Spectral Karyotyping Analysis Research Articles

Identification of recurrent chromosomal breakpoints in multiple myeloma with complex karyotypes by combined G-banding, spectral karyotyping, and fluorescence in situ hybridization analyses

The description of novel chromosomal aberrations in multiple myeloma (MM) remains necessary to fully understand the pathogenesis of this heterogeneous disease. Therefore, we have used spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) with locus-specific probes to characterize the chromosomal abnormalities in 11 MM cases in which G-banding revealed a complex karyotype. SKY refined G-banding karyotypes in all cases. Recurrent breakpoints involved bands Xp11, 8q24, 11q13, 12q13, 13q21, and 14q32. In addition, combined SKY and FISH analyses permitted us to identify a subset of patients harboring 22q11.2 rearrangements not involving the IGL locus. This finding suggests the presence of other gene(s) in band 22q11 that might be implicated in MM pathogenesis. Moreover, band 1p13 was identified as a novel partner of immunoglobulin ( IG) translocations in MM. Finally, using interphase FISH, we have detected interstitial deletions in 13q14 and 17p13, as well as cryptic translocations affecting IGH, which were neither detected by G-banding nor by SKY. The results of the present study suggest the existence of hitherto unknown nonrandom chromosomal changes that may play a role in the pathogenesis of MM. Our findings underline the importance of the combination of banding, SKY, and FISH analyses to increase the accuracy of karyotype interpretation in plasma cell neoplasias. ©2006 Elsevier Inc. All rights reserved.

Deletion (20q) as the sole abnormality in Waldenström macroglobulinemia suggests distinct pathogenesis of 20q11 anomaly

The deletion of the long arm of chromosome 20, or del(20q), is a common cytogenetic abnormality in various myeloid disorders but is less commonly seen in lymphoid neoplasms. Here we report a case of Waldenström macroglobulinemia with del(20q) as the sole cytogenetic anomaly. No translocation including immunoglobulin genes was identified by spectral karyotyping (SKY) analysis. Reviewing all 11 reported cases of plasma cell dyscrasia possessing sole del(20q), including our case, none of 4 cases with del(20q) as an initial anomaly developed myelodysplastic syndrome–acute myeloid leukemia (MDS/AML), but at least 3 cases with del(20q) appearing after chemotherapy developed MDS/AML at or after the time of del(20q). We propose that the del(20q) may have different clinical significance in plasma cell dyscrasia: one is when del(20q) appears at diagnosis and may involve the initial event of oncogenesis, and the other is when del(20q) appears after treatment and is associated with therapy-related and potential MDS/AML risk.

Evaluation of spectral karyotyping (SKY) in biodosimetry for the triage situation following gamma irradiation

Purpose: Biological dosimetry in an acute triage situation of radiation exposure is traditionally performed by scoring unstable dicentric chromosomal aberrations after conventional Giemsa staining, and more recently also by detection of chromosomal translocations after chromosome painting by fluorescence in situ hybridization (FISH). By spectral karyotyping (SKY) each chromosome can be painted in an individual colour, permitting the scanning for structural aberrations throughout the genome in each individual metaphase. Here we have evaluated the performance of SKY analysis in a simulated triage situation after gamma irradiation.Materials and methods: Peripheral leukocytes were irradiated by 60Co (0 – 5 Gy) and analysed by SKY, Giemsa staining and FISH painting of chromosomes 1, 2, and 3.Results: At 1 Gy and higher doses, dicentric aberrations (Dic+) as well as classical one- and two-way translocations were found in increasing and dose-dependent frequencies by SKY. The frequency of dicentrics detected by Giemsa was found to be significantly higher than the total aberrations detected by SKY (p<0.001), but did not differ significantly from that of FISH painting. The difference was mainly attributable to the low sensitivity of SKY to detect Dic+ following frequent lack of acentric fragments with matching chromosomal composition.Conclusions: The findings anticipate that radiation induced chromosomal aberrations may be more complex than expected from conventional and single chromosome painting analyses. While conventional Giemsa staining was found to be the method of choice for the triage situation, it is expected that extended SKY analysis will add to the knowledge of underlying mechanisms for irradiation associated chromosomal aberrations.

