Immunoglobulin heavy chain gene rearrangements and mixed lineage characteristics in acute leukemias with the 11;19 translocation

Although the origin of acute leukemia with the 4;11 translocation has been shown to be an early myeloid progenitor cell or a stem cell with the potential for differentiation into both lymphoid and myeloid lineage, few precise studies on acute leukemia with the 11;19 translocation have thus far been reported. This study focused on the clinical, morphologic, ultrastructural, and immunologic characteristics as well as the DNA in three cases of acute leukemia with the 11;19 translocation. All three patients were infants and showed hyperleukocytosis. The morphologic feature was French-American-British (FAB)-L2 in two patients, in one of which a few monocytoid blasts were also seen by electron microscopy. Cells from the third patient underwent morphologic changes from FAB-L2 at the time of diagnosis to M5b at relapse. Immunologic marker studies revealed that the blast cells from all three patients expressed Ia and B4, but none expressed B1, CALLA(J5), T antigens, or SIg. Cells from one patient simultaneously expressed myeloid antigen (MCS-II) both at diagnosis and relapse. Cells from two patients expressed myeloid antigen after being cultured for a short time in vitro. An analysis of immunoglobulin genes and T-cell receptor genes revealed rearrangements of the heavy chain genes and germ line configurations of the kappa and lambda light chain genes, and of the T-cell receptor beta chain genes. These findings suggest that acute leukemia with the 11;19 translocation has mixed lineage characteristics as a result of leukemogenesis in a stem cell with the potential for both lymphoid and myeloid, especially monocytic, differentiation.     

急性白血病患者混合谱系白血病基因重排的检测

 【摘要】　目的　探讨荧光原位杂交（FISH）技术和多重巢式RT-PCR技术对急性白血病（AL）患者混合谱系白血病（MLL）基因重排检测的价值。方法　采用MLL双色FISH基因探针,应用间期FISH和多重巢式RT-PCR技术对189例AL患者进行检测,同时进行染色体R或G显带。结果　179例AL患者行FISH检测,其中9例（5.03 %）MLL基因重排阳性[无MLL-部分串联重复（PTD）],而经多重巢式RT-PCR检测的189例中16例（8.47 %）MLL基因重排阳性,包括MLL／AF9、MLL／AF10、MLL／AF6、MLL／AF17、MLL／ELL、MLL-PTD,189例患者同时进行染色体R或G显带,其中仅5例（2.65 %）涉及11q23的相互易位。急性淋巴细胞白血病（ALL）（73例）与急性髓系白血病（AML）（116例）中6种常见MLL基因重排的发生率差异均无统计学意义（均P＞0.05）。结论　多重巢式RT-PCR技术是对初诊AL患者进行MLL基因重排筛检的有效方法,不仅能证实常规细胞遗传学易位,还能检测出染色体核型分析和FISH技术均不能检出的 MLL-PTD,为预后判断和治疗方案的选择提供依据。     

Prognostic significance of immunoglobulin and T cell receptor gene rearrangements in patients with acute myeloid leukemia: Taiwan experience.

We investigated the prognostic significance of lymphoid antigen receptor gene rearrangement in patients with newly diagnosed acute myeloid leukemia (AML). Thirty-nine patients were included in the study. Clonal gene rearrangement of immunoglobulin heavy chain (IgH) and T cell receptor beta chain (TCRbeta) was found in leukemic cells in 11 (28.2%) and 10 (25.6%) patients, respectively. Five (12.8%) had both IgH and TCRbeta gene rearrangements. Three of the seven (42.9%) B-lymphoid marker-positive and eight of the 32 (25%) B-lymphoid marker-negative patients had clonal IgH gene rearrangements. Five of the 11 (45.5%) T-lymphoid marker-positive and 5 of the 28 (17.9%) T-lymphoid marker-negative patients had clonal TCRbeta gene rearrangements. All patients were treated with similar regimens. The complete remission rate (62.5% vs 65.2%, p=1.000) and median survival (13 vs 14 months, p=0.366) were similar in patients with and without clonal IgH or TCRbeta gene rearrangements. In conclusion, while clonal rearrangements of IgH or TCRbeta genes were found in AML patients, they did not appear to effect the prognosis.     

