Cyclin D2 dysregulation by chromosomal translocations to TCR loci in T-cell acute lymphoblastic leukemias.

Strong expression of at least one of the three D-type cyclins is common in human cancers. While the cyclin D1 and D3 genes (CCND1 and CCND3) are recurrently involved in genomic rearrangements, especially in B-cell lymphoid neoplasias, no clear involvement of the cyclin D2 gene (CCND2) has been reported to date. Here, we identified chromosomal translocations targeting the CCND2 locus at 12p13, and the T-cell receptor beta (TCRB) or the TCRA/D loci in T-cell acute lymphoblastic leukemias (T-ALLs). Expression analysis demonstrated dramatic cyclin D2 overexpression in the translocated cases (n=3) compared to other T-ALLs (total, n=89). In order to evaluate dysregulation in T-ALL with respect to normal T-cell differentiation, we analyzed CCND2 expression in normal purified human thymic subpopulations. CCND2 levels were downregulated through progression from the early stages of human T-cell differentiation, further suggesting that the massive and sustained expression in the CCND2-rearranged T-ALL cases was oncogenic. Association with other oncogene expression (TAL1, HOXAs, or TLX3/HOX11L2), NOTCH1 activating mutations, and/or CDKN2A/p16/ARF deletion, showed that cyclin D2 dysregulation could contribute to multi-event oncogenesis in various T-ALL groups. This report is the first clear evidence of a direct involvement of cyclin D2 in human cancer due to recurrent somatic genetic alterations.     

Cyclin D1 overexpression is not a specific grouping marker, but may collaborate with CDC37 in myeloma cells

Cyclin D1 is a positive-regulator of the cell cycle and is overexpressed in myeloma cells with t(11;14)(q13;q32). First, we analyzed whether there was a correlation between cyclin D1 overexpression and the presence of Ki67-positive myeloma cells in multiple myeloma (MM). Cyclin D1 overexpression was examined by competitive RT-PCR. Then we found these two markers were present independently in a given case. FISH analysis revealed that cyclin D1 over-expression was caused by t(11;14)(q13;q32) or extra copies of B-cell leukemia/lymphoma-1 (BCL-1/CCND1), and unknown mechanism without them. We compared the gene expression between myeloma cells with cyclin D1 overexpression and those without it using cDNA microarray analysis. Analysis of the expression profiles showed that the significantly up-regulated genes included cyclin D1, cell division cycle 37 (CDC37) and B-cell leukemia/lymphoma-2 (BCL-2), while the down-regulated genes included cyclin D2 and CD9 antigen (p24) in MM cases with cyclin D1 overexpression. However, hierarchical clustering analysis of the data showed that myeloma cells of MM cases with cyclin D1 overexpression could not be distinguished clearly from those without it. Real-time RT-PCR showed that the expression of CDC37 gene was significantly up-regulated in MM patients with cyclin D1 overexpression compared with those without it (p=0.0418). However, there was no significant difference in BCL-2 gene (p=0.5748). These results suggested that MM cases with cyclin D1 overexpression do not constitute a specific group, and cyclin D1 overexpression may not be caused only by abnormality of the BCL-1/CCND1 gene. The CDC37 may collaborate with cyclin D1 in progression of MM.     

Cytogenetic Abnormalities in Multiple Myeloma Patients at a Tertiary Healthcare Center in India