Translocation t(Y;14)(q12;q11): A New Abnormality Associated with r[i(8)(q10)] in T-Prolymphocytic Leukemia Studied by Classic Cytogenetics and Spectral Karyotype.

T-cell prolymphocytic leukemia (T-PLL) is a malignant proliferation of lymphoid cells with a mature postthymic phenotype. The disease is characterized by lymphadenopathy, splenomegaly, skin lesions, and elevated white blood cell count, and it is often resistant to conventional therapy. Classic cytogenetic studies have revealed the presence of complex karyotypes and some recurrent chromosomal abnormalities, of which the most frequent are t(14;14)(q11;q32), inv(14)(q11q32), t(X;14)(q28;q11), i(8)(q10), and t(8;8)(p12;q11). Conventional cytogenetic techniques are insufficient to fully characterize chromosomal abnormalities, especially complex and cryptic aberrations. In this report, we describe a case of T-PLL with t(Y;14)(q12;q11) and a ring chromosome r[i(8)(q10)] studied by classic cytogenetics and spectral karyotyping (SKY). These abnormalities have not been previously described in T-PLL. A 41-year-old man was admitted with night sweats, weight loss, lymphadenopathy and hepatosplenomegaly without skin rash. Peripheral blood counts were: platelets 128 x 109/L and leukocytes 93.6 x 109/L. Hemoglobin levels were 12.5 g/dL. Immunophenotypic studies showed the following results: CD2+, CD3+, CD5+, CD7+, CD4−, CD8+, T-cell receptor (TCR) a/b+, CD1-, and terminal deoxynucleotidyl transferase-negative (TdT-). The patient was treated with fludarabine and CHOP therapy, neither of which resulted in a response. The patient was then treated with alemtuzumab (Campath®) and complete resolution of lymphadenopathy and normalization of blood counts was achieved. Classic cytogenetic and SKY analyses were performed on a 3-day phytohemagglutinin-stimulated culture of peripheral blood lymphocytes. The observed karyotype was abnormal in 18 of the metaphases analyzed: 46, t(X;14)(q28;q11), t(Y;14)(q12;q11), r[i(8)(q10)]. The co-existence of 2 translocations involving both sex chromosomes in P-TLL is a rare occurrence; however, abnormalities involving band Xq28 (the site for the MCTP-1B1 gene which has homology to TCL-1 on 14q32.1) are common in T-PLL. In addition, translocations involving the 14q11-q13 region (TCRa and TCRd) result in juxtaposition of enhancer elements responsible for the expression of TCR genes next to different oncogene loci. This leads to disruption of transcriptional pathways involved in normal T cell development, and ultimately, leukemic transformation. The fibroblast growth factor receptor-1 (FGFR-1) gene, in 8p11, has been implicated in a recurrent breakpoint in a myeloproliferative syndrome associated with T-cell lymphoma and the monocytic leukemia zinc finger (MOZ) gene in acute myelogenous leukemia. The amplification of the q arm of chromosome 8 accompanied by deletion of 8p sequences distal to the breakpoints could mean that two events act synergistically to contribute to the malignant phenotype in T-PLL. It is possible to suggest that the loss of a tumor suppressor gene or activation of an oncogene on 8p cooperates with amplification of the q arm and/or the expression of TCL-1/MTCP-1B1 in T-PLL. Secondary abnormalities of chromosome 8 may play an important role in the development of T-PLL. In this case of T-PLL with rare cytogenetic abnormalities, treatment with alemtuzumab resulted in a clinical response.

A Trangenic Mouse Model of Plasma Cell Malignancy Shows Cytogenetic and Gene Expression Profiles Similar to Human Multiple Myeloma.