Incidence of lineage promiscuity in acute myeloblastic leukemia: diagnostic implications of immunoglobulin and T-cell receptor gene rearrangement analysis and immunological phenotyping

Sixty-nine blood or bone marrow samples from both children and adults with acute myeloblastic leukemia (AML) were investigated to elucidate the frequency of immunoglobulin (IG) and T-cell receptor (TCR)-gene rearrangements. Non-germline configuration for the IG heavy chain (h) gene was detected in the specimens of nine patients of various subtypes according to the French-American-British classification (FAB), including FAB M1, M2, M4 and M5. Rearrangement of the IG kappa chain (k) gene was present in one of these cases which simultaneously revealed a rearranged TCR-beta (b) chain gene. In another two AML samples we found TCR-b gene rearrangements, in one case in combination with an IG-h gene rearrangement. IG-h gene rearrangements were detected in 10 cases, in one case in conjunction with an IG-kappa (k) and TCR-b gene rearrangement. A highly significant correlation between the occurrence of DNA rearrangements of the IG-h locus and nuclear staining with the enzyme terminal deoxynucleotidyl transferase (TdT) and surface expression of the CD 19 and CD 34 antigen could be identified: all 10 TdT positive AML samples rearranged IG-h. Similarly, six out of 69 AML samples exhibited surface expression of CD 19, five of these in combination with CD 34 and all of them rearranged the IG-h gene. The one leukemia with TCR-b gene rearrangement only was TdT positive as well, but did not express CD 19 or CD 34. We conclude that IG-h gene is rearranged in a substantial proportion of AML, strongly associated with a specific immunophenotype (TdT+, CD19+, CD34+), whereas TCR-b gene rearrangement appears more rarely. No positive correlation between occurrence of IG-h and TCR-b gene-rearrangements and one AML FAB-subtype was found, although a clustering of M1 and M4 FAB subtypes in the AML group showing reconstructed IG-h gene became evident.     

The incidence of T-cell receptor gene rearrangements in childhood B-lineage acute lymphoblastic leukemia is related to immunophenotype and fusion oncogene expression

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement is conventionally used for assessment of lymphoid malignant cells. TCR genes rearrangements were reported to occur at high frequency in B-lineage acute lymphoblastic leukemia (ALL). Therefore, we have analyzed 83 children with acute B-lineage ALL (67 de novo patients and 19 relapses) by PCR analysis for clonal IgH, incomplete TCRD (Vdelta2-Ddelta3 and Ddelta2-Ddelta3) and TCRG rearrangements. It was shown that clonal cross-lineage TCR rearrangements were associated with more immature immunophenotype (CD34+, CD117+, CyIgM-) of leukemic cells from patients' bone marrow (BM) samples as compared to cell samples without cross-lineage TCR rearrangements. That was equally detected both in de novo and relapsed cases of disease. Low frequency of clonal TCRG rearrangements was associated with expression of E2A/PBX chimeric oncogene. We suggest that TCRG and TCRD clonal rearrangements in leukemic B-cells are associated with early stages of their differentiation.     

Lack of correlation between immunoglobulin and T cell receptor gene rearrangements and TdT expression in acute myeloid leukaemia.

Thirty-two cases of acute myeloid leukaemia (AML) were examined for expression of terminal deoxynucleotidyl transferase (TdT) and rearrangements of the genes coding for the immunoglobulin heavy chain and the beta chain of the T cell receptor, in order to establish whether these two forms of lineage infidelity are linked. In 17 cases of AML with greater than or equal to 10% TdT+ cells, three cases showed evidence of gene rearrangement, two having clonal rearrangements in the immunoglobulin gene and one with a rearranged T cell receptor gene. Among 15 AML cases without significant numbers of TdT-positive blasts, three cases had rearrangements in both immunoglobulin and T cell receptor genes, while a fourth case had an immunoglobulin gene rearrangement. No relationship was seen between lymphoid gene rearrangements and expression of the lymphoid surface antigens CD7 and CD10. The lack of association between TdT expression and gene rearrangements does not support the concept of an orderly activation of the recombinase machinery in those cases of AML with features of early lymphoid differentiation.     

Rearrangement of T-cell receptor and immunoglobulin heavy chain genes in childhood acute mixed lineage leukaemia

Rearrangement of the beta and gamma chain genes of the TCR gene complex and of the Ig heavy chain genes were examined in three cases of childhood acute mixed lineage leukaemia. Blast cells, classified morphologically as acute lymphoblastic leukaemia (ALL) in one child and acute non-lymphocytic leukaemia (ANLL) in the other two, all co-expressed markers associated with both T (CD7, TdT) and myeloid (CD33) cells. Cytogenetic analysis detected abnormalities associated with myeloid leukaemia. Immunoglobulin heavy chain genes were not rearranged in two patients but a novel rearrangement was seen in the third. No rearrangement of the beta or gamma chains of the T-cell receptor complex were seen. Acute mixed lineage leukaemia may thus arise from a pluripotent precursor cell capable of both lymphoid and myeloid differentiation.     