Objective: Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell neoplasm. Theprognosis of MM patients is dependent on several factors including the patient’s age, the stage of disease and geneticalterations. This study aimed to determine the frequency of common chromosomal abnormalities and their significance inMM patients referred to a tertiary healthcare center in India. Methods: Fluorescence in situ hybridization on interphasenuclei from bone marrow cells using seven MM-specific probes for recurrent aberrations was performed in a total of215 newly diagnosed patients. Results: Chromosomal abnormalities were detected in 161 (74.9%) MM patients inthis study. The most frequent aberration was trisomy(ies) involving only gain of chromosomes in 48 (22.3%) cases.A translocation involving the IGH gene alone or accompanied by trisomy(ies) or by monosomy 13/13q deletion or byboth was registered in 80 (37.2%) patients. Atypical patterns such as a deletion of the IGH variable segment (IGHv)on the derivative chromosome 14 or on the native (normal) chromosome 14, biallelic deletion of IGHv, deletion ofthe IGH constant segment on the rearranged chromosome14 and extra fusions were noticed in 21 (9.8%) patientswith an IGH rearrangement. Monosomy 13/deletion 13q was identified singly or as part of a complex karyotype in74 patients (34.4%). Clonal heterogeneity and additional abnormalities including TP53 deletion and monosomies ofchromosomes 4, 9, 14 and 16 were recorded in 18.6% and 16.3% of patients respectively. Patients with abnormalitiesexhibited plasmacytosis, reduced hemoglobin value and high level of ß2-microglobulin. Conclusions: A lower medianage and a low frequency of IGH translocations particularly t(11;14) and chromosome 13 abnormalities suggest ethnicdiversity. Further investigations on genetic alterations including IGH deletions will contribute to improved insightsinto the biology of myeloma disease, risk stratification and patient management.     

Molecular pathogenesis and a consequent classification of multiple myeloma.

There appear to be two pathways involved in the pathogenesis of premalignant non-immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Nearly half of tumors are nonhyperdiploid, and mostly have one of five recurrent IgH translocations: 16% 11q13 (CCN D1), 3% 6p21 (CCN D3), 5% 16q23 (MAF), 2% 20q12 (MAFB), and 15% 4p16 (FGFR3 and MMSET). The remaining hyperdiploid tumors have multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, and infrequently one of these five translocations. Although cyclin D1 is not expressed by healthy lymphoid cells, it is bi-allelically dysregulated in a majority of hyperdiploid tumors. Virtually all MM and MGUS tumors have dysregulated and/or increased expression of cyclin D1, D2, or D3, providing an apparent early, unifying event in pathogenesis. The patterns of translocations and cyclin D expression (TC) define a novel classification that includes eight groups: 11q; 6p; MAF; 4p; D1 (34%); D1+D2 (6%); D2 (17%); and none (2%). The hyperdiploid D1 group is virtually absent in extramedullary MM and MM cell lines, suggesting a particularly strong dependence on interaction with the bone marrow microenvironment. Despite shared progression events (RAS mutations, MYC dysregulation, p53 mutations, and additional disruption of the retinoblastoma pathway), the phenotypes of MGUS and MM tumors in the eight TC groups is determined mainly by early oncogenic events. Similar to acute lymphocytic leukemia, MM seems to include several diseases (groups) that have differences in early or initiating events, global gene expression patterns, bone marrow dependence, clinical features, prognosis, and response to therapy.     

Identification of novel cryptic translocations involving IGH in B-cell non-Hodgkin's lymphomas

Chromosomal rearrangements involving the immunoglobulin heavy chain gene (IGH) at 14q32 are observed in approximately 50% of patients with B-cell non-Hodgkin's lymphoma (NHL). The 5' end of the IGH gene is located within 8 kb of the telomeric repeats of 14q. Translocations involving the IGH locus and the telomeric band of a partner chromosome are difficult to identify, because most terminal bands of human chromosomes appear pale by conventional G-banding techniques. To determine whether there are cryptic translocations involving the IGH locus, we used dual-color fluorescence in situ hybridization (FISH) of 5' and 3' IGH genomic clones containing the variable sequences, or the J(H) and the 5' constant regions, respectively. We examined cells from 51 patients with B-cell NHL who had a normal karyotype (3 patients), clonal abnormalities not involving 14q32 (35 patients), or alterations of 14q32 other than recurring translocations, i.e., add(14)(q32) (13 patients). FISH detected 17 IGH translocations in 16 of 51 (31%) cases. Of the 13 cases with add(14)(q32), FISH identified the partner chromosome in 9 cases (69%; 3q27, 6 cases; 2p13, 19p13.3, and 18q21.3, 1 case each). Six of thirty-eight (16%) patients without visible alterations of 14q32 and 2 of 13 (15%) patients with an abnormality of one chromosome 14 had masked (5 patients) or cryptic IGH translocations (3 patients), involving 3q27 (3 patients), 5p15.3 (2 patients), 19p13.3 (3 patients), or 14q32 (1 patient; 1 patient had two rearrangements). We identified two novel, recurring, cryptic translocations: t(5;14)(p15.3;q32) (2 patients) and t(14;19)(q32;p13.3) (3 patients). In summary, FISH permitted the detection of cryptic or masked IGH rearrangements in approximately 20% of lymphoma cases without visible rearrangements of 14q32 analyzed retrospectively.     