Multiple myeloma is an incurable plasma cell malignancy for which existing animal models are limited. Human plasma cell tumors are genetically diverse, with no single chromosomal abnormality defining the disease, however, dysregulation of the genes c-myc and bcl-xl are both commonly observed. We have previously shown that targeted expression of c-myc and bcl-xl transgenes in mouse plasma cells produces malignancy which displays features of human myeloma such as localization of tumor cells to the bone marrow and lytic bone lesions. Tumors are also present at extramedullary sites (Cheung et al., J. Clin. Invest. 113: 1763, 2004). Tumors rapidly develop (median 16 weeks) in 100% of mice, and can be adoptively transferred to syngeneic controls using as few as 1 million tumor cells to produce tumors in as few as 10 days. Adoptive transfer of similar cell numbers from younger double transgenic mice, without evidence of malignancy, results in increased tumor latency (>8 weeks) or the absence of tumor formation, suggesting that an accumulation of genetic changes is required for tumor development. In order to understand the specific genetic alterations required for tumor progression and for localization of tumors to the bone marrow vs extramedullary sites, we have undertaken a detailed analysis of plasma cell tumors in myc/bcl-xl mice and have begun to compare them with human multiple myeloma. Analysis of cell surface markers shows the majority of tumors have a plasmablast phenotype, expressing CD138+, B220+, CD38+, and CD19+. This result is confirmed by RT-PCR for B cell and plasma cell specific markers Pax5, Xbp1 and Blimp1, which can be detected in tumor samples. In addition, transcripts for Mip1α, EZH2, and Dusp6, genes shown to be upregulated in human myeloma, can also be detected in the mouse myc/bcl-xl tumors. Spectral karyotype analysis of metaphase chromosomes from primary tumor cell cultures demonstrates that a variety of chromosomal abnormalities are present in mouse tumors, including trisomies and translocations, similar to what is observed in human myeloma. The most frequently aberrant chromosomes are 12 and 16, followed by chromosomes 1 and 4. Interestingly, two common sites for translocations were identified; 12F which corresponds to the mouse immunoglobulin heavy chain locus, and 4D, which corresponds to a genomic region containing genes for plasma cell tumor susceptibility (Bliskovsky et al., PNAS 100:14982, 2003). Further characterization of these translocations are being done to identify the precise breakpoints involved, and analysis of gene expression by RT-PCR and microarray analysis will be correlated to specific chromosomal abnormalities. Additionally, global gene expression profiles from myc/bcl-xl tumor cell cultures have been compared to existing profiles of human myeloma (Zhan et al., Blood 99: 1745, 2002). Our preliminary comparison of gene expression profiles from myc/bcl-xl tumors to human myeloma tumors with high myc expression show the mouse tumors are more similar to human tumors than to normal plasma cells. These data suggest the myc/bcl-xl mouse tumors are similar to a subset of human myelomas, and will provide insight into the specific genes and pathways underlying human disease.

C-Myc rapidly induces acute myeloid leukemia in mice without evidence of lymphoma-associated antiapoptotic mutations

AbstractEctopic expression of c-Myc (Myc) in most primary cell types results in programmed cell death, and malignant transformation cannot occur without additional mutations that block apoptosis. The development of Myc-induced lymphoid tumors has been well studied and supports this model. Myc can be upregulated in acute myeloid leukemia (AML), but its exact role in myeloid leukemogenesis is unclear. To study its role in AML, we used a murine stem cell virus (MSCV) retroviral gene transfer/transplantation system to broadly express Myc in the bone marrow of mice either alone or in combination with antiapoptotic mutations. Myc expression in the context either of Arf/Ink4a loss or Bcl-2 coexpression induced a mixture of acute myeloid and acute lymphoid leukemias (AML+ALL). In the absence of antiapoptotic mutations however, all mice transplanted with MSCV-Myc (100%, n = 110) developed AML exclusively. MSCV-Myc-induced AML was polyclonal, readily transplantable, possessed an intact Arf-p53 pathway, and did not display cytogenetic abnormalities by spectral karyotyping (SKY) analysis. Lastly, we found that Myc preferentially stimulated the growth of myeloid progenitor cells in methylcellulose. These data provide the first direct evidence that Myc is a critical downstream effector of myeloid leukemogenesis and suggest that myeloid progenitors are intrinsically resistant to Myc-induced apoptosis. (Blood. 2005;106: 2452-2461)

A der(13)t(7;13)(p13;q14) with monoallelic loss of RB1 and D13S319 in myelodysplastic syndrome

