The complex translocation (9;14;14) involving IGH and CEBPE genes suggests a new subgroup in B-lineage acute lymphoblastic leukemia

Many subtypes of acute lymphoblastic leukemia (ALL) are associated with specific chromosomal rearrangements. The complex translocation t(9;14;14), a variant of the translocation (14;14)(q11;q32), is a rare but recurrent chromosomal abnormality involving the immunoglobulin heavy-chain (IGH) and CCAAT enhancer-binding protein (CEBPE) genes in B-lineage ALL (B-ALL) and may represent a new B-ALL subgroup. We report here the case of a 5-year-old girl with B-ALL, positive for CD19, CD38 and HLA-DR. A direct technique and G-banding were used for chromosomal analysis and fluorescentin situ hybridization (FISH) with BAC probes was used to investigate a possible rearrangement of the IGH andCEBPE genes. The karyotype exhibit the chromosomal aberration 46,XX,del(9)(p21),t(14;14)(q11;q32). FISH with dual-color break-apartIGH-specific and CEPBE-specific bacterial artificial chromosome (BAC) probes showed a complex t(9;14;14) associated with a deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) and paired box gene 5 (PAX5) at 9p21-13 and duplication of the fusion gene IGH-CEBPE.     

Inverted duplication of JH associated with chromosome 14 translocation and T-cell leukemia in ataxia-telangiectasia.

A specific 14q32 breakpoint is observed in a homologous chromosome 14 translocation [t(14;14)q12q32] occurring in the T-cells of about 10% of patients with ataxia-telangiectasia (AT). To investigate whether the 14q32 breakpoint in AT occurs within the immunoglobulin gene cluster as is frequently detected in B-cell lymphoma, immunoglobulin clones were hybridized to Southern blots of DNA isolated from the T-cells of two AT patients with this chromosome 14 translocation. The 14q32 translocation breakpoints in these patients are apparently not within JH, S mu, C mu, S alpha-1 or -2, or C alpha-1 or -2, but one of the patients has an inverted duplication of at least 26 kilobases (kb) of the C mu region, with an associated 5' flanking deletion. The point of origin of the inverted duplication is within JH near the recombination signal for the J4 gene. This suggests that normal JH recombination mechanisms may have played a role in the development of this 14q32 chromosomal aberration. The presence of AT chromosomal breakpoints near other rearranging genes suggests a role for exaggerated recombination in the pathogenesis of chromosomal instability in AT.     

Frequent chromosome aberrations revealed by molecular cytogenetic studies in patients with aniridia

Seventy-seven patients with aniridia, referred for cytogenetic analysis predominantly to assess Wilms tumor risk, were studied by fluorescence in situ hybridization (FISH), through use of a panel of cosmids encompassing the aniridia-associated PAX6 gene, the Wilms tumor predisposition gene WT1, and flanking markers, in distal chromosome 11p13. Thirty patients were found to be chromosomally abnormal. Cytogenetically visible interstitial deletions involving 11p13 were found in 13 patients, 11 of which included WT1. A further 13 patients had cryptic deletions detectable only by FISH, 3 of which included WT1. Six of these, with deletions <500 kb, share a similar proximal breakpoint within a cosmid containing the last 10 exons of PAX6 and part of the neighboring gene, ELP4. Two of these six patients were mosaic for the deletion. The remaining four had chromosomal rearrangements: an unbalanced translocation, t(11;13), with a deletion including the WAGR (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation) region, and three balanced rearrangements with what appear to be position effect breakpoints 3' of PAX6: (a) a t(7;11) with the 11p13 breakpoint approximately 30 kb downstream of PAX6, (b) a dir ins(12;11) with a breakpoint >50 kb from PAX6, and (c) an inv(11)(p13q13) with a breakpoint >75 kb downstream of PAX6. The proportion and spectrum of chromosome anomalies in familial (4/14, or 28.5%) and sporadic (26/63, or 41%) cases are not significantly different. An unexpectedly high frequency of chromosomal rearrangements is associated with both sporadic and familial aniridia in this cohort.     

Molecular involvement of the pvt-1 locus in a gamma/delta T-cell leukemia bearing a variant t(8;14)(q24;q11) translocation.

