Refinement of lymphoma cytogenetics by the chromosome 18q21 major breakpoint region

A small (2.8-kilobase, kb) major breakpoint region localized to segment 18q21 rearranges in greater than 70% of t(14;18)(q32;q21) lymphomas. This rearrangement interrupts the Bcl-2 gene and introduces it into the Ig locus at 14q32. The rearrangement between the joining region (JH) of Ig on chromosome 14 and the 18q21 region creates a translocation- specific DNA rearrangement. We generated probes that distinguish the 14;18 juncture on the derivative (der) 14 and der (18) chromosomes, providing a molecular approach to t(14;18) identification. Approximately 60% of unselected follicular lymphomas, 20% of diffuse large cell lymphomas, and 50% of adult undifferentiated non-Burkitt lymphomas demonstrated 14;18 rearrangements within the major breakpoint region. Examination of DNA for 14;18 rearrangements resolved the identity of 14q+ chromosomes in two patient's cells that lacked an obvious reciprocal partner. Identification of the exact restriction fragments that mediate translocations complements routine cytogenetics. The detection of DNA rearrangements does not require dividing cells or the presence of an identifiable reciprocal partner and can detect clonal translocation rearrangements when the neoplastic cells are only a minority of all cells present.     

Delineation of the frequently deleted region on chromosome arm 13q in B-cell non-Hodgkin's lymphoma

The loss of a specific chromosomal region provides a clue to the elucidation of the putative tumor suppressor gene implicated in the pathogenesis and progression of tumors. To delineate the specific region(s) involved in lymphomagenesis, we performed a survey of loss of heterozygosity for 11 polymorphic microsatellite loci scattered on variable chromosome arms. We examined 20 primary lymphoma samples, including both indolent and aggressive B-cell non-Hodgkin's lymphoma (B-NHL) and Hodgkin's disease (HD), and found a significant number of B-NHLs with loss of genetic material on chromosome arm 13q at the RB1 locus (50%; 4 of 8 informative cases for the RB1 locus). To specify the 13q deletion and to narrow the critical deleted region, we examined the same 20 lymphomas by intensive microsatellite mapping analysis using 12 microsatellite markers, mapping from 13q12.3 to 13q14. We confirmed the frequent 13q14 deletion to be in the vicinity of the RB1 locus (50% of the informative NHLs for at least 1 of 12 microsatellite loci; 5 of 10 aggressive NHLs and 2 of 4 indolent NHLs, but none of 6 HDs) and determined a subchromosomal region deleted in lymphoma on 13q14 defined by D13S164-D13S273, which is an overlapped region frequently lost in chronic lymphocytic leukemia. Taken together, our data indicate that the 13q alterations are present in a wide variety of NHLs including both indolent and aggressive B-NHLs, suggesting that loss of genetic material at chromosome band 13q14 may play an important role in the formation or development of a wide variety of mature lymphoid malignancies.     

Direct isolation of genes encoded within a homogeneously staining region by chromosome microdissection.

Identification of genes involved in recurring chromosome rearrangements has provided significant insight into the molecular basis of malignancy. We describe here a strategy combining chromosome microdissection and hybrid selection for the direct isolation of chromosome region-specific genes. We modeled this strategy by using sequences recovered from the microdissection of a homogeneously staining region to allow isolation of genes that were overexpressed and present at high copy number within the homogeneously staining region, including the direct isolation of two genes encoded within a 12q homogeneously staining region found in the osteosarcoma cell line OsA-CL. Although first applied to amplified genes, this strategy should be applicable to the isolation of cDNAs from any chromosomal region.     

Ph chromosome in a patient with non-leukemic non-Hodgkin B-cell lymphoma

A standard Philadelphia translocation, t(9;22) (q34;q11), was found in lymph node cells from a patient with non-leukemic non-Hodgkin lymphoma at the time of diagnosis. The rearrangement of the breakpoint cluster region (bcr) was not detected with a bcr-3' probe. The neoplastic clone was of monoclonal B-cell character with E-, CD5-, CD10-, CD13-, CD19+, CD20+, CD21+, CD25-, HLA DR+, and positive surface Ig(kappa). The patient showed no evidence of chronic myelogenous leukemia.     

Cytogenetic and molecular delineation of a region of chromosome 3q commonly gained in marginal zone B-cell lymphoma

BACKGROUND AND OBJECTIVES: Whole or partial trisomy 3 represents the most recurrent chromosomal abnormality occurring in marginal zone B-cell lymphoma (MZBCL), a distinct subtype of B-cell non-Hodgkin's lymphoma (NHL). By conventional cytogenetic analysis, unbalanced translocations involving chromosome 3 and leading to a partial trisomy 3q were identified in a series of 14 MZBCL patients. Fluorescent in situ hybridization (FISH) experiments were then performed to characterize the breakpoints further and to delineate the extent of the 3q gained region more accurately. DESIGN AND METHODS: We studied 14 cases of MZBCL combining cytogenetics and FISH techniques using specific probes for the long arm of chromosome 3, including the chromosome 3 a satellite probe, a representative panel of yeast artificial chromosome (YAC) clones mapping the chromosomal 3q region (3q11.2 to 3q23) and the chromosome 3 subtelomeric (3q29) probe. RESULTS: In the 14 cases, additional chromosome 3q material was found to be involved in different unbalanced translocations with chromosomes 1, 6, 7, 8, 11, 13, 14, 15, 17, 19 and 21, leading to a derivative chromosome. None of the chromosomal abnormality juxtaposed the 3q regions with the heavy and/or light k and l immunoglobulin gene loci. Eight different breakpoints distributed between the 3q11.2 and the 3q13.32 regions were identified and a common 3q13.32 3q29 overrepresented region was delineated. INTERPRETATION AND CONCLUSIONS: These results suggest that this critical region may be of importance in the pathogenesis of MZBCL and support the hypothesis that a gene dosage effect rather than a specific gene disruption may be involved in the development of this disease.     

