Biomedical subjects

A Kallioniemi

Publications and source records attributed to A Kallioniemi.

At least 37 records · Page 2Linked to original sources

PubMed1997

Genetic heterogeneity and clonal evolution underlying development of asynchronous metastasis in human breast cancer.

To understand the genetic basis and clonal evolution underlying metastatic progression of human breast cancer in vivo, we analyzed the genetic composition of 29 primary breast carcinomas and their paired asynchronous metastases by comparative genomic hybridization and fluorescence in situ hybridization. The mean number of genetic changes by comparative genomic hybridization was 8.7 +/- 5.3 in primary tumors and 9.0 +/- 5.7 in their metastases. Although most of the genetic changes occurred equally often in the two groups, gains of the Xq12-q22 region were enriched in the metastases. According to a statistical analysis of shared genetic changes and breakpoints in paired specimens, 20 of the metastases (69%) showed a high degree of clonal relationship with the corresponding primary tumor, whereas the genetic composition of 9 metastases (31%) differed almost completely from that of the paired primary tumors. In both groups, however, chromosome X inactivation patterns suggested that the metastatic lesions originated from the same clone as the primary tumor. Fluorescence in situ hybridization analysis with probes specific to metastatic clones usually failed to find such cells in the primary tumor sample. In conclusion, detailed characterization of the in vivo progression pathways of metastatic breast cancer indicates that a linear progression model is unlikely to account for the progression of primary tumors to metastases. An early stem line clone apparently evolves independently in the primary tumor and its metastasis, eventually leading to multiple, genetically almost completely different, clones in the various tumor locations in a given patient. The resulting heterogeneity of metastatic breast cancer may underlie its poor responsiveness to therapy and explain why biomarkers of prognosis or therapy responsiveness measured exclusively from primary tumors give a restricted view of the biological properties of metastatic breast cancer.

Adenocarcinoma↗

PubMed1997

Increased copy number at 17q22-q24 by CGH in breast cancer is due to high-level amplification of two separate regions.

Studies by comparative genomic hybridization (CGH) have defined a chromosomal site at 17q22-q24 that is often overrepresented in breast cancer, neuroblastoma, and several other tumor types. Due to the limited resolution and dynamic range of CGH, it remain unclear whether this gain reflects high-level amplification of small subregion(s) or low-level gain of most of the distal 17q. We used 32 physically mapped 17q probes to construct more accurate copy number profiles for 14 breast cancer cell lines by interphase fluorescence in situ hybridization (FISH). Six cell lines (43%) showed an increased copy number of the 17q-22q24 region by CGH, and seven (50%) by FISH. FISH copy number profiles had a substantially higher dynamic range than did CGH profiles. FISH revealed two independent, highly amplified regions (A and B) at 17q23, separated by about 5 Mb of non-amplified DNA. These regions were distinctly telomeric from the ERBB2 gene locus. However, region A was often co-amplified with ERBB2, whereas B was amplified in cell lines that showed no ERBB2 amplification. We conclude that distal 17q gains recently discovered in breast cancer by CGH are due to high-level amplifications of two different regions at 17q23. This chromosomal region has previously been reported to undergo allelic loss and therefore was thought to harbor a tumor suppressor gene. The present FISH data provide support for the presence, and a starting point for the positional isolation, of 17q23 genes whose upregulation by amplification may play a role in the progression of breast cancer and many other tumor types.

Breast Neoplasms↗

PubMed1997

Genome screening by comparative genomic hybridization.

Comparative genomic hybridization (CGH) provides a molecular cytogenetic approach for genome-wide scanning of differences in DNA sequence copy number. The technique is now attracting wide-spread interest, especially among cancer researchers. The rapidly expanding database of CGH publications already covers about 1500 tumors and is beginning to reveal genetic abnormalities that are characteristic of certain tumor types or stages of tumor progression. Six novel gene amplifications, as well as a locus for a cancer-predisposition syndrome, have been discovered based on CGH data. CGH has now been established as a first-line screening technique for cancer researchers and will serve as a basis for ongoing efforts to develop high-resolution next-generation genome scanning, such as the microarray technology.

