Slug/Pcad pathway controls epithelial cell dynamics in mammary gland and breast carcinoma.

Mammary gland morphogenesis results from the coordination of proliferation, cohort migration, apoptosis and stem/progenitor cell dynamics. We showed earlier that the transcription repressor Slug is involved in these functions during mammary tubulogenesis. Slug is expressed by a subpopulation of basal epithelial cells, co-expressed with P-cadherin (Pcad). Slug-knockout mammary glands showed excessive branching, similarly to Pcad-knockout. Here, we found that Slug unexpectedly binds and activates Pcad promoter through E-boxes, inducing Pcad expression. We determined that Pcad can mediate several functions of Slug: Pcad promoted clonal mammosphere growth, basal epithelial differentiation, cell-cell dissociation and cell migration, rescuing Slug depletion. Pcad also promoted cell migration in isolated cells, in association with Src activation, focal adhesion reorganization and cell polarization. Pcad, similarly to Slug, was required for in vitro 3D tubulogenesis. Therefore, Pcad appears to be responsible for epithelial-mesenchymal transition-linked plasticity in mammary epithelial cells. In addition, we found that genes from the Slug/Pcad pathway components were co-expressed and specifically correlated in human breast carcinomas subtypes, carrying pathophysiological significance.

Transcription Factor Networks derived from Breast Cancer Stem Cells control the immune response in the Basal subtype.

Breast cancer is the most common cancer in women worldwide and metastatic dissemination is the principal factor related to death by this disease. Breast cancer stem cells (bCSC) are thought to be responsible for metastasis and chemoresistance. In this study, based on whole transcriptome analysis from putative bCSC and reverse engineering of transcription control networks, we identified two networks associated with this phenotype. One controlled by SNAI2, TWIST1, BNC2, PRRX1 and TBX5 drives a mesenchymal or CSC-like phenotype. The second network is controlled by the SCML4, ZNF831, SP140 and IKZF3 transcription factors which correspond to immune response modulators. Immune response network expression is correlated with pathological response to chemotherapy, and in the Basal subtype is related to better recurrence-free survival. In patient-derived xenografts, the expression of these networks in patient tumours is predictive of engraftment success. Our findings point out a potential molecular mechanism underlying the balance between immune surveillance and EMT activation in breast cancer. This molecular mechanism may be useful to the development of new target therapies.

Cyclin L1 (CCNL1) gene alterations in human head and neck squamous cell carcinoma.

We evaluated the expression and amplification of cyclin L1 (CCNL1) gene, a potential oncogene localised in the commonly amplified 3q25-28 region, in human head and neck squamous cell carcinomas (HNSCCs). Overexpression was observed in 55 out of 96 cases (57%) and amplification in nine out of 35 tumours (26%) with no relationships to the clinico-pathological parameters. The Cyclin L1 antibody we developed labels nuclear speckles in tumour cells compatible with a role for CCNL1 in RNA splicing.

Primary tumour genetic alterations and intra-tumoral heterogeneity are maintained in xenografts of human colon cancers showing chromosome instability.

Evaluation of the role of clonal heterogeneity in colon tumour sensitivity/resistance to drugs and/or in conferring metastatic potential requires an adequate experimental model in which the tumour cells maintain the initial genetic alterations and intra-tumoral heterogeneity through maintenance of the genetic clones present in the initial tumour. Therefore, we xenografted subcutaneously into nude mice seven human colonic tumours (from stages B1 to D) that showed chromosome instability and transplanted them sequentially for up to 14 passages. Maintenance after xenografting of the genetic alterations present in the initial tumours was scored by allelotype studies targeting 45 loci localized on 18 chromosomes. We show that xenografting does not alter the genetic or the histological profiles of the tumours even after 14 passages. Screening of the entire genome of one tumour by comparative genome hybridization also showed overall stability of the alterations between the initial and the xenografted tumour. In addition, intra-tumoral heterogeneity was maintained over time, suggesting that no clonal selection occurred in the nude mice. The observation that some loci showed partial allelic imbalance in the initial tumour but loss of heterozygosity after the first passage in nude mice when all the normal cells were lost may allow identification of interesting genetic defects that could be involved in tumour expansion. Thus, sequential xenografts of colon tumours will provide a powerful model for further study of tumour clonality and for the identification of genetic profiles responsible for differential resistance to therapeutic treatments. Our data also suggest that tumour expansion can result from alterations in several distinct genetic pathways.