A highly malignant human T-cell receptor (TCR) gamma/delta+ T-cell leukemia was shown to have a productive rearrangement of the TCR delta locus on one chromosome 14 and a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on the other chromosome 14. Chromosome walking coupled with pulsed-field gel electrophoretic (PFGE) analysis determined that the TCR J delta 1 gene fragment of the involved chromosome was relocated approximately 280 kb downstream of the c-myc proto-oncogene locus found on chromosome band 8q24. This rearrangement was reminiscent of the Burkitt's lymphoma variants that translocate to a region identified as the pvt-1 locus. Sequence comparison of the breakpoint junctions of interchromosomal rearrangements in T-cell leukemias involving the TCR delta-chain locus revealed novel signal-like sequence motifs, GCAGA(A/T)C and CCCA(C/G)GAC. These sequences were found on chromosome 8 at the 5' flanking site of the breakpoint junction of chromosome 8 in the TCR gamma/delta leukemic cells reported here and also on chromosome 1 in T-cell acute lymphocytic leukemia patients carrying the t(1;14)(p32;q11) rearrangement. These results suggest that (i) during early stages of gamma delta T-cell ontogeny, the region 280 kb 3' of the c-myc proto-oncogene on chromosome 8 is fragile and accessible to the lymphoid recombination machinery and (ii) rearrangements to both 8q24 and 1p32 may be governed by novel sequence motifs and be subject to common enzymatic mechanisms.     

Growth of large chromosomally abnormal T cell clones in ataxia telangiectasia patients is associated with translocation at 14q11

Summary Human T cell malignancies often show chromosome breaks at 14q11, within the chain locus of the human T cell antigen receptor, with translocation of the distal portion of 14 to one of several sites. In patients with ataxia telangiectasia (A-T) the majority of T cell chromosome translocations associated with this disorder appear to occur at the sites of the T cell antigen receptor genes 7p14, 7q35, and 14q11 and may result in clone formation. In three large proliferating A-T T cell clones we have observed (including one which became malignant) and in most T cell tumours reported, the clonal chromosome exchange involves one breakpoint at 14q11 with the second breakpoint occurring in a gene not involved in the immunoglobulin supergene family. Our observations on A-T patients confirm the suggestion that chromosome exchanges involving either t(7;14)(p14;q11), t(7;14)(q35;q11), inv(7) (p14q35), or t(7;7)(p14;q35) confer only a small proliferative advantage on T cells in vivo without the capacity for malignant transformation and that the potential for malignant change is not a feature of all these rearrangements, but is restricted to cells or clones with other chromosome exchanges.     

Molecular cytogenetic aberrations in Tunisian patients with multiple myeloma identified by cIg-FISH in fixed bone marrow cells

Cytogenetic studies in multiple myeloma (MM) are hampered by the hypo-proliferative nature of plasma cells. In order to circumvent this problem, we have used a combination of immunolabeling of cytoplasmic Ig light chains (λ or κ) and FISH (cIg-FISH), which allowed a comprehensive detection of the most common and/or recurrent molecular cytogenetic aberrations on fixed bone marrow cells of 70 Tunisian patients. Translocations involving the chromosome 14q32 region were observed in 32 cases (45.7%), including 18 cases with a t(11;14), 8 cases with a t(4;14), and 2 cases with a t(14;16). Deletions of the 13q14 region (D13S319/RB1) were detected in 18.6%, and deletions of the 17p13 region (TP53) in 5.7% of the cases, respectively. Of all patients with a D13S319/RB1 deletion, 61.5% also carried a 14q32 translocation, whereas TP53 deletions were associated with a t(11;14) in 2 cases (50%) and a D13S319 deletion in 1 case (25%). Our results suggest that there is a correlation between the presence of 14q32 translocations and chromosome 13q14 deletions in MM patients and that cIg-FISH is more sensitive as compared to conventional karyotyping in detecting molecular cytogenetic abnormalities in this disease.     

Molecular cytogenetic analysis of follicular lymphoma (FL) provides detailed characterization of chromosomal instability associated with the t(14;18)(q32;q21) positive and negative subsets and histologic progression