Rapid amplification of immunoglobulin heavy chain switch (IGHS) translocation breakpoints using long-distance inverse PCR.

Molecular cloning of immunoglobulin heavy chain (IGH) translocation breakpoints identifies genes of biological importance in the development of normal and malignant B cells. Long-distance inverse PCR (LDI-PCR) was first applied to amplification of IGH gene translocations targeted to the joining (IGHJ) regions. We report here successful amplification of the breakpoint of IGH translocations targeted to switch (IGHS) regions by LDI-PCR. To detect IGHS translocations, Southern blot assays using 5' and 3' switch probes were performed. Illegitimate Smu rearrangements were amplified from the 5' end (5'Smu LDI-PCR) from the alternative derivative chromosome, and those of Sgamma or Salpha were amplified from the 3' end (3'Sgamma or 3'alpha LDI-PCR) from the derivative chromosome 14. Using a combination of these methods, we have succeeded in amplifying IGHS translocation breakpoints involving FGFR3/MMSET on 4p16, BCL6 on 3q27, MYC on 8q24, IRTA1 on 1q21 and PAX5 on 9p13 as well as BCL11A on 2p13 and CCND3 on 6p21. The combination of LDI-PCR for IGHJ and IGHS allows rapid molecular cloning of almost all IGH gene translocation breakpoints.     

Multicolor interphase cytogenetics for the study of plasma cell dyscrasias

Specific chromosomal abnormalities such as chromosome 13 deletions and some translocations affecting the immunoglobulin heavy chain (IGH) gene, namely t(4;14)(p16;q32) and t(14;16)(q32;q23) have been associated with an adverse prognosis in multiple myeloma. Conventional cytogenetic techniques fail to detect these aberrations in the majority of cases. Thus, we have developed a novel set of interphase fluorescence in situ hybridization (I-FISH) assays targeting those regions frequently lost on chromosome 13 as well as those oncogenes most recurrently involved in translocations with the IGH locus in multiple myeloma, i.e. IRTA1/2 (1q21), FGFR3/MMSET (4p16), CCND3 (6p21), IRF4 (6p25), CCND1 (11q13), MAF (16q23), and MAFB (20q12). The probes were combined in a multicolor fashion to develop novel multicolor I-FISH (MI-FISH) assays, whose validity and applicability was evaluated in negative controls and in a series of 13 plasma cell neoplasias. Additionally, a combination of the novel MI-FISH assays with staining for the plasma cell-specific antigen VS38c by means of multicolor FICTION (M-FICTION, fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms) allowed us to selectively analyze the plasma cell compartment, and thereby to increase the assay sensitivity.     

Overexpression of cyclin D1 and cortactin is primarily independent of gene amplification in salivary gland adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) of the salivary glands exhibits persistent growth, invasion and metastasis. Chromosome 11q13 amplification is a frequent event associated with tumor progression in a number of carcinomas and is associated with poor prognosis. Two genes within the 11q13 amplicon that are overexpressed as a result of 11q13 amplification are the cell cycle regulatory protein cyclin D1 (CCND1) and cortactin (CTTN), a protein involved cell motility and invasion. To determine the expression and gene status of cyclin D1 and cortactin in ACC, we evaluated 39 ACC cases by immunohistochemistry (IHC) for cyclin D1 and cortactin expression. Amplification of CCND1 and CTTN was determined by fluorescent in situ hybridization (FISH). Cyclin D1 overexpression was present in 90% (35/39) and cortactin expression in 62% (24/39) of evaluated cases, although CCND1 and CTTN levels were elevated in only two cases (5%) as determined by FISH. Our results indicate that chromosome 11q13 amplification is uncommon in ACC, but that cyclin D1 and cortactin are frequently overexpressed and may therefore contribute to the growth and invasive potential of ACC.     