Animals↗

PubMed1996

Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in human breast cancer.

DNA amplification at 20q13.2 is common in breast cancer, correlates with poor prognosis, and may reflect location of an important oncogene. Recently, other regions along 20q were also found to undergo amplification. Here, amplification levels and patterns of co-amplification were analyzed by interphase fluorescence in situ hybridization at 14 loci along 20q in 146 uncultured breast carcinomas and 14 cell lines. Three regions were independently amplified in uncultured tumors: RMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases), PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%). Co-amplifications involving two or three of these regions were seen in 11 of the 19 highly amplified tumors. The results suggest that three distinct nonsyntenic regions along 20q may be important and that complex chromosomal rearrangements underlie their frequent co-amplification in breast cancer.

Breast Neoplasms↗

PubMed1996

Gene Copy Number Analysis by Fluorescence in Situ Hybridization and Comparative Genomic Hybridization

Fluorescence in situ hybridization (FISH) with gene- and locus-specific probes provides a rapid means to assess copy numbers of specific sequences in individual interphase nuclei. Recent technical improvements have made FISH applicable to the analysis of both fresh and archival tissue specimens in research as well as in diagnostic laboratories. FISH is limited to analysis of one or a few loci at a time, making genome-wide surveys impractical. Comparative genomic hybridization (CGH) was developed as a means to screen entire genomes for DNA sequence copy number changes. CGH is based on the cohybridization of differentially labeled test and reference DNAs to normal metaphase chromosomes. Measurement of the test to reference fluorescence ratios along all chromosomes provides information on chromosomal regions that are over- or underrepresented in the test genome. The use of these two techniques will be illustrated in the analysis of genetic changes in solid tumors. The techniques are complementary to one another and have proven to be highly useful for identification of previously unknown genetic changes and genes that play an important role in tumor progression.

Journal Article↗

PubMed1996

Amplifications of oncogene erbB-2 and chromosome 20q in breast cancer determined by differentially competitive polymerase chain reaction.

A new method of measuring gene copy number in small samples of DNA was used to measure amplification of the erbB-2 gene and of chromosome 20q in breast cancer. This method, termed 'differentially competitive polymerase chain reaction' (DC-PCR) combines the advantages of two other techniques for measuring amplification by PCR, namely differential PCR and competitive PCR. The DC-PCR methodology was evaluated for sensitivity and specificity by comparing amplification of erbB-2 measured by DC-PCR with that obtained by fluorescence in situ hybridization (FISH) for 42 cases or Southern blotting and/or slot blot analysis for 34 cases. There was over 90 percent concordance with both FISH and Southern blotting and/or slot blot analysis. DC-PCR was used to further characterize the newly described amplicon at chromosome 20q. By analyzing DNA from 10 breast cancer cell lines at 7 different loci, we identified a potential common region of amplification of approximately 5 centimorgans at chromosome 20q13 bordered by loci D20S52 and RMC20C100-S1. One hundred and seventeen cases of primary breast cancer were evaluated for amplification at these two loci. Amplification at one or more loci, defined as > 1.5 fold higher copy number than that of normal DNA, was found in 25 cases (21%). Sixteen cases were amplified at only one of the two probes (12 cases for RMC20C001-S1 and 4 cases for D20S52), suggesting that the target gene lies between the two markers or that there are two independent target genes within a small chromosome region.

Blotting, Southern↗

PubMed1995

Gains and losses of DNA sequences in osteosarcomas by comparative genomic hybridization.

