Tumor samples of 53 patients with t(11;14)-positive mantle cell lymphomas (MCLs) were analyzed by matrix-based comparative genomic hybridization (matrix-CGH) using a dedicated DNA array. In 49 cases, genomic aberrations were identified. In comparison to chromosomal CGH, a 50% higher number of aberrations was found and the high specificity of matrix-CGH was demonstrated by fluorescence in situ hybridization (FISH) analyses. The 11q gains and 13q34 deletions, which have not been described as frequent genomic aberrations in MCL, were identified by matrix-CGH in 15 and 26 cases, respectively. For several genomic aberrations, novel consensus regions were defined: 8p21 (size of the consensus region, 2.4 megabase pairs [Mbp]; candidate genes: TNFRSF10B, TNFRSF10C, TNFRSF10D); 10p13 (2.7 Mbp; BMI1); 11q13 (1.4 Mbp; RELA); 11q13 (5.2 Mbp; CCND1); 13q14 (0.4 Mbp; RFP2, BCMSUN) and 13q34 (6.9 Mbp). In univariate analyses correlating genomic aberrations and clinical course, 8p- and 13q14- deletions were associated with an inferior overall survival. These data provide a basis for further studies focusing on the identification of pathogenetically or clinically relevant genes in MCL.

Multicolor fluorescence in situ hybridization (FISH) assays are nowadays indispensable for a precise description of complex chromosomal rearrangements. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole chromosome painting probes in multiplex-FISH (M-FISH) and spectral karyotyping (SKY). Since then different approaches for chromosomal differentiation based on multicolor-FISH (mFISH) assays have been described. Predominantly, they have been established to characterize marker chromosomes identified in conventional banding analysis. Their characterization is of high clinical impact and is the requisite condition for further molecular investigations aimed at the identification of disease-related genes. Here we present a review on the available mFISH methods including their advantages, limitations and possible applications.

Intratumor heterogeneity in chromosomal aberrations is believed to represent a major challenge in the treatment of cancer. The aim of our work was to assess the chromosomal heterogeneity of advanced cervical carcinomas and to distinguish aberrations that had occurred at a late stage of the disease from early events. A total of 55 biopsies, sampled from 2-4 different sites within 20 tumors, were analyzed by use of comparative genomic hybridization. Heterogeneous aberrations were identified as those present in at least 1 of the biopsies and which were not seen, nor seen as a tendency, in the others of the same tumor. The homogeneous aberrations were those seen in all biopsies of the tumor. The most frequent homogeneous aberrations were gain of 3q (65%), 20q (65%) and 5p (50%), indicating that these are early events in the development of the disease. Chromosomal heterogeneity was observed in 11 tumors. The most frequent heterogeneous aberrations were loss of 4p14-q25 (60% of 10 cases with this aberration), and gain of 2p22-pter (50% of 6 cases), 11qcen-q13 (33% of 9 cases) and 8q (27% of 11 cases), suggesting that these events promote progression at a later stage. Many of the heterogeneous regions contained genes known to influence the prognosis of cervical cancer, such as 7p (EGFR), 8q (c-MYC), 11qcen-q13 (CCND1) and 17q (ERBB2). Three evolution sequences for the subpopulations in the heterogeneous tumors were identified: a serial, a parallel and a mixed sequence. In 2 tumors with a serial sequence, it was indicated that the aberrations +8 and -X had occurred after the other heterogeneous aberrations and hence were the aberrations most recently formed. Our results suggest pronounced chromosomal instability in advanced cervical carcinomas. Moreover, aggressive and treatment-resistant subpopulations may emerge at a late stage and possibly contribute to a poor prognosis of the advanced stages.