We analyzed a cohort of 61 follicular lymphomas (FL) with an abnormal G-banded karyotype by spectral karyotyping (SKY) to better define the chromosome instability associated with the t(14;18)(q32;q21) positive and negative subsets of FL and histologic grade. In more than 70% of the patients, SKY provided additional cytogenetic information and up to 40% of the structural abnormalities were revised. The six most frequent breakpoints in both SKY and G-banding analyses were 14q32, 18q21, 3q27, 1q11-q21, 6q11-q15 and 1p36 (15-77%). SKY detected nine additional sites (1p11-p13, 2p11-p13, 6q21, 8q24, 6q21, 9p13, 10q22-q24, 12q11-q13 and 17q11-q21) at an incidence of >10%. In addition to the known recurring translocations, t(14;18)(q32;q21) [70%], t(3;14)(q27;q32) [10%], t(1;14)(q21;q32) [5%] and t(8;14)(q24;q32) [2%] and their variants, 125 non-IG gene translocations were identified of which four were recurrent within this series. In contrast to G-banding analysis, SKY revealed a greater degree of karyotypic instability in the t(14;18) (q32;q21) negative subset compared to the t(14;18)(q32;q21) positive subset. Translocations of 3q27 and gains of chromosome 1 were significantly more frequent in the former subset. SKY also allowed a better definition of chromosomal imbalances, thus 37% of the deletions detected by G-banding were shown to be unbalanced translocations leading to gain of genetic material. The majority of recurring (>10%) imbalances were detected at a greater (2-3 fold) incidence by SKY and several regions were narrowed down, notably at gain 2p13-p21, 2q11-q21, 2q31-q37, 12q12-q15, 17q21-q25 and 18q21. Chromosomal abnormalities among the different histologic grades were consistent with an evolution from low to high grade disease and breaks at 6q11-q15 and 8q24 and gain of 7/7q and 8/8q associated significantly with histologic progression. This study also indicates that in addition to gains and losses, non-IG gene translocations involving 1p11-p13, 1p36, 1q11-q21, 8q24, 9p13, and 17q11-q21 play an important role in the histologic progression of FL with t(14;18)(q32;q21) and t(3q27).     

Molecular characterization of a ring chromosome 14 showing that the PI locus is centromeric to the D14S1 and IGH loci

Summary Molecular characterization of a ring chromosome 14 was carried out in a patient with the 46,XX,r(14) karyotype. The breakpoints shown by chromosome banding were within bands p11 and q32. Using molecular probes for the immunoglobulin heavy chain (IGH), D14S1 and PI loci located at 14q32, we showed that the IGH and D14S1 loci, located at 14q32.2 and 14q32.2, respectively, were deleted on the ring chromosome 14, but that the PI locus was not. Therefore, the chromosomal break lies between PI and D14S1. These results show that the order of these chromosome 14 markers is cen-PI-D14S1-IGH, in keeping with multipoint linkage data. Further molecular characterization of ring 14 chromosomes should lead to a detailed understanding of the molecular events and clinical consequences of the gene deletion associated with such chromosomal aberrations.     

NOTCH2 Is Neither Rearranged nor Mutated in t(1;19) Positive Oligodendrogliomas

The combined deletion of 1p and 19q chromosomal arms is frequent in oligodendrogliomas (OD) and has recently been shown to be mediated by an unbalanced t(1;19) translocation. Recent studies of 1p/19q co-deleted OD suggest that the NOTCH2 gene is implicated in oligodendrocyte differentiation and may be involved in this rearrangement. The objective of the present study was to analyze the NOTCH2 locus either as a chromosomal translocation locus that may be altered by the 1p/19q recurrent rearrangement or as a gene that may be inactivated by a two hit process. We performed an array-CGH analysis of 15 ODs presenting 1p/19q co-deletion using a high-density oligonucleotide microarray spanning 1p and 19q pericentromeric regions with 377 bp average probe spacing. We showed that the 1p deletion extends to the centromere of chromosome 1 and includes the entire NOTCH2 gene. No internal rearrangement of this gene was observed. This strongly suggests that the t(1;19) translocation does not lead to an abnormal NOTCH2 structure. The analysis of the entire NOTCH2 coding sequence was performed in four cases and did not reveal any mutation therefore indicating that NOTCH2 does not harbor genetic characteristics of a tumor suppressor gene. Finally, the detailed analysis of chromosome 19 pericentromeric region led to the identification of two breakpoint clusters at 19p12 and 19q11–12. Interestingly, these two regions share a large stretch of homology. Together with previous observations of similarities between chromosome 1 and 19 alphoid sequences, this suggests that the t(1;19) translocation arises from complex intra and interchromosomal rearrangements.This is the first comprehensive deletion mapping by high density oligo-array of the 1p/19q co-deletion in oligodendroglioma tumors using a methodological approach superior to others previously applied. As such this paper provides clear evidence that the NOTCH2 gene is not physically rearranged by t(1;19) translocation of oligodendroglioma tumors.     