A human RNA polymerase II subunit is encoded by a recently generated multigene family.

BACKGROUND: The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes. RESULTS: While those characterized so far for the human (h) RPB are also unique, we show that hRPB subunit 11 (hRPB11) is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex; the second generates polypeptides hRPB11balpha and hRPB11bbeta through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR). Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11balpha is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G), and encodes a single polypeptide which is identical to subunit hRPB11a. CONCLUSIONS: The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.

A simple specific pattern of chromosomal aberrations at early stages of head and neck squamous cell carcinomas: PIK3CA but not p63 gene as a likely target of 3q26-qter gains.

Low-grade head and neck squamous cell carcinomas without lymph node involvement or distant metastasis (N(0)M(0)) were screened for chromosomal imbalances by comparative genomic hybridization (CGH). pT(1-2) tumors contain a low number of aberrations (average number, 4.3; 15 cases), in contrast to pT(3) tumors (average number, 11.8; 6 cases), and exhibit a specific CGH pattern, affecting three chromosomes: partial or total 3q gain and/or 3p loss (73% of cases), 8q gain (47%), and 11q13 gain (27%). Thus, these changes represent early events in the pathogenesis of low-grade tumors. Cytogenetic exploration of chromosome 3 aberrations in head and neck cell lines suggests that the formation of an isochromosome 3q is one intermediate mechanism leading to 3p losses and/or 3q gains. On the long arm of chromosome 3, most of tumors exhibit low-level gains of large segments, involving systematically the 3q26-qter area, but with two alternative smallest region overlaps at 3q26 and 3q28-qter. We decided to refine the mapping of 3q26-qter gains by using fluorescence in situ hybridization on tumor nuclei, with clones containing two outstanding positional and functional candidate genes, PIK3CA and p63, located respectively at 3q26 and at 3q28. Although PIK3CA or p63 were preferentially gained in few cases (4 of 45), both genes were over-represented in 27 of 45 low-grade N(0)M(0) carcinomas analyzed by CGH or fluorescence in situ hybridization. To evaluate the relative contribution of PIK3CA and p63 in the pathogenesis of head and neck carcinomas displaying a 3q gain, we measured their respective transcription levels in tumors with previously determined gene copy number. DNp63, the predominant p63 transcript, is overexpressed in tumors compared with normal tissues, but its expression level is independent to gene copy number. In contrast, a significant PIK3CA overexpression is associated with increased gene dosage. These results indicate that PIK3CA, contrary to DNp63, may participate to the progression of head and neck tumors consequent to a low-level 3q over-representation. Interestingly, survival analysis using CGH suggested, in accordance with previous data, that 3q26 gain, the locus of PIK3CA, could predict clinical outcome for early disease tumors. This prompts us to pursue 3q26 (or PIK3CA) prognostic evaluation in a larger population of head and neck squamous cell carcinomas.

Genomic structure and chromosomal mapping of the gene coding for ICBP90, a protein involved in the regulation of the topoisomerase IIalpha gene expression.