Our aim was to identify chromosomal regions that are likely to harbor previously unknown genes with an important role in the genesis of osteosarcoma. Comparative genomic hybridization was used to screen for losses and gains of DNA sequences along all chromosome arms in 11 tumors. Extensive genetic aberrations, with an average of 11 changes/tumor (range, 1-20), were found in 10 of the 11 specimens. High level amplifications of small chromosomal regions were detected in eight tumors. These involved the 12q12-q13 region (known to contain the SAS-MDM2 locus) and several previously unreported amplification sites such as 17p11-p12, 3q26, and Xq12. When all DNA sequence gains were evaluated, the gains at 8q and Xp were most common (45%). The most common losses of DNA sequences were seen at 2q, 6q, 8p, and 10p (36%). In conclusion, despite the very complex pattern of genetic changes in osteosarcomas, certain chromosomal regions appear to be affected more often than others. Most of these regions have not previously been reported to be implicated in osteosarcomas and may thus highlight locations of novel genes with an important role in the development and progression of these tumors.

Chromosome Aberrations↗

PubMed1995

A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis.

The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously.

Base Sequence↗

PubMed1995

Computer image analysis of comparative genomic hybridization.

We describe and evaluate the image-processing and analysis techniques we have developed for the quantitative analysis of comparative genomic hybridization (CGH; Science 258:818, 1992). In a typical CGH application, two genomic DNA samples are simultaneously hybridized to metaphase chromosomes and detected with different fluorochromes. The primary data in CGH are contained in the intensity ratios of the fluorochromes as a function of position on the chromosomes, which reflect variation in DNA copy number ratio between the two DNA samples. Analysis involves chromosome segmentation, intensity normalization, background corrections, and calculation of the fluorescence intensity profiles and the ratio profile along the chromosome's length. Profiles from several copies of the same chromosome in different metaphases are averaged to reduce the noise. Confidence intervals are calculated and displayed for the mean profiles. The techniques were evaluated by examining the variability found in comparisons of two normal genomic DNAs, where the ratio was expected to be constant, and by measuring the ratios obtained for cell lines with cytogenetically documented copy number changes involving several chromosomal segments. The limits of sensitivity of CGH analysis were investigated by simulation. Guidelines for the interpretation of CGH data and indications of areas for future development of the analytical techniques are also presented.

Cell Line↗

PubMed1995

Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization.

Comparative genomic hybridization (CGH) makes it possible to detect losses and gains of DNA sequences along all chromosomes in a tumor specimen based on the hybridization of differentially labeled tumor and normal DNA to normal human metaphase chromosomes. In this study, CGH analysis was applied to the identification of genomic imbalances in 26 bladder cancers in order to gain information on the genetic events underlying the development and progression of this malignancy. Losses affecting 11p, 11q, 8p, 9, 17p, 3p, and 12q were all seen in more than 20% of the tumors. The minimal common region of loss in each chromosome was identified based on the analysis of overlapping deletions in different tumors. Gains of DNA sequences were most often found at chromosomal regions distinct from the locations of currently known oncogenes. The bands involved in more than 10% of the tumors were 8q21, 13q21-q34, 1q31, 3q24-q26, and 1p22. In conclusion, these CGH data highlight several previously unreported genetic alterations in bladder cancer. Further detailed studies of these regions with specific molecular genetic techniques may lead to the identification of tumor suppressor genes and oncogenes that play an important role in bladder tumorigenesis.

Chromosome Mapping↗

PubMed1995

Comparative genomic hybridization in clinical cytogenetics.

We report the results of applying comparative genomic hybridization (CGH) in a cytogenetic service laboratory for (1) determination of the origin of extra and missing chromosomal material in intricate cases of unbalanced aberrations and (2) detection of common prenatal numerical chromosome aberrations. A total of 11 fetal samples were analyzed. Seven cases of complex unbalanced aberrations that could not be identified reliably by conventional cytogenetics were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome-specific probes. Four cases representing common prenatal numerical aberrations (trisomy 21, 18, and 13 and monosomy X) were also successfully diagnosed by CGH. We conclude that CGH is a powerful adjunct to traditional cytogenetic techniques that makes it possible to solve clinical cases of intricate unbalanced aberrations in a single hybridization. CGH may also be a useful adjunct to screen for euchromatic involvement in marker chromosomes. Further technical development may render CGH applicable for routine aberration screening.