<p>Persistant infection with human papillomavirus (HPV) of the uterine cervix is related with cytological atypia (SIL), the oncogenic potential of which is unclear in a given time point of monitoring. HPV-induced genetic instability result in polyploidization as well as in low frequency random chromosome aberrations in squamous cells. In the present work we analyzed whether highly polyploid/aneuploid cells reflect genomic changes at the chromosomal level. 13 samples with the cytological diagnosis of HSIL were analyzed for HPV type and nuclear DNA content measured by laser scanning cytometry (LSC). Hyperdiploid cells with &gt;5c and with &gt;9c DNA content were further analyzed for numerical aberrations of the chromosomes 3 and 17 by fluorescence in situ hybridization (FISH) following repositioning. Cells with &gt;5c DNA content were found more frequently than cells with &gt;9c DNA content (5-98 and 1-44 cells, respectively). The FISH analysis demonstrated frequent polysomies, however, the rate of aneusomy (other than 2, 4, 8 or 16 chromosome copies) was significantly higher in cells with &gt;9c DNA content than in cells with &gt;5c DNA content or the normal diploid cells. The imbalance of chromosome 3 and 17 copy number was also increased in cells with &gt;9c DNA content. Moreover, in three out of the 13 analyzed HSIL samples, recurrent abnormal chromosome 3/17 ratio was demonstrated in a significant part of the cells, indicating a common origin of these cells. Highly polyploid/aneuploid cells in HSIL accumulate cytogenetic aberrations detectable by FISH analysis. These cells may reflect early changes with tumorigenic potential in a very concentrated fashion.</p>

Chromosome 17 is severely rearranged in breast cancer. Whereas the short arm undergoes frequent losses, the long arm harbors complex combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic array-comparative genomic hybridization and of associated RNA expression changes by cDNA arrays. We built a genomic array covering the entire chromosome at an average density of 1 clone per 0.5 Mb, and patterns of gains and losses were characterized in 30 breast cancer cell lines and 22 primary tumors. Genomic profiles indicated severe rearrangements. Compiling data from all samples, we subdivided chromosome 17 into 13 consensus segments: 4 regions showing mainly losses, 6 regions showing mainly gains, and 3 regions showing either gains or losses. Within these segments, smallest regions of overlap were defined (17 for gains and 16 for losses). Expression profiles were analyzed by means of cDNA arrays comprising 358 known genes at 17q. Comparison of expression changes with quantitative anomalies revealed that about half of the genes were consistently affected by copy number changes. We identified 85 genes overexpressed when gained (39 of which mapped within the smallest regions of overlap), 67 genes underexpressed when lost (32 of which mapped to minimal intervals of losses), and, interestingly, 32 genes showing reduced expression when gained. Candidate genes identified in this study belong to very diverse functional groups, and a number of them are novel candidates.

Telomere length was evaluated by terminal repeat fragment method (TRF) in 50 patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) arising from MDS and in 21 patients with untreated primary AML to ascertain, whether telomere erosion was associated with progression of MDS towards overt leukemia. Heterogeneity of TRF among MDS FAB subgroups (P=0.004) originated from its shortening in increased number of patients during progression of the disease. Chromosomal aberrations were present in 32% MDS patients with more eroded telomeres (P=0.022), nevertheless a difference between mean TRF in the subgroups with normal and abnormal karyotype diminished during progression of MDS. A negative correlation between individual TRF and IPSS value (P=0.039) showed that telomere dynamics might serve as a useful prognostic factor for assessment of an individual MDS patient’s risk and for decision of an optimal treatment strategy.

A number of genetic abnormalities have been detected in multiple myeloma (MM) using cytogenetic techniques. The prominent abnormalities are deletions of 13q and translocations affecting the IgH locus on 14q32. The recurrence of chromosomal abnormalities in MM suggests a specific role for them concerning its pathogenesis. We performed comparative genomic hybridization (CGH) on samples from 53 patients with MM and 4 with monoclonal gammopathies of undetermined significance. In 31 cases (54%), normal ratio profiles were found, whereas 26 cases (46%) had aberrant profiles. The most common aberrations were gains of 9p (n = 14), 11 (n = 9), and 21q (n = 5) and loss of 22 (n = 7). In earlier reports on cytogenetics of lymphomas, gains of 9p are described as characteristic of primary mediastinal B-cell lymphoma, but the consensus region is smaller than in the present study (9p23pter vs 9p13pter). Therefore, we suggest a stronger genetic affinity between MM and primary mediastinal B-cell lymphoma than MM and other B-cell lymphomas. To support this suggestion, more molecular cytogenetic techniques and expression analyses have to be performed.