荧光原位杂交技术在多发性骨髓瘤患者del(13q14)检测中的应用

 目的　了解多发性骨髓瘤（MM）患者中13号染色体缺失情况,分析13号染色体部分缺失在MM中的临床价值。方法　采用荧光原位杂交（FISH）技术检测38例MM患者骨髓标本中Rb-1基因和13q14位点的缺失;采用Fisher确切概率法分析13号染色体部分缺失与患者确诊时临床特征间关系。结果　在38例MM患者中,13号染色体部分缺失20 例,其中仅Rb-1基因缺失4例,仅13q14位点缺失2例,两位点同时缺失14例。Fisher确切概率法分析显示13号染色体长臂缺失（del 13q14）与患者血清乳酸脱氢酶升高及国际分期系统（ISS）有关。结论　Rb-1基因和13q14位点的缺失在MM中均较为常见,13号染色体部分缺失对MM的生物学行为有一定影响,del (13q14)在 MM中的意义有待进一步探讨;FISH是一种在分析 MM患者13号染色体异常方面较为快速、准确和敏感的方法。     

Identification of CCND3 and BYSL as candidate targets for the 6p21 amplification in diffuse large B-cell lymphoma.

PURPOSE: Increases in gene dosage through DNA amplification represents a common feature of many tumors and can result in the up-regulation of tumor-promoting genes. Our recent genome-wide, array-based comparative genomic hybridization analysis of 66 cases of diffuse large B-cell lymphoma found that genomic gain of 6p21 was observed in as many as 17 cases, including 14 cases with low-level copy number gain and three cases with high-level copy number gains (amplifications). EXPERIMENTAL DESIGN AND RESULTS: To identify the target gene(s) for 6p21 amplification, we constructed a detailed amplicon map at the region of genomic amplification with the aid of high-resolution contig array-based comparative genomic hybridization glass slides, consisting of contiguously ordered bacterial artificial chromosome/P1-derived artificial chromosome clones covering 3 Mb throughout the 6p21 amplification region. Alignment of the amplifications identified a minimally overlapping 800 kb segment containing 15 genes. Quantitative expression analysis of the genes from both patient samples and the SUDHL9 cell line revealed that CCND3 and BYSL (1.9 kb telomeric to the CCND3 gene locus) are the targets of 6p21 genomic gain/amplification. CONCLUSIONS: Although it is known that t(6;14)(p21;q32) induces aberrant overexpression of CCND3 in B-cell malignancies, we were able to show that CCND3, which encodes the cyclin D family member protein that controls the G1-S phase of cell cycle regulation, can also be a target of genomic gain/amplification. Overexpression of CCND3 through genomic amplification is likely to lead to aberrant cell cycle control, although the precise biological role of BYSL with respect to tumorigenesis remains to be determined.     