Chromosome Aberrations↗

PubMed1995

c-myc copy number gains in bladder cancer detected by fluorescence in situ hybridization.

Amplification and overexpression of c-myc have been suggested as prognostic markers in human cancer. To assess the role of c-myc gene copy number alterations in bladder cancer, 87 bladder tumors were examined for c-myc aberrations by fluorescence in situ hybridization. Dual labeling hybridization with a repetitive pericentromeric probe specific for chromosome 8 and a probe for the c-myc locus (at 8q24) was performed to analyze c-myc copy number in relation to chromosome 8 copy number on a cell by cell basis. A clear-cut c-myc amplification (up to 40 to 150 copies per cell) was found in 3 tumors. There was a low level c-myc copy number increase in 32 of the remaining 84 tumors. There was no association of low level c-myc copy number increase with c-myc protein overexpression. This suggests that a c-myc gene copy number gain as detected by fluorescence in situ hybridization does not necessarily reflect a disturbed c-myc gene function but may indicate a structural chromosome 8 abnormality including gain of distal 8q. The strong association of low level c-myc (8q) gains with tumor grade (P < 0.0001), stage (P < 0.0001), chromosome polysomy (P < 0.0001), p53 protein expression (P = 0.0019), p53 deletion (P = 0.0403), and tumor cell proliferation (Ki67 labeling index; P = 0.0021) is consistent with a role of chromosome 8 alterations in bladder cancer progression.

Chromosomes, Human, Pair 8↗

PubMed1995

Amplification of chromosomal region 20q13 in invasive breast cancer: prognostic implications.

Amplification of the chromosome 20q13 region was recently discovered in breast cancer by comparative genomic hybridization and subsequently further defined by fluorescence in situ hybridization with specific probes. The target gene of the amplification remains unknown. Here, fluorescence in situ hybridization with a cosmid probe for the minimal region of amplification (RMC20C001) was used to study 20q13 amplification in 132 primary breast carcinomas and 11 metastases. The size of the amplicon was studied with four flanking probes. Thirty-eight (29%) primary tumors and 3 (27%) metastases showed increased copy number of the RMC20C001 probe (>1.5-fold relative to the p-arm control). Nine (6.8%) of the primary tumors were highly (>3-fold) amplified. Although the size and location of the amplified region varied from one tumor to another, only the RMC20C001 probe was consistently amplified. 20q13 amplification was significantly associated with a high histological grade (P = 0.01), DNA aneuploidy (P = 0.01), and high S-phase fraction (P = 0.0085). High-level amplification was also associated with short disease-free survival of patients with node-negative breast cancer (P = 0.002). We conclude that high-level 20q13 amplification may be an indicator of poor clinical outcome in node-negative breast cancer and that this chromosomal region is likely to contain a gene with an important role in breast cancer progression. A large definitive study is warranted to assess the independent prognostic value of 20q13 amplification.

Adult↗

PubMed1994

Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes.

Studies by comparative genomic hybridization have indicated that a major new locus for DNA amplification in breast cancer is 20q13 and suggested that this genetic event is associated with aggressive clinical behavior. We used interphase fluorescence in situ hybridization with anonymous cosmid probes and gene-specific P1 clones to determine the minimal common region of increased copy number and to study involvement of known genes at 20q13. Based on high-level copy number increases (3 to 10-fold) found with one or more probes in 5 of 14 (35%) breast cancer cell lines and in 3 of 36 (8%) primary tumors, the critical region was narrowed to approximately 1.5 megabases at 20q13.2 defined by fractional length pter values 0.81-0.84. Previously known genes were excluded as candidates, implying that this chromosomal region harbors a novel oncogene that contributes to the malignant progression of breast cancer.