We have recently identified a novel CCAAT box binding protein (ICBP90) involved in the regulation of topoisomerase IIalpha gene expression. We have observed that it is expressed in non-tumoral proliferating human lung fibroblast cells whereas in HeLa cells, a tumoral cell line, ICBP90 was still present even when cells were at confluence. In the present study, we have determined the ICBP90 gene structure by screening of a human placenta genomic library and PCR analysis. We report that the ICBP90 gene spans about 35.8 kb and contains six coding exons named A to F. In the 5' upstream sequence of the region containing the coding exons, two additional exons (I and II) were found. Additionally, an internal splicing site was found in exon A. A promoter region, including three putative Sp1 binding sites between exons I and A, was identified by transient transfection. Northern blot analysis of several cancer cell lines revealed the existence of two ICBP90 mRNA species of 5.1 and 4.3 kb that are transcribed from the gene. The relative amounts of these mRNAs depended on the cell type. In MOLT-4 cells and Burkitt's lymphoma Raji cells, the 4.3 kb or the 5.1 kb transcripts were mainly observed, respectively. In other cell lines, such as HL-60 cells, chronic myelogenous leukaemia K-562, lung carcinoma A549, HeLa or colorectal SW480, both 4.3 and 5.1 kb forms of ICBP90 mRNA could be detected. Interestingly, western blot analysis showed several ICBP90 protein bands in HeLa but only a single band in MOLT-4 cell extracts. Taken together our results are consistent with the ICBP90 gene exhibiting alternative splicing and promoter usage in a cell-specific manner.

Diagnosis of aneuploidy in archival, paraffin-embedded pregnancy-loss tissues by comparative genomic hybridization.

OBJECTIVE: To evaluate the detection of aneuploidy in archival tissues from miscarriages by a method that uses microdissection and DNA extraction of villus cells from paraffin blocks, followed by universal DNA amplification and comparative genomic hybridization (CGH). DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Nine archival tissues from cases of spontaneous abortion with trisomy 16 (two cases), trisomy 21 (three cases), trisomy 22 (two cases), triploidy (one case), and monosomy X (one case). INTERVENTION(S): Villus DNA was extracted from microdissected, formalin-fixed, paraffin-embedded tissues. Aneuploidy was detected by CGH after universal amplification of the DNA with the use of degenerate oligonucleotide-primed polymerase chain reaction. MAIN OUTCOME MEASURE(S): Detection of aneuploidy in archival pregnancy-loss tissues using CGH. RESULT(S): In all nine cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH. In six of nine cases, the chromosomal abnormality detected by conventional cytogenetic analysis was identified by CGH: trisomy 16 (2/2), trisomy 21 (3/3), and trisomy 22 (1/2). One case of each of the following was not detectable: triploidy (1/1), monosomy X (1/1), and trisomy 22 (1/2). CONCLUSION(S): We propose CGH as a method for determination of aneuploidy in pregnancy-loss archival tissues when conventional cytogenetic analysis is unsuccessful or when it was not performed when fresh tissue was available.

Genetic abnormalities detected by comparative genomic hybridization in a human endometriosis-derived cell line.

Comparative genomic hybridization (CGH) was used in parallel with fluorescence in-situ hybridization (FISH) and conventional karyotyping to perform a genome-wide survey of DNA gains and losses in the endometriosis-derived permanent cell line, FbEM-1. The cytogenetic analysis showed a complex karyotype with numerical changes and multiple chromosome aberrations, including the der(1) complement marker exhibiting a large homogenous staining region (HSR). The chromosomal rearrangement interpreted as der(5) t(5;6)(q34;p11) was found in the majority of the metaphases indicating a clonal abnormality. Repeated CGH experiments demonstrated over-representation of chromosomes 1, 2, 3, 5, 6p, 7, 16, 17q, 20, 21q and 22q, while chromosomes 6q, 9, 11p, 12, 13q, 18 and X were under-represented. Using DNA from the original endometriotic tissues, including a peritoneal implant and ovarian endometrioma, CGH analysis revealed loss of DNA copy number on 1p, 22q and chromosome X, while gain was found on chromosomal arms 6p and 17q. FISH analysis confirmed that the gain at 17q includes amplification of the proto-oncogene HER-2/neu in 16% of the FbEM-1 nuclei and in 12% of cells from the primary ovarian endometrioma tissue. These findings demonstrate that FbEM-1 cells share certain molecular cytogenetic features with the original tissue and suggest that chromosomal instability is important in the development of endometriosis.