Deletions or translocations of chromosome band 13q14, the locus of the retinoblastoma gene ( RB1), have been observed in a variety of hematological malignancies including myelodysplastic syndrome (MDS). We describe here a novel unbalanced translocation der(13)t(7;13)(p13;q14) involving 13q14 in a patient with MDS. A 66-year-old woman was diagnosed as having MDS, refractory anemia with excess of blasts (RAEB-1) because of 7.4% blasts and trilineage dysplasia in the bone marrow cells. G-banding and spectral karyotyping analyses showed complex karyotypes as follows: 46,XX,der(6)t(6;7)(q11;?),der(7)del(7)(?p13)t(6;7)(q?;q11)t(6;13)(q?;q?),der(13)t(7;13)(p13;q14). Fluorescence in situ hybridization (FISH) analyses demonstrated that one allele of the RB1 gene and the microsatellite locus D13S319, located at 13q14 and telomeric to the RB1 gene, was deleted. Considering other reported cases, our results indicate that submicroscopic deletions accompanying 13q14 translocations are recurrent cytogenetic aberrations in MDS. The RB1 gene or another tumor suppressor gene in the vicinity of D13S319, or both, may be involved in the pathogenesis of MDS with 13q14 translocations by monoallelic deletion.

Recurrent chromosomal rearrangements at bands 8q24 and 11q13 in gastric cancer as detected by multicolor spectral karyotyping.

To identify chromosomal translocations specific to gastric cancer (GC), spectral karyotyping (SKY) analysis was performed on established cell lines and cancerous ascitic fluids. SKY analysis of 10 established cell lines and seven cancerous ascitic fluid samples identified recurrent chromosomal breakpoints and translocations in GC, several of which involved chromosomal loci of oncogenes or tumor suppressor genes. A total of 630 chromosomal breaks were identified. Chromosome no.8 was the most frequently involved in rearrangements (65 breaks), followed by chromosomes no.11 (53), no. 1 (49), no. 7 (46), no. 13 (37), no. 3 (36), no. 17 (33), and no. 20 (29). Frequent breakpoints were detected in 8q24.1 (30 breaks), 11q13 (29), 13q14 (16), 20q11.2 (14), 7q32 (13), 17q11.2 (13), 18q21 (12), 17q23 (9), 18q11.2 (9). SKY analysis identified a total of 242 chromosomal rearrangements including 190 reciprocal and non-reciprocal translocations. The recurrent combinations of chromosomal bands involved in translocations were 8q24.1 and 13q14 (3 cases), 8q24.1 and 11q13 (3), 11q13 and 17q11.2 (2), and 18q11.2 and 20q11.2 (2). Our study validated the ability of SKY to characterize in detail the chromosomal rearrangements in solid tumors and derived cell lines. Moreover, fluorescence in situ hybridization helped to identify the insertions, translocations, and homogeneously staining regions of MYC and CCND1 gene loci. The non-random co-localization of certain cytogenetic bands suggests the importance of chromosomal translocations in gastric carcinogenesis, by serving as landmarks for the cloning of GC causing genes.

Chromosomal aberrations in cell lines derived from thyroid tumors spontaneously developed in TRβ PV/PV mice

The etiology and genetic alterations of follicular thyroid carcinoma are not well understood. By targeting a mutation (PV) into the thyroid hormone receptor β gene (TRβPV mouse), we created a knock-in mutant TRβ PV/PV mouse that spontaneously develop follicular thyroid carcinoma with progression to metastasis similar to human follicular thyroid carcinoma. This mouse model provides a valuable tool to ascertain the nature and the extent of genomic rearrangements that occur during carcinogenesis of the thyroid. Spectral karyotyping analysis (SKY) of seven cell lines derived from thyroid tumors developed in TRβ PV/PV mice showed that all of them had abnormal karyotypes, with chromosome number ranging from near-diploid (39–42 chromosomes) to hypotetraploid (63–79 chromosomes). These seven cell lines also exhibited a variety of structural chromosomal aberrations, including common recurrent translocations and deletions. This SKY analysis shows that the development and progression of follicular thyroid carcinoma in knock-in TRβ PV/PV mutant mice comprise recurrent structural and numerical genomic changes, some of which mimic those described in human thyroid cancer.