Breast Neoplasms↗

PubMed1994

Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) and specific DNA probes for peri-centromeric repeat regions and unique sequence loci have made it possible to study chromosomal aberrations from interphase tumor nuclei. Large-scale retrospective studies on the prognostic value of interphase cytogenetics would become feasible if these techniques were readily applicable to nuclei from archival formalin-fixed tumor tissues. We describe here an improved technique for interphase FISH analysis of tumors that have been extensively fixed in formalin. The protocol aims at improving probe penetration and hybridization efficiency by inducing chromatin decondensation and swelling of the nuclei with a heat treatment in a 90 degrees C glycerol solution prior to hybridization. Using this cell pretreatment, FISH results on the detection of chromosome copy number aberrations and amplification of the c-erbB-2 oncogene from formalin-fixed, paraffin-embedded tissues were highly concordant with those from fresh tissues. In contrast to previously described methods, separate adjustments of denaturation or proteinase K digestion are not required for each sample. This method facilitates retrospective analyses of large series of tumors and is also useful for applying FISH to routine diagnostic purposes using formalin-fixed material.

Breast Neoplasms↗

PubMed1994

Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization.

Comparative genomic hybridization was applied to 5 breast cancer cell lines and 33 primary tumors to discover and map regions of the genome with increased DNA-sequence copy-number. Two-thirds of primary tumors and almost all cell lines showed increased DNA-sequence copy-number affecting a total of 26 chromosomal subregions. Most of these loci were distinct from those of currently known amplified genes in breast cancer, with sequences originating from 17q22-q24 and 20q13 showing the highest frequency of amplification. The results indicate that these chromosomal regions may contain previously unknown genes whose increased expression contributes to breast cancer progression. Chromosomal regions with increased copy-number often spanned tens of Mb, suggesting involvement of more than one gene in each region.

Breast Neoplasms↗

PubMed1994

Physical mapping of chromosome 17 cosmids by fluorescence in situ hybridization and digital image analysis.

We used fluorescence in situ hybridization and digital image analysis to localize cosmids along human chromosome 17. Seventy-one cosmids were selected at random from a chromosome 17 library constructed from a partial Sau3AI digest of flow-sorted chromosomes from a mouse-human hybrid cell line. Sixty-three of these (89%) gave a signal only on chromosome 17. The 40 cosmids producing the most distinct hybridization signals in metaphase and interphase cells were precisely mapped using digital image analysis. An additional 20 cosmids, previously mapped by linkage analysis, were also mapped. The order of these probes determined by metaphase mapping was consistent with the order determined by linkage analysis.

Chromosome Mapping↗

PubMed1994

Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors.

Comparative genomic hybridization (CGH) is a powerful new method for molecular cytogenetic analysis of cancer. In a single hybridization, CGH provides an overview of DNA sequence copy number changes (losses, deletions, gains, amplifications) in a tumor specimen and maps these changes on normal chromosomes. CGH is based on the in situ hybridization of differentially labeled total genomic tumor DNA and normal reference DNA to normal human metaphase chromosomes. After hybridization and fluorescent staining of the bound DNAs, copy number variations among the different sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target metaphase chromosomes. CGH is in particular useful for analysis of DNA sequence copy number changes in common solid tumors where high-quality metaphase preparations are often difficult to make, and where complex karyotypes with numerous markers, double minutes, and homogeneously stained chromosomal regions are common. CGH only detects changes that are present in a substantial proportion of tumor cells (i.e., clonal aberrations). It does not reveal translocations, inversions, and other aberrations that do not change copy number. At present, CGH is a research tool that complements previous methods for genetic analysis. CGH will advance our understanding of the genetic progression of cancer and highlight important genomic regions for further study. Direct clinical applications of CGH are possible, but will require further development and validation of the technique. We describe here our recent optimized procedures for CGH, including DNA labeling, hybridization, fluorescence microscopy, digital image analysis, data interpretation, and quality control, emphasizing those steps that are most critical. We will also assess sensitivity and resolution limits of CGH as well as discuss possible future technical improvements.

Chromosome Aberrations↗