Digital object identifier (DOI): 10.1309/5KWK-P6UK-GNXX-HMYH

BACKGROUND AND OBJECTIVES: Nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) showed recurrent rearrangement of the BCL6 which is gene detected in 48% of cases analyzed by interphase-fluorescent in situ hybridization (FISH). These findings point to a critical role for BCL6 in the development of this distinct Hodgkin's lymphoma. We present our results of metaphase-FISH analyses aimed at identifying and characterizing BCL6-related chromosomal translocations in NLPHL. DESIGN AND METHODS: Four NLPHL cases with available metaphase spreads obtained either at the time of diagnosis or during progression to diffuse large B-cell lymphoma (DLBCL) were collected. Extensive metaphase-FISH analysis was performed to identify the affected partner chromosomes and reciprocal breakpoints. RESULTS: Each of the analyzed NLPHL cases showed a different type of BCL6 rearrangement that included the t(3;22)(q27;q11) targeting immunoglobulin (IG) alpha chain locus, complex t(3;7;3;1) involving the 7p12/Ikaros gene region, t(3;9)(q27;p13) affecting an unknown gene in vicinity of PAX5, and t(3;4)(q27;q32) showing the alternative 3q27 breakpoint outside BCL6 and possibly, an internal deletion of BCL6. Retrospective interphase-FISH analysis of 2 cases with subsequent DLBCL showed the same type of BCL6 translocation as in NLPHL samples. INTERPRETATION AND CONCLUSIONS: The spectrum of BCL6 aberrations targeting IG as well as non-IG loci in NLPHL is similar to that found in DLBCL. These findings further support the hypothesis of a germinal center B-cell-derived origin of NLPHL and of a relationship between these two lymphoma entities. This latter issue is additionally illustrated in two NLPHL patients who subsequently developed DLBCL and showed the same type of BCL6 rearrangements in both tumors.

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.

The 8p11-21 region is a frequent target of alterations in breast cancer and other carcinomas. We surveyed 34 breast tumor cell lines and 9 pancreatic cancer cell lines for alterations of this region by use of multicolor fluorescence in situ hybridization (M-FISH) and BAC-specific FISH. We describe a recurrent chromosome translocation breakpoint that targets the NRG1 gene on 8p12. NRG1 encodes growth factors of the neuregulin/heregulin-1 family that are ligands for tyrosine kinase receptors of the ERBB family. Breakpoints within the NRG1 gene were found in four of the breast tumor cell lines: ZR-75-1, in a dic(8;11); HCC1937, in a t(8;10)(p12;p12.1); SUM-52, in an hsr(8)(p12); UACC-812, in a t(3;8); and in two of the pancreatic cancer cell lines: PaTu I, in a der(8)t(4;8); and SUIT-2, in a del(8)(p). Mapping by two-color FISH showed that the breaks were scattered over 1.1 Mb within the NRG1 gene. It is already known that the MDA-MB-175 breast tumor cell line has a dic(8;11), with a breakpoint in NRG1 that fuses NRG1 to the DOC4 gene on 11q13. Thus, we have found a total of seven breakpoints, in two types of cancer cell lines, that target the NRG1 gene. This suggests that the NRG1 locus is a recurring target of translocations in carcinomas. PCR analysis of reverse-transcribed cell line RNAs revealed an extensive complexity of the NRG1 transcripts but failed to detect a consistent pattern of mRNA isoforms in the cell lines with NRG1 breakpoint.

The detection of disseminated tumor cells (DTCs) in the hematopoetic system is important for various reasons. It is essential for tumor staging. According to the International Neuroblastoma Staging System (INSS) only the cytomorphological examination of bone marrow smears is accepted despite the fact that an infiltrate below 0.1%, can hardly be detected and even infiltrates of more than 10% are sometimes overlooked. Another important aspect is the monitoring of the disease response to cytotoxic drugs by quantifying DTCs. Moreover, bone marrow aspirates represent an ideal source to determine the genetic and biological make up of DTCs at diagnosis and during follow up. Key issues that can be tested on DTCs are: determination of the proliferation capacity, the apoptotic rate, the drug sensitivity etc. The prerequisite for such a bone-marrow diagnosis, however, is the unequivocal identification of disseminated tumor cells. Thus, in order to avoid false positive and false negative results, which are a risk in bone-marrow diagnostics, a system was developed to distinguish tumor cells from non-neoplastic cells and to facilitate the gain of insights into the biological make-up of tumor cells more easily.