应用荧光原位杂交技术检测多发性骨髓瘤

 目的　应用荧光原位杂交（FISH）技术检测多发性骨髓瘤（MM）常见基因异常。方法　采用1q21／RB1、D13S319／p53、IGH序列特异性基因探针,应用FISH技术对按照WHO诊断标准确诊的44例初诊MM患者进行检测,并与常规细胞遗传学技术（吉姆萨显带）检测结果及临床参数对比。结果　FISH技术检测44例初诊MM患者中32例（72.7 %）基因异常,其中1q21扩增11例（25.0 %）,RB1缺失17例（38.6 %）,D13S319缺失16例（36.4 %）,p53缺失6例（13.6 %）,IGH基因重排19例（43.2 %）。检出1种异常者10例（22.7 %）,同时有2种异常者11例（25.0 %）,3种异常者8例（18.2 %）,4种异常者3例（6.8 %）。44例应用常规吉姆萨显带检测16例无分裂象,28例有分裂象者中检出异常核型2例（7.14 %）,FISH技术基因异常检出率明显高于常规吉姆萨显带技术,差异有统计学意义（P＜0.05）。基因异常与临床参数比较结果显示,β2-微球蛋白（β2-MG）与IGH基因重排呈正相关（P＜0.05）,骨髓浆细胞数与D13S319缺失呈正相关（P＜0.05）,CRE与p53缺失及IGH基因重排呈正相关（P＜0.05）,CRP与p53缺失呈正相关（P＜0.05）。基因异常与MM分型、分期、年龄分组之间无明显相关性（P＞0.05）。结论　MM最常见的基因异常是IGH基因重排以及RB1和D13S319缺失,其次是1q21扩增,p53缺失最少。FISH可检测出与预后密切相关的累及基因位点的微缺失,异常克隆检出率较常规细胞遗传学技术显著提高,FISH可作为MM患者的常规检查,以评估预后,指导临床治疗。     

Involvement of the CCND1 gene in hairy cell leukemia

BACKGROUND:: Previous results suggested increased mRNA expression of CCND1in hairy cell leukemia (HCL). The CCND1 gene is involved inthe t(11;14)(q13;q32) chromosomal rearrangement, a characteristicabnormality in mantle cell lymphoma (MCL). We and others reportedthat, in contrast to other B-cell lymphomas, almost all MCLhave overexpression of the CCND1 gene with a good correlationbetween RNA and protein analysis. Recent studies showed thatoverexpression of the cyclin Dl protein can be easily detectedby immunohistochemistry (IHC) on formalin-fixed, paraffin embeddedtissues. PATIENTS AND METHODS:: To investigate whether the CCND1 gene is involved in HCL, weperformed IHC on a series of 22 cases using formalin-fixed paraffinembedded splenectomy specimens. For IHC the sections were boiledin citrate buffer. The presence of rearrangements within theBCL-1 locus and the CCND1 gene was analyzed in 13 of 22 casesby Southern blot analysis using all available break point probes.Expression of CCND1 was analyzed at the mRNA level (Northernblot) and protein level (IHC). RESULTS:: Overexpression of the cyclin Dl protein using IHC was observedin all cases, with strong expression in 5 cases. Pre-existingB- and T-cell areas of the spleen did not express significantlevels of the cyclin D1 protein. Seven of 9 cases analyzed byboth IHC and Northern blotting showed overexpression of theCCND1 gene with both methods. No genomic abnormalities wereobserved in any of the 13 cases studied by Southern blot analysis.Additionally, no 11q13 abnormalities were detected by bandinganalysis of 19 of 22 cases. CONCLUSIONS:: The elevated levels of CCND1 mRNA and protein in conjunctionwith the absence of overt rearrangements within the BCL-1 locusdistinguish HCL from MCL and other B-cell malignancies. Thissuggests that activation of the CCND1 gene in HCL is due tomechanisms other than chromosomal rearrangement. BCL-1, CCND1, cyclin D1, HCL, immunohistochemistry, RNA, t(11;14)     

Molecular characterization of a t(11;14)(q23;q32) chromosome translocation in a B-cell lymphoma

We have analyzed the molecular features of a t(11;14)(q23;q32) chromosome translocation of a cell line established from a B-cell lymphoma. Somatic hybrid cells carrying the 11q- and/or 14q+ chromosome(s) were produced in order to map the breakpoints. Southern blot analyses of DNAs from these hybrid cell lines together with various probes from the IGH locus on chromosome 14 and the ETS-1 and CD3 genes on chromosome 11 showed that the breakpoints of the translocation occurred between the constant regions of the C phi gamma and C gamma 2 genes on chromosome 14 and between the CD3 and ETS-1 genes on chromosome 11. The t(11;14)(q23;q32) translocation does not seem to involve the same mechanism that is responsible for translocations occurring at the immunoglobulin heavy chain joining segment (JH).     