Clonal relationship of tumor nodules in hepatocellular carcinoma: a hierarchical clustering analysis of comparative genomic hybridization data

Information on the clonal relationship between tumor nodules in patients diagnosed with hepatocellular carcinoma (HCC) holds prognostic significance in the prediction of recurrence and postoperative treatment. Here, we investigated the clonal relationships in 11 focal nodules from 5 patients with HCC by comparative genomic hybridization (CGH) and spectral karyotyping. CGH analysis indicated tumor nodules in 5 (100%) of 5 cases to share similar patterns of genomic imbalances, suggesting nodules to be clonally related. This clonal relationship was further substantiated in 2 cases, where spectral karyotyping analysis indicated identical structural rearrangements between focal tumors. Few studies have attempted to differentiate multicentric growth from intrahepatic dissemination in HCC using CGH analysis; however, data interpretation remained subjective. In this study, we have extended our data to include published CGH findings on 57 nodules derived from 19 HCC cases. Simulation of CGH findings by unsupervised 2-way hierarchical clustering indicated tumor nodules in 20 (83.3%) of 24 cases to cluster concordantly, signifying a high incidence of clonal similarity. In sum, bioinformatic analysis of genomic profiles may represent a reliable research tool in analyzing clonal relationships among tumor nodules.

Establishment of a new human epithelioid sarcoma cell line, FU-EPS-1: Molecular cytogenetic characterization by use of spectral karyotyping and comparative genomic hybridization

A small number of human epithelioid sarcoma cell lines have been reported, but their characterization at a molecular cytogenetic level is not well known. In this study, a new permanent human cell line, FU-EPS-1, derived from a metastatic epithelioid sarcoma developing in the axillary lymph node of a 21-year-old man is described. This cell line was characterized by use of immunocytochemistry, conventional G-banding analysis, spectral karyotyping (SKY) and comparative genomic hybridization (CGH). FU-EPS-1 cells were round, polygonal or spindle shaped with an abundant cytoplasm, and have been maintained continuously in vitro for over 100 passages during more than 15 months. Histologic features of the heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original tumor, revealing a multinodular proliferation of eosinophilic epithelioid and spindle cells. Both in vitro and in vivo, the cells were immunopositive for cytokeratins (AE1/AE3 and CAM5.2), epithelial membrane antigen, vimentin and CD34, but were negative for desmin, S-100 protein, CD31 or factor VIII-related antigen. By G-banding and SKY analyses, FU-EPS-1 revealed a hyperdiploid karyotype with the following chromosomal abnormalities: +i(5)(p10), -8, +13, der(13)t(8;13)(q?;p11), +der(19)t(9;19)(?;?) and del(22)(q13). In addition, CGH analysis identified gains of 5p, 9q, 19q and 22q and a loss of 8p. This study demonstrates the value of molecular cytogenetic techniques such as SKY and CGH in defining genomic alterations in greater detail. The FU-EPS-1 cell line will be exceedingly useful for biologic and molecular pathogenetic studies of human epithelioid sarcoma.

Chromosomal instability and phenotypic plasticity during the squamous–spindle carcinoma transition: association of a specific T(14;15) with malignant progression

In mouse epidermal carcinogenesis, the latest stage of malignant progression involves the transition from squamous cell carcinoma to a highly aggressive type of tumor with spindle morphology. In this work, we have isolated a minor epithelial cell subpopulation (CarC-R) contained in the highly malignant spindle carcinoma cell line CarC. CarC-R exhibited a drastic reduction in tumorigenicity when compared with CarC, but CarC-R-induced tumors were mainly sarcomatoid, although they subsequently reverted to the epithelial phenotype when tumor explants were recultured in vitro. Several single-cell clones with either stable epithelial or fibroblastic phenotypes were isolated from an explanted CarC-R tumor (CarC-RT). All these cell lines contained the same specific point mutation in H-Ras codon 61, but while CarC spindle cells had lost the normal H-Ras allele, it was retained in CarC-R- and CarC-RT-derived cell lines. Furthermore, CarC cells have inactivated p16INK4a and p19INK4a/ARF transcription, while CarC-R and CarC-RT clones expressed p19 mRNA and protein but not p16. Altogether, these results suggest that CarC-R represents a precursor stage to CarC in malignant progression. Spectral karyotyping analysis revealed that CarC-R was highly aneuploid and contained many chromosomal abnormalities. In contrast, CarC had a diploid or tetraploid modal chromosome number and contained a specific T(14;15) translocation in all of the analysed metaphases. The T(14;15) translocation was present in only a minority (1.9%) of CarC-R cells, but it was widely spread in CarC-RT and its derived cell clones, regardless of their epithelial or fibroblastic phenotype, indicating that T(14;15) segregates with malignancy.