Detection of DNA copy number changes in human endometriosis by comparative genomic hybridization.

Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.

Analysis of ovarian borderline tumors using comparative genomic hybridization and fluorescence in situ hybridization.

It is unclear whether ovarian borderline tumors (tumors of low malignant potential) are independent entities or whether they are part of a continuum of tumor progression that culminates in ovarian carcinoma. Little is known about genetic abnormalities in borderline tumors because of the difficulty of growing them in culture for chromosome studies, and because the low ratio of tumor to nontumor cells can interfere with molecular genetic examination. To circumvent these problems, we performed comparative genomic hybridization (CGH) on 10 serous borderline tumors from nine patients, using microdissection to enrich the samples for tumor DNA and reduce contamination from stromal and inflammatory cells. CGH analysis revealed that three of the tumors had detectable chromosomal imbalances, whereas seven were in a balanced state. In those tumors with imbalances, the number of abnormalities ranged from 3-6 per tumor. Additional studies by fluorescence in situ hybridization (FISH) on disaggregated nuclei confirmed the imbalances detected by CGH, revealed one tumor to be hypertriploid, and indicated that the remaining tumors were diploid and in a balanced state. All abnormalities observed in the aneuploid cases are consistent with chromosomal aberrations previously reported for ovarian carinomas, providing further evidence that some borderline tumors are part of a continuum of tumor progression. These results also suggest that there may be different mechanisms leading to borderline tumor formation, including one associated with multiple chromosomal imbalances, and others that do not involve imbalances detectable by CGH. Genes Chromosomes Cancer 25:307-315, 1999.

A recurring pattern of chromosomal aberrations in mammary gland tumors of MMTV-cmyc transgenic mice.

Mice carrying the MMTV-cmyc transgene develop mammary tumors at 9 to 12 months of age. Little is known about karyotypic changes in this model of human breast cancer. We have developed and applied molecular cytogenetic techniques to study chromosomal aberrations that occur in these tumors, namely, comparative genomic hybridization and spectral karyotyping. Cell lines from eight tumors were established and analyzed, four of which carried a heterozygous p53 mutation. All of the tumor cell lines revealed increases in ploidy and/or multiple numerical and structural chromosomal aberrations. No consistent differences were observed between cmyc/p53+/+ and cmyc/p53+/- tumors, suggesting that cmyc induces karyotype instability independent of p53 status. Loss of whole chromosome (Chr) 4 was detected in five of the eight tumors. Parts of Chr 4 are syntenic to human 1p31-p36, a region that is also deleted in human breast carcinomas. Four tumors carried translocations involving the distal portion of Chr 11 (syntenic to human chromosome arm 17q), including two translocations T(X;11), with cytogenetically identical breakpoints. We compare the pattern of chromosomal aberrations with human breast cancers, find similarities in several syntenic regions, and discuss the potential of an interspecies cytogenetic map of chromosomal gains and losses.

Comparative genomic hybridization and loss of heterozygosity analyses identify a common region of deletion at 15q11.1-15 in human malignant mesothelioma.

Comparative genomic hybridization analysis was performed to identify chromosomal imbalances in 24 human malignant mesothelioma (MM) cell lines derived from untreated primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. The most frequent underrepresented segments were 22q (58%) and 15q1.1-21 (54%); other recurrent sites of chromosomal loss included 1p12-22 (42%), 13q12-14 (42%), 14q24-qter (42%), 6q25-qter (38%), and 9p21 (38%). The most commonly overrepresented segment was 5p (54%). DNA sequence amplification at 3p12-13 was observed in two cases. Whereas some of the regions of copy number decreases (i.e., segments in 1p, 6q, 9p, and 22q) have previously been shown to be common sites of karyotypic and allelic loss in MM, our comparative genomic hybridization analyses identified a new recurrent site of chromosomal loss within 15q in this malignancy. To more precisely map the region of 15q deletion, loss of heterozygosity analyses were performed with a panel of polymorphic microsatellite markers distributed along 15q, which defined a minimal region of chromosomal loss at 15q11.1-15. The identification of frequent losses of a discrete segment in 15q suggests that this region harbors a putative tumor suppressor gene whose loss/inactivation may contribute to the pathogenesis of many MMs.