We describe a patient initially diagnosed with a chronic myeloproliferative disorder in the accelerated phase. Cytogenetic analysis showed the presence of two independent clones. One clone contained a typical Philadelphia (Ph) chromosome due to t(9;22)(q34;q11), as the sole abnormality which was proven molecularly to result in the b2a2-BCR/ABL fusion. The other clone displayed a complex karyotype with several structural and numerical aberrations including trisomy 11 and 22 but lacking a t(9;22) or any other structural abnormalities involving chromosomes 9 and 22. Fluorescence in situ hybridization demonstrated that the t(9;22) was present only in cells with two copies of chromosomes 11 and 22. In contrast, cells with trisomies 11 and 22 lacked evidence for a BCR/ABL fusion. Based on the genetic findings, simultaneous chronic and acute myelocytic leukemias were diagnosed rather than a blastic phase of a chronic myelocytic leukemia.

<p>The BRCA2 gene is mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair and transcriptional regulation. Here we identify a protein, EMSY, which binds BRCA2 within a region (exon 3) deleted in cancer. EMSY is capable of silencing the activation potential of BRCA2 exon 3, associates with chromatin regulators HP1beta and BS69, and localizes to sites of repair following DNA damage. EMSY maps to chromosome 11q13.5, a region known to be involved in breast and ovarian cancer. We show that the EMSY gene is amplified almost exclusively in sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, EMSY amplification is associated with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic value. The remarkable clinical overlap between sporadic EMSY amplification and familial BRCA2 deletion implicates a BRCA2 pathway in sporadic breast and ovarian cancer.</p>

Diagnostic possibilities of CGH and M-FISH techniques for detection of submicroscopic chromosomal imbalancies were compared on the basis of two cases of t(X;Y) and one case of marker chromosome. In cases with t(X;Y), the sequences specific for chromosome Y were detected by PCR and CGH, but the localisation of these sequences on the short arm of chromosome X was confirmed by the FISH technique, employing two Yp-specific probes for SRY and TSPY genes. Significant differences between above cases were revealed in the size of Yp chromosome fragments translocated on chromosome X. An extra material of chromosome marker could not be identified by classical banding and FISH techniques and it was only CGH and M-FISH techniques that enabled detecting the chromosomal origin of the marker. The applied CGH technique enabled finding subtle chromosomal imbalancies in the presented cases with a resolution of approximately 3 Mbp.

We report on the fourth known case with an interstitial deletion in 15q21. In the present case the breakpoints have been determined by GTG-banding, microdissection and the recently developed multicolor banding (MCB) technique as 15q21.1-q21.3. Common features in all four cases are mental retardation, growth retardation, a beak-like nose with hypoplastic alae nasi and a thin upper lip. Additional frequent features are small hands and feet, hypotonia, low hair implantation, low set ears, clinodactyly and obesity. The possibility that a critical region for a new microdeletion-syndrome is situated in 15q21 is discussed.

<p>The sensitive detection of bone marrow involvement is crucial for tumor staging at diagnosis and for monitoring of the therapeutic response in the patient's follow-up. In neuroblastoma, only conventional cytomorphological techniques are presently accepted for the detection of bone marrow involvement, yet since the therapeutic consequences of the bone marrow findings may be far-reaching, the need for highly reliable detection methods has become evident. For this purpose, we developed an automatic immunofluorescence plus FISH (AIPF) device which allows the exact quantification of disseminated tumor cells and the genetic verification in critical cases. In this study, the power of the immunofluorescence technique is compared with conventional cytomorphology. 198 samples from 23 neuroblastoma patients (stages 4 and 4s) at diagnosis and during follow-up were investigated. At diagnosis, 45.6% of the samples (26 of 57) which were positive by AIPF investigation were negative by cytomorphology. During follow-up, 74.2% (49 of 66) of AIPF-positive samples showed no cytological signs of tumor cell involvement. False negative morphological results were found in up to 10% of tumor cell content. A tumor cell infiltrate below 0.1% was virtually not detectable by conventional cytomorphology. Using the sensitive immunofluorescence technique, the analysis of only two instead of four puncture sites did not lead to false negative results. Thus, the immunofluorescence technique offers an excellent tool for reliable detection and quantification of disseminated tumor cells at diagnosis and during the course of the disease.</p>