T-cell receptor gamma and delta gene rearrangements in T-cell acute lymphoblastic leukemia in Indian patients

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal lymphoid malignancy and junctional sequences of rearranged T-cell receptor (TCR) represent the best suitable marker to study clonality in these patients. A sensitive, non-radioactive, and rapid approach of PCR coupled with heteroduplex analysis was used to analyse clonality of TCR gamma and delta gene rearrangements in 26 Indian T-ALL patients. Amongst TCR gamma gene family, VgammaI-Jgamma1.3/2.3 sequences were most utilized (53.9%) while from TCRdelta repertoire Vdelta1-Jdelta1 sequences were preferentially rearranged (23.1%) in these patients. 19.2% of Indian T-ALL patients demonstrated both clonal TCR gamma and delta gene rearrangements along with surface expression of TCRgammadelta. Although the majority of T-ALL patients showed surface expression of TCRalphabeta, the small fraction (19.2%) of TCRgammadelta+ T-ALL represent a distinct subgroup which needs further evaluation.     

Phenotypic and genotypic lineage switch of a lymphoma with shared chromosome translocation and T-cell receptor gamma gene rearrangement.

A case of non-Hodgkin's lymphoma showed a phenotypic and genotypic cell lineage switch twice during nine years of his clinical history; first, T-cell type, pleomorphic small cell lymphoma developed, followed by B-cell type, diffuse centroblastic/centrocytic lymphoma, and finally T-zone lymphoma without follicles again developed, from which AST-1 cultured cell line was established. Karyotype analysis demonstrated a shared abnormal chromosome, der(1)t(1;?)(p36;?), among the first relapsed B-cell tumor, the second relapsed T-cell tumor and AST-1 cell line. Furthermore, T-cell receptor (TCR) gamma gene rearrangement bands of the same size were observed in the first relapsed B-cell tumor and the second relapsed T-cell tumor as well as AST-1 cell line. These results suggested that both relapsed tumors of different cell lineages are derived from a common malignant clone, presumably a committed lymphoid stem cell. A unique translocation, t(2;14)(q37;q11.2), which may involve TCR delta/alpha gene complex, was observed in the second relapsed tumor and AST-1 cells. To attempt to isolate the breakpoint of this translocation, the configuration of TCR delta/alpha gene complex was studied. The result showed that two rearrangements of TCR alpha gene detected with J alpha probes were the products of the normal TCR rearrangement process, and were not involved in the translocation at this region. This patient, together with the AST-1 cell line, provided us a unique opportunity to study the development and clonal evolution of malignant lymphoma.     

BCR translocation to derivative chromosome 2: a new case of chronic myeloid leukemia with a complex variant translocation and Philadelphia chromosome

The well-known typical fusion gene BCR/ABL is observed in connection with a complex translocation event in 5-8% of cases of chronic myeloid leukemia (CML). The present study described an exceptional CML case with complex chromosomal aberrations not previously observed. Aberrations included a translocated BCR to the derivative chromosome 2 [der(2)] that also involved a four-chromosome translocation, implying chromosomal regions 1p32 and 2q21, besides 9q34 and 22q11.2, which were characterized by molecular cytogenetics.     

NPM1基因突变的急性髓系白血病免疫表型研究

 【摘要】 目的 研究伴有NPM1基因突变的急性髓系白血病(AML)患者的免疫表型特征。方法 采用流式细胞术检测237例初诊AML患者的免疫表型,并采用实时定量PCR方法对NPM1基因突变进行筛查,比较NPM1突变型和野生型患者的免疫表型差异。结果 NPM1突变型患者占所有AML患者的19.0 %（45／237）,与野生型相比其CD34、CD117、HLA-DR、CD15、CD19表达降低（均P＜0.05）。在正常核型AML患者中,NPM1突变率37.7 %（40／106）,突变型患者CD34、HLA-DR、CD15和CD7表达降低（均P＜0.05）。伴NPM1突变的正常核型AML患者的免疫表型在M1中表现为CD34、HLA-DR、CD7表达降低（均P＜0.05）,在M2中为HLA-DR表达降低和CD9表达升高（均P＜0.05）,在M5中为CD117表达降低（P＜0.05）。结论 NPM1突变能对AML患者的免疫表型特征造成影响,不同FAB分型的AML患者免疫表型改变存在差异。     