Identification of de novo chromosomal markers and derivatives by spectral karyotyping.

Despite major advances in molecular cytogenetics during the past decade and the important diagnostic role that fluorescence in situ hybridization (FISH) plays in the characterization of chromosomal abnormalities, the usefulness of this technique remains limited by the number of spectrally distinguishable fluorochromes or fluorochrome combinations. Overcoming this major limitation would allow one to use FISH to screen the whole human genome for chromosomal aberrations which, until recently, was possible only through conventional karyotyping. A recently described molecular cytogenetics technology, 24-color FISH using spectral karyotyping (SKY), permits the simultaneous visualization of all human chromosomes in 24 different colors. Most chromosomal aberrations detected during cytogenetic evaluation can be resolved using routine cytogenetic techniques alone or in combination with single- or dual-color FISH. However, some cases remain unresolved, in particular de novo supernumerary marker chromosomes and de novo unbalanced structural rearrangements. These findings cause particular diagnostic and counseling concerns when detected during prenatal diagnosis. The purpose of this report is to demonstrate the application of SKY in the characterization of these de novo structural chromosomal abnormalities. Eight cases are described in this report. SKY has considerable diagnostic applications in prenatal diagnosis because of its reliability and speed. The identification of the chromosomal origin of markers and unbalanced translocations provides the patient, physician, and genetic counselor with better predictive information on the phenotype of the carrier.

Cytogenetic and molecular characterization of random chromosomal rearrangements activating the drug resistance gene, MDR1/P-glycoprotein, in drug-selected cell lines and patients with drug refractory ALL.

Drug resistance, both primary and acquired, is a major obstacle to advances in cancer chemotherapy. In vitro, multidrug resistance can be mediated by P-glycoprotein (PGY1), a cell surface phosphoglycoprotein that acts to efflux natural products from cells. PGY1 is encoded by the MDR1 gene located at 7q21.1. Overexpression of MDR1 has been demonstrated in many cancers, both in patient tumors and in cell lines selected with a variety of chemotherapeutic agents. Recent studies in drug-selected cell lines and patients samples have identified hybrid mRNAs comprised of an active, but apparently random, gene fused 5' to MDR1. This observation indicates that random chromosomal rearrangements, such as translocations and inversions, leading to "capture" of MDR1 by constitutively expressed genes may be a mechanism for activation of this gene following drug exposure. In this study, fluorescence in situ hybridization (FISH) using whole chromosome paints (WCP) and bacterial artificial chromosome (BAC)-derived probes showed structural rearrangements involving 7q in metaphase and interphase cells, and comparative genomic hybridization (CGH) revealed high levels of amplification at chromosomal breakpoints. In an adriamycin-selected resistant colon cancer line (S48-3s/Adr), WCP4/WCP7 revealed t(4;7)(q31;q21) and BAC-derived probes demonstrated that the breakpoint lay between MDR1 and sequences 500-1000 KB telomeric to it. Similarly, in a subline isolated following exposure to actinomycin D (S48-3s/ActD), a hybrid MDR1 gene composed of heme oxygenase-2 sequences (at 16p13) fused to MDR1 was identified and a rearrangement confirmed with WCP7 and a subtelomeric 16p probe. Likewise, in a paclitaxel-selected MCF-7 subline where CASP sequences (at 7q22) were shown to be fused to MDR1, WCP7 showed an elongated chromosome 7 with a homogeneously staining regions (hsr); BAC-derived probes demonstrated that the hsr was composed of highly amplified MDR1 and CASP sequences. In all three selected cell lines, CGH demonstrated amplification at breakpoints involving MDR1 (at 7q21) and genes fused to MDR1 at 4q31, 7q22, and 16p13.3. Finally, in samples obtained from two patients with drug refractory ALL, BAC-derived probes applied to archived marrow cells demonstrated that a breakpoint occurred between MDR1 and sequences 500-1000 KB telomeric to MDR1, consistent with a random chromosomal rearrangement. These results support the proposal that random chromosomal rearrangement leading to capture and activation of MDR1 is a mechanism of acquired drug resistance.