ALK activation by the CLTC-ALK fusion is a recurrent event in large B-cell lymphoma

We present 3 cases of large B-cell lymphoma (LBCL) with a granular cytoplasmic staining for anaplastic lymphoma kinase (ALK). All of the cases showed striking similarities in morphology and immunohistochemical profile characterized by a massive monomorphic proliferation of CD20-/CD138+ plasmablast-like cells. In one of the cases, initially diagnosed as a null-type anaplastic large cell lymphoma (ALCL), the B-cell phenotype became evident only at recurrence. Fluorescent in situ hybridization (FISH) and molecular studies led to the detection of a CLTC-ALK rearrangement in all 3 cases, without any evidence of full-length ALK receptor expression. The associated t(2;17)(p23;q23) was demonstrated in the karyotype of 2 cases. Although a similar CLTC-ALK aberration was previously identified in ALK-positive T-/null cell ALCL and inflammatory myofibroblastic tumor, its association with ALK-positive LBCL seems to be specific and intriguing.     

Ph chromosome and B-cell malignancy-associated chromosomal aberrations in non-leukaemic immunoblastic B-cell lymphoma

A 62-year-old female with a microK phenotypic immunoblastic B-cell lymphoma with bone marrow involvement but without leukaemia is reported. Bone marrow cells, cytogenetically studied at diagnosis, showed a Philadelphia chromosome due to a (9p;22q) translocation, deleted chromosomes 3 and 6, a 14q+ marker, and two extra chromosomes 18. The Ph chromosome is previously only once reported in a well-characterized B-cell lymphoma, whereas the latter aberrations are common findings in B-cell malignancies.     

Localization of the human pim oncogene (PIM) to a region of chromosome 6 involved in translocations in acute leukemias.

The human homolog, hpim, of the murine pim-1 gene, which is activated in murine T-cell lymphomas by insertion of retrovirus proviral genomes in the pim-1 region, has been molecularly cloned; the cloned probe has been used to map the hpim locus to human chromosome region 6p21 by somatic cell hybrid analysis and chromosomal in situ hybridization. The hpim gene is expressed as a 3.2-kilobase mRNA in various human cell lines of hematopoietic lineage, most dramatically in the K562 erythroleukemia cell line, which contains a cytogenetically demonstrable rearrangement in the 6p21 region. A characteristic chromosome anomaly, a reciprocal translocation t(6;9)(p21;q33), has been described in myeloid leukemias and could involve the hpim gene.     

Distribution of breakpoint within the breakpoint cluster region (bcr) in chronic myelogenous leukemia with a complex Philadelphia chromosome translocation

We analyzed, by Southern blot hybridization, the site of breakpoint within the breakpoint cluster region (bcr) in six patients with a complex Philadelphia chromosome (Ph) translocation and in 23 unselected patients with a standard Ph. The breakpoint was found within the 5.8 kb bcr in all 29 patients. When the bcr was subdivided into four parts, fragments I-IV, based on the restriction enzyme sites, among the six patients with a complex Ph, two had a breakpoint at fragment I, three at fragment II, and one at fragment III. This distribution of breakpoints in patients with a complex Ph did not differ significantly from that in patients with a standard Ph. A deletion of an allele within the bcr was found in three patients (50%) with a complex Ph and in three (13%) with a standard Ph. The internal bcr deletion may be more common in patients with a complex Ph.     

Type-C virus-like particles in a human B-cell lymphoma cell line

Type-C virus-like particles (VLPs) were found in an Epstein-Barr (EB) virus-infected human B-cell lymphoma cell line, SP-50B, that was established from a patient with non-Hodgkin lymphoma. The cell line continuously produces a small number of type-C VLPs, 150-200 nm in diameter, over 1 year. SP-50B cells were negative for HTLV-I and HTLV-II antigens and did not contain the HTLV-I genome. In addition, two EB virus nuclear antigen (EBNA)-positive B-cell lines, SP-54-Cord and SP-57-CLL, were established from human cord blood and chronic lymphocytic leukemia (CLL), respectively, by coculture with lethally irradiated SP-50B cells. Type-C VLPs with the same morphology were also found in both cell lines.     