从门静脉癌栓建立肝癌细胞株及对其细胞遗传特征的研究

背景和目的：肝细胞癌（hepatocellular carcinoma,HCC)预后差的原因是术后复发率高和早期过门静脉转移。本研究建立门静脉癌栓肝癌细胞株,并探讨其分子细胞遗传特征。方法：从HCC患者门脉癌栓中获得取肝癌细胞进行细胞培养,对培养成功的细胞（H4M）进行染色体G带染色后分析其核型和对比基因组杂交（comparative genomic hybridization,CGH)结果;用差异PCR检测周期素D1基因。结果：H4M细胞核型为超3倍体,染色体数为71－78,其中有一条标记染色体含有一长的均染区（hsr)。H4M主要的细胞遗传学改变为8p缺失和11q13高拷贝数扩增。周期素D1基因CCND1明显扩增。结论：染色体8p丢失和11q13高拷贝数扩增与H4M细胞的转移特性相关。周期素D1基因CCND1扩增可能是染色体11q13扩增的原因。     

Frequent occurrence of CCND1 deregulation in patients with early stages of plasma cell dyscrasia

Chromosomal translocations involving the immunoglobulin heavy chain gene ( IgH ) and nonrandom protooncogene loci are the hallmark of genetic alterations found not only in multiple myeloma (MM), but also in premalignant stages of MM, including monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM). We studied the frequency of IgH (14q32) rearrangements and their partner chromosomes in 16 Japanese patients with MGUS (13 cases), and SMM (3 cases) by means of interphase double-color fluorescence in situ hybridization (DCFISH) applied to purified plasma cells and using CD138-bead selection. IgH rearrangement was recognized in nine of the patients (56.3%). Protooncogene loci juxtaposed to IgH were identified in seven cases including CCND1 (11q13) in six cases and FGFR3 (4p16) in one. Four out of the six t(11;14)-positive cases showed nuclear staining of the cyclin D1 protein, whereas none of the seven t(11;14)-negative cases did. Moreover, neither MUM1 (6p25)- IgH nor MAFB (20q11)- IgH fusion signals were observed. This suggests to us that cyclin D1 deregulation due to the presence of t(11;14) is involved in the early development of plasma cell neoplasms, and that this event alone is not enough for the development of symptomatic myeloma. (Cancer Sci 2003; 94: 350-354)     

Fluorescence in situ hybridization analysis of chromosome aberrations in 60 Chinese patients with multiple myeloma

Conventional cytogenetic analysis is often hampered owing to the low mitotic index of multiple myeloma (MM) cells in bone marrow samples of MM. Interphase fluorescence in situ hybridization (I-FISH) analysis combined with magnetic-activated cell sorting (MACS) has substantially enhanced the sensitivity of cytogenetic analysis. Here, we used I-FISH to explore the incidence of chromosomal abnormalities in 60 Chinese patients with newly diagnosed MM. Five different specific probes for the regions containing 13q14.3 (D13S319), 14q32 (IGHC/IGHV), 1q21, 1p12, and 17p13 were used to detect chromosomal aberrations, and LSI IGH/CCND1, LSI IGH/FGFR3, and LSI IGH/MAF probes were further applied to detect t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23) in patients with 14q32 rearrangement. Fifty of the patients (83.3%) had at least one type of abnormalities regarding the regions analyzed. Nine patients (15%) had one abnormality; 10 patients (16.7%) had two abnormalities; 31 patients (51.7%) had three or more abnormalities. The most frequent abnormality in the patients was illegitimate IgH rearrangement (70%), followed by 13q14 deletion (63.3%), 1q21 amplification (61.7%), 1p12 deletion (33.3%), and 17p13 deletion (13.3%). These aberrations are not randomly distributed, but strongly interconnected. Patients with 17p13 deletion or t(4;14)(p16;q32) had significant higher ??2-microglobulin level (P?<?0.05). However, all these abnormalities had no correlation with age, gender, disease stage, and Ig isotype; yet, it was showed that the frequencies of the individual chromosomal abnormalities were very high. Taken together, MACS in combination with I-FISH may be a promising tool to detect the molecular cytogenetic abnormalities of MM.