Pattern of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements in childhood acute lymphoblastic leukemia in India

In 120 cases of acute lymphoblastic leukemia (median age 8 years), IgH chain gene was rearranged in 99% B-Cell Precursor (BCP) ALLs and 13% T-ALLs. One or the other TCR locus was rearranged not only in all T-ALLs, but also in 87% of BCP-ALLs. TCR-beta rearrangement in BCP-ALL was associated with a higher mean age at presentation (8.7 vs. 6.2 years, P=0.008), lower mean platelet counts (61.2x10(9)/l vs. 103.7x10(9)/l, P=0.003) and a poorer DFS (% cummulative survival 0 vs. 88.9+/-10.5, P=0.004). TCR-gamma rearrangement in T-ALL was associated with a higher mean WBC count (186.3x10(9)/l vs. 63. 4x10(9)/l, P=0.002). Also, the pattern of rearrangement of these genes appeared to be different from the West; viz. TCR-beta rearrangement in a higher proportion of BCP-ALLs (58%, 95% confidence intervals 45-69%), invariable deletion of Cgamma1 and only monoallelic rearrangement for TCR-delta locus. This repertoire of gene rearrangement may have a bearing on the poor treatment outcome reported previously from our geographic region.     

Methylation status of T-cell receptor beta-chain gene in B precursor acute lymphoblastic leukemia: correlation with hypomethylation and gene rearrangement.

Epigenetic changes may play a role in genetic alterations in cancer cells, but little is known about this phenomenon. In this study we examined the correlation between rearrangement and methylation status of the T-cell receptor (TCR) beta-chain gene in 23 patients with B precursor acute lymphoblastic leukemia (ALL). In B precursor ALL, all patients had a CCmeGG sequence in the C beta 2 region, a pattern similar to that observed in normal mature B-cells. Approximately 55% of patients with B precursor ALL exhibiting a hypomethylated CCGG sequence at the J beta 1 region showed rearrangement of this region. Furthermore, the same allele of rearranged J beta 1 always contained an unmethylated sequence in the region, although another allele without rearrangement contained methylated J beta 1. By contrast, none of the patients had a rearrangement in the J beta 1 region without hypomethylation. Therefore, rearrangement of the J beta 1 region may link to the hypomethylation status of this region. A close association between hypomethylation of the J beta 1 region may promote accessibility to a putative common recombinase to produce TCR J beta 1 rearrangement. In contrast, about 45% of patients with a hypomethylated J beta 1 did not show rearrangement in this region, thus allowing categorization of B precursor ALL patients into subtypes, according to the combination of TCR beta-chain gene rearrangement and hypomethylation status, especially in the J beta 1 region.     

Immunoglobulin and T-cell receptor gene rearrangements in acute lymphoblastic leukemia--a higher incidence of double rearrangements in patients with myeloid antigen expression.

Among 160 patients who were diagnosed as having acute lymphoblastic leukemia (ALL) by French American British (FAB) criteria, 32 patients (20%) expressed myeloid-associated antigens on leukemic blasts (My+ALL). Correlation of immunophenotype with rearrangement of immunoglobulin (Ig) heavy chain and T-cell receptor (TCR) beta chain genes was performed on 73 of these patients (21 were My+ALL). Rearrangements of both Ig and TCR genes (double rearrangements) were detected in 24 patients, including three (19%) of 16 T-lineage ALL. 17 (33%) of 52 B-lineage ALL, and four of five ALL expressing both B and T-cell surface markers. Also a higher incidence of double rearrangements in My+ALL was found as compared with My-ALL (43% vs 29%). This difference was more evident when only B-lineage ALL was considered (50% in My+ patients vs 24% in My- patients). However the difference is not statistically significant yet possibly due to the small number of patients in the study. Further studies on more patients are needed to confirm this. In My-B-lineage ALL, rearrangements of TCR beta chain gene were restricted to certain subgroups (Groups III & IV) of patients who expressed CD10 surface antigens but lacked cytoplasmic Ig. In My+ B-lineage ALL, rearrangements of TCR beta chain gene could be found in various subgroups studied (Groups II through V). Cross-lineage gene rearrangement in My+ALL may involve mechanisms different from those in My-ALL.     