Inhibition of human B-cell lymphoma growth by CD40 stimulation

CD40 is a molecule present on B lymphocyte lineage cells that is important in B-cell differentiation and activation. Signaling through CD40 has been shown to exert costimulatory signals on normal B cells resulting in proliferative and differentiation responses. Examination of several B-cell lymphomas showed cell-surface expression of the CD40 molecule. Incubation of these lymphomas with anti-CD40 antibodies resulted in significant growth inhibition in vitro. Cross-linking of the CD40 antibodies resulted in even greater inhibition of proliferation. A recombinant soluble human CD40 ligand was also shown to inhibit lymphoma proliferation. When various human B-cell lymphomas were transferred into mice with severe combined immune deficiency, the treatment of the mice with anti-CD40 antibodies resulted in significant increases in survival showing that anti-CD40 is efficacious after in vivo administration. Thus, CD40 stimulation by either the antibody or soluble ligand directly inhibits human B-cell lymphoma growth and therefore, may be of significant clinical use in their treatment.     

应用染色体显微切割技术筛选人小细胞肺癌多药耐药相关基因的实验研究

目的应用染色体显微切割技术筛选人小细胞肺癌多药耐药基因。方法在倒置显微镜下,显微切割人小细胞肺癌(small cell lung cancer,SCLC)多药耐药(multidrug-resistance,MDR)细胞系NCI-H446/CDDP特异性畸变染色体并构建该染色体片段DNA微文库,然后采用菌落斑点杂交、反Northern Blot及Northern Blot等方法,筛选人SCLC MDR相关基因。结果筛选出25个在耐药细胞中上调表达的基因或DNA片段。在已测序的20个DNA序列中,3个分别为人类2号和5号染色体BAC片段DNA序列,在全长库中未检索到其对应的相似序列,可能代表了某个基因或者该序列位于基因变异丰富的3’端,而无法查到与其它物种基因的同源性。其它17个序列与已知基因存在95%～100%的同源性。已知基因包括硫氧还蛋白、Bcl-2、TRAF、神经酰胺等基因。结论多个基因可能构成网络调控系统,参与人SCLC MDR相关基因的形成,但其具体的调控机制,则仍需大量的研究探索。     

Analysis of a T-cell tumor-specific breakpoint cluster at human chromosome 14q32.

Cytogenetic abnormalities involving chromosome 14 band q32 are consistently observed in human T-cell tumors. Patients with ataxia-telangiectasia (AT) are especially prone to development of these tumors, which frequently carry either inversion inv(14)(q11;q32) or translocation t(14;14) (q11;q32) chromosomes. We have previously shown that the cytogenetic breakpoints of one t(14;14)(q11;q32) chromosome and two inv(14)(q11;q32) chromosomes in T-cell tumors from AT and non-AT patients join the T-cell receptor alpha chain locus, at chromosome band 14q11, with a region(s) at 14q32 centromeric of the immunoglobulin heavy chain variable region (VH) gene IGHV. We now show that these two inv(14) breakpoints are linked by 2.1 kb of germ-line 14q32 DNA and that the three breakpoints define, by in situ hybridization analysis, a single locus at chromosome band 14q32.1 located about 15-20 million base pairs on the centromeric side of the IGH locus. Sequence analysis of the 14q32.1 breakpoint regions indicates that abnormal recombination does not universally result from mistaken V-D-J joining (D = diversity region; J = joining region). Therefore, we invoke a tumor selection model to describe the role of the 14q32.1 locus in tumor development.     

Natural killer susceptibility of human cells may be regulated by genes in the HLA region on chromosome 6.

Natural killer (NK) cells exist in each individual in the absence of any intentional immunization. They are able to kill a wide range of targets from tumoral as well as from normal origin. However, their exact physiologic role is not clearly understood. In this study we report results about a human Epstein-Barr virus-transformed B-cell line from which variants perturbed in the expression of HLA molecules have been derived. Our results indicate that in these cell lines an inverse relationship exists between expression of HLA antigens and susceptibility to NK lysis. The original cell line is highly resistant to NK lysis. On the contrary, the variant perturbed in class I antigen expression is highly susceptible. Variant perturbed in class II antigen expression is intermediate in susceptibility. Interferon, which induces HLA class I expression and NK resistance in the unrelated classical K-562 target cells, does not change either HLA expression or NK susceptibility in the variant cell lines. The difference between the original cell line and the variants does not reside in the ability to be bound by NK effectors. Our results suggest a different role for HLA molecules. By some unknown mechanism discussed here, the presence of HLA molecules at the surface of a cell would prevent this cell from being killed by NK cells. The loss of this "good health" signal would lead to the elimination of the cell through NK lysis.     

SNP rs6457327 in the HLA region on chromosome 6p is predictive of the transformation of follicular lymphoma

Inherited risk determinants for follicular lymphoma (FL) have recently been described in the immune gene-rich human leukocyte antigen region on chromosome 6p. The known importance of host immune response to FL survival led us to evaluate these germline factors in FL outcome. We confirm the association of single nucleotide polymorphisms rs10484561 (P = 3.5 × 10??) and rs6457327 (P = .008) with risk of FL and demonstrate that rs6457327 predicts both time to (P = .02) and risk of (P < .01) FL transformation independently of clinical variables, including the Follicular Lymphoma International Prognostic Index.