Characterization of a B-lymphocyte t(2;14) (p11;q32) translocation from an ataxia telangiectasia patient conferring a proliferative advantage on cells in vitro

Patients with the recessively inherited disorder ataxia telangiectasia (AT) are particularly prone to the development of both B-cell and T-cell tumours. Specific translocations involving T-cell gene rearrangements and an unknown locus 3' of IGH have been described in AT T-cell clone and tumour cells. We describe here a t(2;14)(p11;q32) translocation which was observed in nonmalignant short-term-cultured B lymphocytes from an AT patient. In vivo, the clone of cells grew from 1% to 6% of the total cell population over a period of 2 yr. Clonal translocations may therefore be associated with AT B cells, as well as AT T cells. B lymphocytes were transformed with Epstein-Barr virus, and the t(2;14) translocation cell was cellularly cloned. Using Southern filter analysis and in situ hybridisation to define more clearly the positions of the breakpoints, we show that the translocation at 14q32 involves a deletion within the IGH chain gene of at least J1, J2, DQ52, and sequences 1.5 kb 5' of DQ52 and that the breakpoint is either adjacent to the non-deleted JH sequences or upstream of these sequences, within the D or V regions, but proximal to all members of the VHII family of genes. The breakpoint at 2p11 is outside and proximal to IGK with respect to the centromere in an unknown gene. Sub-lines with an initially low proportion of translocation cells eventually became monoclonal in vitro for these cells. This suggests they have a growth advantage in vitro.     

Immunoglobulin heavy chain and alpha T-cell receptor genes rearrange in T-cell leukemia associated with a t(14;14) (q11.2;q32) translocation

We report here a cytogenetic and molecular analysis of two cases of T-cell leukemia with t(14;14) (q11.2;q32). Through in situ hybridization and Southern blotting, using radioactively labeled immunoglobulin heavy chain (IGH) and alpha T-cell receptor (TCRA) gene probes, we found in both tumors that the loci of both IGH and TCRA were rearranged. Molecular analysis of the t(14;14) clearly demonstrated that in some tumors rearrangements of the IGH and TCRA genes are associated with interchromosomal exchanges that result in specific chromosome translocations that confer a proliferative advantage and predisposition to leukemic transformation. The implication of these rearrangements for normal and neoplastic T-cell development is discussed.     

Cytogenetic investigations in a family with ataxia telangiectasia

Summary Cytogenetic findings on a family with ataxia telangiectasia (A-T) in which three of four sibs were affected are described. The affected individuals had approximately twice the level of spontaneous chromosome breakage of a normla control, while the parents and the normal sib had no significant increase. Lymphocytes from all three A-T homozygotes showed specific stable chromosomal rearrangements involving chromosomes 7 and 14. All of these abnormalities involved breakage at the usual four sites associated with A-T (7p14, 7q35, 14q12, and 14q32). Two rearrangements detected in the eldest and most severely affected patient were clones, one of which [t(14;14)(p11;q12)] is not commonly found in A-T cells. No chromosomal rearrangements were encountered in lymphocytes from the control, the parents, or the normal sib. Lymphocytes from the A-T patients also were found to be 7–11 times more sensitive to the induction of chromatid aberrations by X-irradiation than control cells. Lymphocytes from the parents and normal sib showed a moderately increased frequency of X-ray induced aberrations compared with that of the control.     

Cytogenetic investigations in three cell types of a Saudi family with ataxia telangiectasia

Summary Chromosomal analyses were performed on lymphocytes, fibroblasts and lymphoblastoid cell lines derived from a Saudi family with ataxia telangiectasia (AT). The three siblings of a consanguineous marriage were all affected. The lymphocytes of the AT homozygotes (probands) showed an increase of 2- to 6-fold and 4- to 8-fold respectively, in the frequency of spontaneous and X-ray-induced chromosomal aberrations compared with controls, while the parents (obligate heterozygotes) of the patients showed no notable difference. The unirradiated lymphocytes from the oldest AT sibling, an 11-year-old boy (AT1), showed specific rearrangements involving chromosomes 7 and 14 [t(7;14)(q35;q12)] and 12 and 14 [t(12;14)(q23;q12)] in two different clones. The most severely affected sibling was a 9-year-old girl (AT2) who presented with a clone showing a novel rearrangement involving chromosomes 14 and 17, namely: del(14) (q31q32) and dup(17)(q21–q24). The lymphocytes from the third sibling, a 2-year-old boy (AT3), showed a t(2;14)(p24;q12). In addition, an inv(14)(q12q32) was observed in all three AT patients, while inv(7)(p14q35) was found only in patients 2 and 3. The lymphocytes from the AT parents and controls showed normal karyotypes. The breakpoints involving chromosomes 2,12 and 17, observed in our studies, have rarely been reported in other series of AT patients. No non-random chromosomal rearrangements were observed either in the skin fibroblasts or in the lymphoblastoid cell lines derived from the AT patients, although all cell lines showed an increase in both spontaneous and radiation-induced chromosomal breaks per cell. The present study constitutes the first report on a cytogenetic analysis of a Saudi family with three AT siblings.     