Comparative genomic hybridization as part of a new diagnostic strategy in childhood hyperdiploid acute lymphoblastic leukemia.

The detailed definition of karyotype changes associated with hyperdiploid acute lymphoblastic leukemia (ALL) is a precondition for their exploitation in minimal residual disease studies with fluorescence in situ hybridization analysis (FISH). In addition, certain karyotype patterns may have different prognostic implications. We have therefore used comparative genomic hybridization (CGH) to analyze the quantitative karyotype abnormalities in 14 cases of hyperdiploid ALL and correlated the results with those obtained by flow cytometry and conventional cytogenetic analyses. Despite an overall good agreement between the karyotypes obtained by classical banding techniques and CGH, we came across at least one karyotype discrepancy per case. Clarification of the discordant findings with fluorescence in situ hybridization (FISH) showed that all stem lines had been correctly defined by CGH. In eight cases, however, cytogenetic analyses revealed structural abnormalities that were undetectable by CGH. The other discrepancies were mainly due to a cytogenetic misinterpretation of similar sized and shaped chromosomes. Based on these findings we present a new diagnostic strategy for childhood ALL that includes flow cytometry and classical cytogenetics as well as CGH for the analysis of aneuploid cases and FISH to resolve the unavoidable discrepancies.

Comparative genomic hybridization detects frequent overrepresentation of chromosomal material from 3q26, 8q24, and 20q13 in human ovarian carcinomas.

We used comparative genomic hybridization (CGH) to identify recurrent chromosomal imbalances in tumor DNA from 25 malignant ovarian carcinomas and two ovarian tumors of low malignant potential (LMP). Many of the carcinoma specimens displayed numerous imbalances. The most common sites of copy number increases, in order of frequency, were 8q24.1, 20q13.2-qter, 3q26.3-qter, 1q32, 20p, 9p21-pter, and 12p. DNA amplification was identified in 12 carcinomas (48%). The most frequent sites of amplification were 8q24.1-24.2, 3q26.3, and 20q13.2-qter. Other recurrent sites of amplification included 7q36, 17q25, and 19q13.1-13.2. The most frequent sites of copy number decreases were 5q21, 9q, 17p, 17q12-21, 4q26-31, 16q, and 22q. Underrepresentation of 17p was observed in six of 16 stage III/IV tumors, but in none of seven stage I/II tumors, suggesting that this change may be a late event associated with the transition of ovarian carcinomas to a more metastatic disease. Overrepresentation of 3q26.3-qter, 5p14-pter, 8q24.1, 9p21-pter, 20p, and 20q13.2-qter and underrepresentation of 4q26-31 and 17q12-21 also tended to be more common in advanced-stage tumors. All ten grade 3 tumors had copy number increases involving 8q24.1, compared to only three of nine grade 2 tumors. Overrepresentation of 3q26.3-qter and 20q13.2-qter was also observed at a higher frequency in high-grade tumors. One of the two LMP tumors displayed chromosomal alterations, which consisted of overrepresentation of 5p and 9p only. Taken collectively, these findings and data from other CGH studies of ovarian cancers define a set of small chromosome segments that are consistently over- or underrepresented and, thus, highlight sites of putative oncogenes and tumor suppressor genes that contribute to the pathogenesis of these highly malignant neoplasms.

Previously hidden chromosome aberrations in T(12;15)-positive BALB/c plasmacytomas uncovered by multicolor spectral karyotyping.

