive precursor T-cell (lymphoblastic) neoplasms that represent discrete stages surrounding the TCR gene rearrangement process. Significantly, the delta TCR gene showed rearrangement in most (13 of 19) of these T cells, and in addition it was rearranged in two cells displaying no rearrangement for any other TCR gene. Our survey showed three types of delta gene rearrangements associated with cell-surface TCR expression that presumably represent usage of three V delta genes. This analysis demonstrates (1) a major subclass of human precursor T-cell neoplasms belonging to the gamma/delta T-cell receptor-rearranging subtype; (2) a narrow repertoire of human V delta gene usage; and (3) the utility of delta gene rearrangements as a diagnostic clonal marker in precursor T lymphoblastic neoplasms.     

Analysis of clonal rearrangements of the Ig heavy chain locus in acute leukemia

Clonal rearrangements of the Ig heavy chain (IGH) locus occur in nearly all cases of B-cell precursor acute leukemia (BCP-ALL). Some of these rearrangements may be detected by polymerase chain reaction (PCR) using VH gene framework III (FRIII) and JH consensus primers. However, about 20% of BCP-ALLs fail to amplify with this technique. To determine the causes of these PCR failures and to investigate any possible association with specific subgroups of disease, we analyzed 72 acute leukemias of defined immunophenotype and cytogenetics, comparing FRIII with VH-family leader-specific PCR methods and Southern blotting. Of 37 BCP-ALL cases, 6 (16.2%) failed totally to amplify with FRIII and JH primers. None of these cases amplified with VH leader primers. Additionally, all cases retained germline VH6 genes and 5 of 11 rearranged alleles amplified with a consensus DH primer, indicating that these rearrangements represented biallelic DH-JH recombinations. Among the 6 FRIII and VH leader PCR-negative BCP-ALL cases, there was no common immunophenotype or consistent cytogenetic abnormality, although all showed structural chromosomal abnormalities and 3 of 5 successfully karyotyped had abnormalities of chromosome 12p. 13 cases with t(9;22)(q34;q11) Philadelphia chromosome-positive [Ph+]) and IGH rearrangements (9 BCP-ALL and 4 biphenotypic cases) were also analyzed. Of 23 rearranged IGH alleles, 19 (82%) were positive by FRIII PCR, and all 4 remaining alleles were amplified by VH leader primers. Use of the leader primers in these Ph+ cases also detected 3 additional clonal rearrangements that were not anticipated from Southern blotting; such unexpected bands were not observed in 21 other Ph- cases. The additional bands represented "new" and unrelated VH rearrangements rather than VH-VH replacement events. We conclude that biallelic DHJH rearrangements occur in a subgroup of BCP-ALL; in these cases, the activation of the full VHDHJH recombination mechanism had not occurred. Therefore, these cases of BCP-ALL were arrested at an early stage of B- cell differentiation. In contrast, all Ph+ BCP-ALLs and biphenotypic acute leukemias, which may represent the transformation of multipotent hemopoietic stem cells, had undergone VHDHJH recombination. Of 9 Ph+ BCP-ALL cases, 3 also showed ongoing VHDHJH rearrangement, reflecting the persistent expression of the VHDHJH recombinase. Finally, sequence analysis of 33 rearranged VHDHJH genes showed that only 3 including 2 Ph+ BCP-ALL maintained an intact open-reading frame. Loss of the open- reading frame occurred not only because of out-of-frame VHDH and DHJH joining, but also because of VH gene mutation and deletion. These data show that most BCP-ALLs may represent the neoplastic transformation of BCPs destined to die in the bone marrow.     

Modeling the evolution of ETV6-RUNX1-induced B-cell precursor acute lymphoblastic leukemia in mice

The t(12;21) translocation that generates the ETV6-RUNX1 (TEL-AML1) fusion gene, is the most common chromosomal rearrangement in childhood cancer and is exclusively associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The translocation arises in utero and is necessary but insufficient for the development of leukemia. Single-nucleotide polymorphism array analysis of ETV6-RUNX1 patient samples has identified multiple additional genetic alterations; however, the role of these lesions in leukemogenesis remains undetermined. Moreover, murine models of ETV6-RUNX1 ALL that faithfully recapitulate the human disease are lacking. To identify novel genes that cooperate with ETV6-RUNX1 in leukemogenesis, we generated a mouse model that uses the endogenous Etv6 locus to coexpress the Etv6-RUNX1 fusion and Sleeping Beauty transposase. An insertional mutagenesis screen was performed by intercrossing these mice with those carrying a Sleeping Beauty transposon array. In contrast to previous models, a substantial proportion (20%) of the offspring developed BCP-ALL. Isolation of the transposon insertion sites identified genes known to be associated with BCP-ALL, including Ebf1 and Epor, in addition to other novel candidates. This is the first mouse model of ETV6-RUNX1 to develop BCP-ALL and provides important insight into the cooperating genetic alterations in ETV6-RUNX1 leukemia.