Identification of a ring chromosome with spectral karyotyping in a pleural synovial sarcoma

The origin of a ring chromosome in a monophasic synovial sarcoma of the diaphragmatic pleura of an 18-year-old man was investigated using spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). Conventional cytogenetic analysis revealed the following clonal karyotypic abnormalities: 47,Y,t(X;18)(p11.2;q11.2),t(11;19)(q12;q13.4),+12,−13,+r[6]. The SYT-SSX1 fusion transcript was detected with reverse transcriptase-polymerase chain reaction analysis. SKY analysis suggested that the ring chromosome was composed of material from chromosome 8. Subsequent FISH analysis with a whole-chromosome 8 paint probe confirmed the SKY results. This study demonstrates the usefulness of SKY as an adjunct for determining the chromosomal composition of ring chromosomes.

SKY and FISH analysis of radiation-induced chromosome aberrations: A comparison of whole and partial genome analysis

For a retrospective dose estimation of human exposure to ionising radiation, a partial genome analysis is routinely used to quantify radiation-induced chromosome aberrations. For this purpose, fluorescence in situ hybridisation (FISH) with whole chromosome painting probes for selected chromosomes is usually applied covering about 20% of the whole genome. Since genome-wide screening techniques like spectral karyotyping (SKY) and multiplex FISH (mFISH) have been developed the detection of radiation-induced aberrations within the whole genome has now become feasible. To determine the correspondence between partial and whole genome analysis of radiation-induced chromosome aberrations, they were measured comprehensively in this study using in vitro irradiated blood samples from three donors. We were able to demonstrate that comparable results can be detected with both approaches. However, complex aberrations might be misinterpreted by partial genome analysis. We therefore conclude that whole genome analysis by SKY is useful especially in the high dose range to correct aberration data for complex exchange aberrations.

The Antitumor Enediyne C-1027 Alters Cell Cycle Progression and Induces Chromosomal Aberrations and Telomere Dysfunction

This study examined the extent of chromosome instability induced in cultured human colon carcinoma HCT116 cells by the antitumor radiomimetic enediyne antibiotic C-1027. Spectral karyotype analysis showed frequent intrachromosomal fusions and fragmentations 26 hours after addition of as little as 0.035 nmol/L C-1027. When the concentration was increased to 0.14 nmol/L C-1027, 92% of cells showed chromosomal aberrations compared with only 2.9% after treatment with an equivalent growth inhibitory dose of ionizing radiation (20 Gy). Thus, chromosome misrejoining was associated to a much greater extent with C-1027-induced than with ionizing radiation-induced cell growth inhibition. Despite these aberrations, a large fraction of C-1027-treated cells progressed into G1. Comet analysis showed that these extensive chromosomal anomalies were not due to increased induction or reduced repair of C-1027-induced compared with ionizing radiation-induced strand breaks. Fluorescence in situ hybridization analysis showed that misrejoining of telomere repeats (i.e., chromosomes joined end to end at their telomeres or fused together after complete loss of telomere sequences) was observed within 26 hours of C-1027 addition. The extreme cytotoxicity of C-1027 may reflect both induction and erroneous repair of DNA double-strand break in the whole genome and/or in subgenomic targets such as telomere sequences.

Interstitial loss and gain of sequences on chromosome 22 in meningiomas with normal karyotype