Molecular cytogenetics of IGH rearrangements in non-Hodgkin B-cell lymphoma

Rearrangements involving the IGH gene have been identified in about 50% of non-Hodgkin B-cell lymphomas (NHLs) and correlated to clinically relevant subgroups. However, the detection rate largely varied with the technique used. We analyzed the incidence of IGH rearrangements using several fluorescence in situ hybridization (FISH) techniques on metaphases obtained from 96 patients with nodal NHL. An IGH rearrangement was identified in 71 cases (74%). A t(14;18)(q32;q21) was found in 37 of the 42 follicular lymphomas (88.1%) studied and a t(11;14)(q13;q32) in 12 of the 14 mantle cell lymphomas (85.7%). IGH rearrangements were identified in 21 of the 40 diffuse large B-cell lymphomas (52.5%), including seven t(14;18)(q32;q21) and four t(3;14)(q27;q32). Conventional cytogenetics was uninformative in several cases. However, the complemented analysis using 24-color FISH, chromosomal whole paints, telomeric probes and locus specific identifiers enabled us to characterize complex and/or masked IGH translocations in follicular lymphomas and mantle cell lymphomas and to identify all the chromosomal partners involved in IGH rearrangements in diffuse large B-cell lymphomas. This study shows the interest of using metaphase FISH in addition to conventional cytogenetics. Following banding techniques, FISH with the IGH dual color probe can be the first approach in NHL, after which chromosome painting and 24-color FISH can be used to identify the chromosomal partners involved in IGH rearrangements. The identification of these genes is of utmost importance for a better understanding of the molecular mechanisms involved in the genesis of lymphoma.     

Delineation of the frequency and boundary of chromosomal copy number variations in paediatric neuroblastoma

Neuroblastoma, the most common solid tumour in early childhood, is characterized by very frequent chromosomal copy number variations (CNVs). While chromosome 2p amplification, 17q gain, 1p and 11q deletion in human neuroblastoma tissues are well-known, the exact frequencies and boundaries of the chromosomal CNVs have not been delineated. We analysed the publicly available single nucleotide polymorphism (SNP) array data which were originally generated by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, defined the frequencies and boundaries of chromosomes 2p11.2 – 2p25.3 amplification, 17q11.1-17q25.3 gain, 1p13.3-1p36.33 deletion and 11q13.3-11q25 deletion in neuroblastoma tissues, and identified chromosome 7q14.1 (Chr7:38254795-38346971) and chromosome 14q11.2 (Chr14:21637401-22024617) deletion in blood and bone marrow samples from neuroblastoma patients, but not in tumour tissues. Kaplan Meier analysis showed that double deletion of Chr7q14.1 and Chr14q11.2 correlated with poor prognosis in MYCN gene amplified neuroblastoma patients. In conclusion, the oncogenes amplified or gained and tumour suppressor genes deleted within the boundaries of chromosomal CNVs in tumour tissues should be studied for their roles in tumourigenesis and as therapeutic targets. Focal deletions of Chr7q14.1 and Chr14q11.2 together in blood and bone marrow samples from neuroblastoma patients can be used as a marker for poorer prognosis and more aggressive therapies.     

The chromosome breakpoint at 14q32 in an ataxia telangiectasia t(14;14) T cell clone is different from the 14q32 breakpoint in Burkitts and an inv(14) T cell lymphoma

Summary The T cell receptor chain gene locus and the immunoglobulin heavy chain gene locus (IgH) have previously been mapped to the q11 and q32 positions respectively of the human chromosome 14. Both of these sites are also common breakpoints in lymphocytes from ataxia telangiectasia (A-T) patients. Using in situ hybridisation we show that the 14q32 breakpoint in an A-t non-leukaemic T cell clone with t(14;14) translocation, lies outside the IgH locus and proximal to it with respect to the centromere. The 14q11-14qter segment of the homologous chromosome 14 carrying the constant gene region of the chain locus is translocated to this 14q32 position.