The pattern of clonal immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements in Chinese adult acute lymphoblastic leukemia patients

We have studied forty Chinese adult ALL patients at newly diagnosis, using standard primers and protocols of BIOMED-2 multiplex PCR, to determine the feasibility of Ig and TCR gene rearrangements as diagnostic and patient-specific MRD-RQ-PCR targets for molecular monitoring. Clonal IGH, IGK, IGL, TCRB, TCRG and TCRD rearrangements were found in 86%, 22%, 9%, 19%, 77%, 55% of adult patients with B-lineage ALL, respectively. While in T-ALL, clonal IGH, TCRB, TCRG and TCRD rearrangements were detected in 6%, 83%, 78%, 33% of patients. Several specialties in the pattern of Ig/TCR gene rearrangements in Chinese adult ALL patients in comparison with those reported for children and adult patients in other countries have been noted. These results are useful for further MRD-RQ-PCR detection and quantification for all patients.     

Detection of acute leukemia cells with mixed lineage leukemia (MLL) gene rearrangements by flow cytometry using monoclonal antibody 7.1.

Translocations involving 11q23 are among the most common genetic abnormalities in hematologic malignancies, occurring in approximately 5-10% of acute lymphoblastic leukemia (ALL) and 5% of acute myeloblastic leukemia (AML). In 11q23 translocations, the mixed lineage leukemia (MLL) gene on chromosome 11, band q23, is usually disrupted. The human homologue of the rat NG2 chondroitin sulfate proteoglycan molecule, as detected by the monoclonal antibody (moab) 7.1, was shown to be expressed on leukemic cells with MLL rearrangements of children with acute leukemia. We further investigated the reactivity of the moab 7.1 on 533 cell samples of adults (n = 215) and children (n = 318) with acute leukemias (271 AML, 217 B-lineage ALL, 37 T-lineage ALL, eight CD7+ CD56+ myeloid/natural killer cell precursor acute leukemias) by flow cytometry. In AML, 38 samples were positive for moab 7.1 ('20%-cut-off-level'). These moab 7.1-positive AML cases revealed a myelomonocytic-differentiated immunophenotype with coexpression of the NK cell marker CD56 in 33 of 38 cases. Two of eight cell samples of the recently described CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia entity reacted with moab 7.1. In ALL, 35 samples mostly of the pro-B-ALL subtype (33 pro-B-ALL, one common-ALL, one pre-B-ALL) were positive for moab 7.1. 58 (81%) of 72 samples with MLL rearrangements were positive for moab 7.1 including 28/31 with a t(4;11), 16/17 with a t(9;11), 3/5 with a t(11;19), and 2/6 with a del(11)(q23). All moab 7.1-positive ALL (n = 34) and childhood AML (n = 17) cases revealed MLL rearrangements as detected by Southern blot analysis and RT-PCR. However, 11 adults with AML, and one adult with moab 7.1-positive CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia were negative for MLL rearrangements as proved by Southern blot analysis. We conclude that moab 7.1 is a sensitive but not entirely specific marker for the identification of 11q23-associated AML and ALL by flow cytometry in children and adults.     

Characterization of acute lymphoblastic leukemia of childhood by immunoglobulin and T-cell receptor gene patterns.

Molecular biological studies of immunoglobulin (Ig) and T-cell antigen receptor (TCR) genes provide novel approaches to the identification and characterization of the acute lymphoblastic leukemias (ALL). Such studies greatly enhance our understanding of both the cells of origin in these diseases and the order of assembly of immune receptor genes in B-cells and T-cells. The patterns of Ig and TCR genes in B-cell precursor and T-cell ALL of childhood and ALL of infancy are heterogeneous though generally distinctive. The vast majority of cases of B-cell precursor ALL of childhood rearrange Ig heavy (H) chain genes, and 40-50% rearrange Ig light (L) chain genes. In contrast, in ALL of infancy, Ig genes are frequently germline, indicating generally less mature cells of origin in younger patients. Similarly, the vast majority of cases of T-cell ALL of childhood rearrange TCR delta, gamma, and beta genes and approximately one-half rearrange TCR alpha. TCR gene rearrangements are very common in cases of B-cell precursor ALL, but in patterns different from T-cells. In contrast, T ALL cells only infrequently rearrange Ig genes, and TCR gamma rearrangements are not found in ALL of infancy. The demonstration of lineage non-restricted Ig and TCR gene rearrangements raises questions about lymphocyte development and about the 'precursor' nature of ALL. The identification of generally distinctive patterns of these genes creates a foundation for their utilization as markers of minimal and preclinical disease. The extent to which specific immune receptor gene patterns correlate with clinical outcome in ALL warrants further study.