In nearly half of sporadic low grade meningiomas no chromosome aberration can be detected. In the majority of the other half chromosome 22 is lost. In higher grade meningiomas this loss is followed by characteristic secondary chromosome aberrations. Regarding the molecular findings in Schwannomas, homozygous loss or mutation of the NF2 gene located on chromosome 22, was supposed also to be the primary event in meningioma development. However, in nearly all high grade but in only a minority of low grade meningiomas the loss of the NF2 protein is observed. Therefore, both the hypothetical combined heterozygous loss of or inactivation of two or more tumour suppressor genes (at least one of them located on chromosome 22) or the homozygous loss of a regulatory gene on chromosome 22 different from NF2 was discussed. In search for microdeletions or/and structural recombinations of chromosome 22 we investigated primary cell cultures of 43 meningiomas by conventional G-banding (26 without, 17 with loss of chromosome 22). Twenty-seven tumours were analysed with spectral karyotyping (SKY) and 16 with fluorescence in situ hybridisation (FISH) with DNA probes for the chromosomal regions of 22q11.2, 22q11.23q12.1, 22q12.1 and 22q13.3. SKY analysis confirmed G-banding data for chromosome 22 and could specify marker chromosomes and translocations containing material from chromosome(s) 22. Confirming our assumption microdeletions on chromosome 22 were detected by FISH in 6/8 cytogenetically non-aberrant meningiomas. Surprisingly, in 2/8 cases we observed gains of the 22q13.3 and in 2/8 gains of the 22q12.1 region. Here we present first evidence for an uncommon mechanism during early meningioma development at least for a meningioma subgroup: i) duplication and translocation of sequences from chromosome 22 to different chromosomes. ii) deletion of the original sequences on chromosome 22, resulting in disomy again (only visible as translocation in metaphase FISH). iii) loss of chromosome 22.

Transcriptional profiling on chromosome 19p indicated frequent downregulation of ACP5 expression in hepatocellular carcinoma

Chromosomal rearrangements unraveled by spectral karyotyping (SKY) indicated frequent chromosome 19 translocations in hepatocellular carcinoma (HCC). In an effort to characterize the aberrant 19 rearrangements in HCC, we performed positional mapping by fluorescence in-situ hybridization (FISH) in 10 HCC cell lines. SKY analysis indicated structural rearrangements of chromosome 19 in 6 cell lines, 4 of which demonstrated recurring 19p translocations with different partner chromosomes. Using fluorescence-labeled BAC probes, physical mapping indicated a breakpoint cluster between 19p13.12 and 19p12. A corresponding transcriptional mapping by cDNA array on 19p suggested the differential expression of a single downregulated gene ACP5 (tartrate-resistant acid phosphatase type 5). Quantitative RT-PCR confirmed the reduced expression of ACP5 and indicated a strong correlation of its repressed expression only in cell lines that contain a 19p rearrangement (p = 0.004). We further examined the expression of ACP5 in a cohort of 82 primary tumors and 74 matching nonmalignant liver tissues. In the primary HCC examined, a reduction of ACP5 transcripts by 2 to as much as 1,000-fold was suggested in 67% of tumors (55/82 cases). When compared to adjacent nonmalignant tissues, 46% of tumors (34/74 cases) demonstrated a lower expression level (p = 0.015). On closer examination, a high significance of ACP5 repression was suggested in the cirrhotic HCC subgroup that was derived from chronic hepatitis B infected patients (55%; 30/54 cases; p = 0.001). Functional examination of ACP5 ectopic expression in HCC cells further demonstrated a significant growth inhibitory effect of ACP5 on tumor cell survival (p < 0.001). In our study, the novel finding of common ACP5 downregulation in HCC may provide basis for further investigations on the role of acid phosphatase in hepatocarcinogenesis.

Genetic and Expression Profiles of Squamous Cell Carcinoma of the Head and Neck Correlate with Cisplatin Sensitivity and Resistance in Cell Lines and Patients

The choice of treatment for squamous cell carcinoma of the head and neck (SCCHN) is still primarily based on the tumor-node-metastasis classification. However, it is reasonable to believe that biological profiles of SCCHN may be independently associated with response to therapy. The aim of the present study was to examine genetic changes and gene expression profiles that might correlate with sensitivity to cisplatin [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] in 10 SCCHN cell lines. Five cisplatin-sensitive and five cisplatin-resistant cell lines [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] were studied by comparative genomic hybridization, spectral karyotyping, and cDNA microarray analysis (21,632 sequence-validated human cDNA; confirmation by reverse transcriptase-PCR for selected genes). For the MET proto-oncogene, which showed low expression in the chemosensitive cell lines, we did immunohistochemical staining on SCCHN of 29 patients who received induction chemotherapy. The five cisplatin-resistant cell lines showed significantly more genetic imbalances (regions of loss and amplification) and chromosomal abnormalities by comparative genomic hybridization and spectral karyotyping, respectively, than did the five cisplatin-sensitive cell lines. Microarray studies identified approximately 60 genes that clearly distinguish between the two groups of cell lines. Some of these genes are known to be involved in tumor progression, metastasis, and drug resistance. We identified low expression of c-met (immunohistochemistry) as a predictive factor for complete response in nondiploid tumors (P = 0.026). We conclude that cisplatin sensitivity and resistance are related to distinctive differences in the genetic and expression profiles in individual SCCHN tumor cell lines and in SCCHN patients. The genes we have identified may serve as potential targets for novel treatment strategies.