The majority of BALB/c mouse plasmacytomas harbor a balanced T(12;15) chromosomal translocation deregulating the expression of the proto-oncogene c-myc. Recent evidence suggests that the T(12;15) is an initiating tumorigenic mutation that occurs in early plasmacytoma precursor cells. However, the possible contribution of additional chromosomal aberrations to the progression of plasmacytoma development has been largely ignored. Here we use multicolor spectral karyotyping (SKY) to evaluate 10 established BALB/c plasmacytomas in which the T(12;15) had been previously detected by G banding. SKY readily confirmed the presence of this translocation in all of these tumors and in three plasmacytomas newly identified secondary cytogenetic changes of the c-myc-deregulating chromosome (Chr) T(12;15). In addition, numerous previously unknown aberrations were found to be scattered throughout the genome, which was interpreted to reflect the general genomic instability of plasmacytomas. Instability of this sort was not uniform, however, because only half of the tumors were heavily rearranged. Seven apparent hot spots of chromosomal rearrangements (40% incidence) were identified and mapped to Chrs 1B, 1G-H, 2G-H1, 4C7-D2, 12D, 14C-D2, and XE-F1. Two of these regions, Chr 1B and Chr 4C7-D2, are suspected to harbor plasmacytoma susceptibility loci; Pctr1 and Pctr2 on Chr 4C7-D2 and as yet unnamed loci on Chr 1B. These results suggest that secondary chromosomal rearrangements contribute to plasmacytoma progression in BALB/c mice. To evaluate the biological significance of these rearrangements, SKY will be used in follow-up experiments to search for the presence of recurrent and/or consistent secondary cytogenetic aberrations in primary BALB/c plasmacytomas.

Isolation of osteosarcoma-associated amplified DNA sequences using representational difference analysis.

Comparative genomic hybridization analysis of a primary osteosarcoma and its metastasis revealed two regions of DNA amplification, one at 17p11.2-12 and one at 19q12-13. Subsequent representational difference analysis of the primary tumor resulted in the isolation of two distinct tumor-amplified DNA fragments originating from chromosome 19. A YAC clone corresponding to one of the two isolated DNA fragments was used for fluorescence in situ hybridization on normal human lymphocyte metaphases and tumor-derived nuclei. This resulted in the localization of this YAC to 19q12-13.1 and confirmed the amplification status of the isolated fragment in the tumors. The availability of such RDA-isolated sequences may be instrumental in the search for genes relevant for tumor development.

Analysis of genetic alterations in uterine leiomyomas and leiomyosarcomas by comparative genomic hybridization.

Uterine leiomyomas are the most prevalent tumor type in women of reproductive age and are the most common reason for hysterectomies. Although uterine leiomyomas are considered to be benign, they are a major public health concern for women. In contrast, leiomyosarcomas are rare but highly malignant uterine tumors. They may arise in uteri with preexisting leiomyomas and histologically sometimes resemble leiomyomas, thus causing controversy about whether leiomyosarcomas arise within leiomyomas. In this study, we used comparative genomic hybridization (CGH) to identify genetic alterations unique to each tumor type and alterations that are common between the two tumors. We analyzed 14 cases of uterine leiomyomas and eight cases of uterine leiomyosarcomas. Only two of the 14 leiomyomas exhibited genetic alterations, and those were restricted to gains on chromosomes 14 and 19 and losses on chromosomes 1 and 4. In addition, 68 leiomyomas were examined for loss of heterozygosity on chromosomes 1 and 4, and only three tumors exhibited any losses. In contrast, all eight leiomyosarcomas showed gains and losses of DNA by CGH, and in many cases multiple changes were observed. The most commonly observed genetic aberration, occurring in five tumors, was gains on both arms of chromosome 1, suggesting that this chromosome contains loci involved in the development of leiomyosarcoma. Our results do not provide evidence for the progression from benign leiomyoma to malignant leiomyosarcoma. Moreover, the large number of random chromosomal alterations in the leiomyosarcomas suggests that increased genetic instability plays a role in the formation of these tumors.