Primary effusion lymphoma of the pericardial cavity carrying t(1;22)(q21;q11) and t(14;17)(q32;q23)

We report a case of primary effusion lymphoma (PEL) in a 75-year-old woman without human immunodeficiency virus or hepatitis C virus, which presented as fever, chest pain, and pericardial effusion. The lymphoma cells were positive for CD20 and CD79a, and were negative for CD3 and CD10. Genomic human herpes virus 8 (HHV-8) and Epstein-Barr virus were not detected in the lymphoma cells. Cytogenetic analysis showed complex abnormalities by the G-banding technique, and spectral karyotyping (SKY) analysis provided more detailed characterization of the chromosomal aberrations, including t(1;22)(q21;q11) and t(14;17)(q32;q23). We did not detect C-MYC gene rearrangement or BCL-2 expression. She was treated successfully with six courses of the CHOP regimen. The present case demonstrated a rare category of PEL that is not associated with HHV-8 or C-MYC gene rearrangement. In addition, SKY analysis disclosed cryptic chromosomal abnormalities involving 1q21 and 17q23.

Recurrent Chromosomal Rearrangements and a Homozygous Deletion Spanning 1-Mb Region at 10p11.2 in Adult T-Cell Leukemia/Lymphoma.

Cytogenetic analyses and their correlation with clinical features are still limited in mature T-cell malignancies. The cytogenetics of adult T-cell leukemia/lymphoma (ATLL) is complicated by many structural and numerical abnormalities of chromosomes. Thus, it is occasionally difficult to define karyotype of ATLL only by chromosomal banding method. Using multicolor spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH), we performed molecular-cytogenetic studies on 38 patients with adult T-cell leukemia/lymphoma (ATLL) and 7 established cell lines. In SKY analysis, chromosomal bands involved in rearrangements were identified based on the inverted images of DAPI staining. Chromosome no.7 (25 cases) and no.14 (30) were frequently involved in structural rearrangements including translocations, inversions, insertions, isochromosomes, and deletions. Breakages frequently occurred at 10p11-13 (21 cases, 46.6%), and 14q11.2 (16, 35.6%), 14q32 (12, 26.7%), 7q22 (10, 22.2%), 9q22 (10, 22.2%), 13q14 (9, 20.0%), 22p11 (8, 17.7%), 8p11 (7, 15.5%), 18p11 (7, 15.5%), 21q22 (7, 15.5%) and 19q13 (6, 13.3%). Recurrent partners associated with 10p11.2-13 rearrangements were 21q22 (3 cases), 13q14 (2) and 14q32.1 (2). Partner chromosomal breakpoints involving 14q11.2 rearrangements were 14q32 (3 cases), 8p11.2-21 (3), and 11q13.3 (2), while those involved in 14q32 rearrangements were 14q11 (3) and 10p11-13 (2). Using FISH with 37 bacterial artificial chromosome clones, we analyzed breakpoints at 10p11.2-13 in two cell lines KOB4 and KK1 and one patient (HH) by walking a segment ranging from 10p13 to 10p11.2. Among 37 BAC clones used as FISH probes, 9 were assigned to 10p15, 3 to 10p14-13, and the remaining 25 to 10p11.2-12. Large genomic deletions were detected at 10p11.2 on der(10) in two cell lines. In patient HH, a homozygous deletion was identified at 10p11.2, spanning the region of 1 Mb in size. Chromosome 14q32 translocation was associated with shorter survival (median survival time; 4.2 months vs. 8.0 months), although statistically not significant. As for 10p11.2-13 and 14q11.2 rearrangements, there was no significant difference of survival between patients with and without respective abnormalities. In conclusion, SKY and FISH analysis identified recurrent chromosomal rearrangements and a homozygous deletion at 10p11.2 in ATLL. Our molecular analysis is now focusing on this genomic region to clone genes associated with development of ATLL.