Biomedical subjects

R Karhu

Publications and source records attributed to R Karhu.

At least 37 records · Page 2Linked to original sources

PubMed1997

Quality control of CGH: impact of metaphase chromosomes and the dynamic range of hybridization.

With the recent rapid expansion in the use of the comparative genomic hybridization (CGH) technique, increased attention to quality control is essential. In the present study, we show that despite optimization and standardization of metaphase preparation techniques and the commercial availability of metaphase spreads, batch-to-batch variability of the preparations remains a significant problem. To facilitate reliable CGH analysis despite this variability, we have developed a rapid denaturation test to assess the quality of the preparations without hybridization and quantitative image analysis criteria for assuring the day-to-day quality of CGH experiments, including sensitivity, specificity, and dynamic range. Monitoring the dynamic range of the hybridizations was found to be particularly critical for achieving sensitive and reliable CGH results. This reliability can be achieved, for example, by hybridization of a green-labeled normal male DNA against red-labeled female DNA and monitoring of the green:red ratio of the X chromosome in relation to that of the autosomes.

Chromosome Banding↗

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

Distinct somatic genetic changes associated with tumor progression in carriers of BRCA1 and BRCA2 germ-line mutations.

BRCA1 and BRCA2 mutations confer increased risk for development of breast cancer, but a number of additional, currently largely unknown, somatic genetic defects must also accumulate in the breast epithelial cells before malignancy develops. To evaluate the nature of these additional somatic genetic defects, we performed a genome-wide survey by comparative genomic hybridization on breast cancers from 21 BRCA1 mutation carriers, 15 BRCA2 mutation carriers, and 55 unselected controls. The total number of genetic changes was almost two times higher in tumors from both BRCA1 and BRCA2 mutation carriers than in the control group. In BRCA1 tumors, losses of 5q (86%), 4q (81%), 4p (64%), 2q (40%), and 12q (40%) were significantly more common than in the control group (7-13%). BRCA2 tumors were characterized by a higher frequency of 13q (73%) and 6q (60%) losses and gains of 17q22-q24 (87%) and 20q13 (60%) as compared to the prevalence of these changes in the control group (12-18%). In conclusion, accumulation of somatic genetic changes during tumor progression may follow a unique pathway in individuals genetically predisposed to cancer, especially by the BRCA1 gene. Activation or loss of genes in the affected chromosomal regions may be selected for during tumor progression in cells lacking functional BRCA1 or BRCA2. Identification of such genes could provide targets for therapeutic intervention and early diagnosis.

Adult↗

PubMed1997

Frequent loss of the 11q14-24 region in chronic lymphocytic leukemia: a study by comparative genomic hybridization. Tampere CLL Group.

The genetic basis and molecular pathogenesis of chronic lymphocytic leukemia (CLL) and the molecular mechanisms responsible for its progression remain poorly understood. Here, karyotyping techniques specifically optimized for CLL, comparative genomic hybridization (CGH), and fluorescence in situ hybridization were used to search for CLL-specific genetic aberrations. CGH and karyotyping both revealed copy number changes in 12 of the 25 CLL cases (48%) analyzed. Loss at 11q emerged as the most common aberration (6 cases), followed by a gain of chromosome 12 (4) and loss at 13q (3). Concordance between CGH and G-banding was found in 23 of the 25 cases (92%), which is more than reported in a recent similar CGH study of CLL. Owing to the basic differences in G-banding and CGH, however, their simultaneous clinical application is recommended. The frequent loss of 11q14-24 suggests that this chromosomal region deserves further attention as a candidate locus involved in the pathogenesis of CLL.

Aged↗

PubMed1997

Comparative genomic hybridization reveals frequent gains of 20q, 8q, 11q, 12p, and 17q, and losses of 18q, 9p, and 15q in pancreatic cancer.

Comparative genomic hybridization (CGH) was used to screen for genomic imbalances in 24 exocrine pancreatic carcinomas, including II low-passage cell lines (4-8 subcultures) and 13 uncultured samples. Aberrations were found in all cell lines and in seven of the 13 biopsies. The most frequent changes in the cell lines were gains of 20q (91%), 11q (64%), 17q (64%), 19q (64%), 8q, 12p, 14q, and 20p (55%), and losses of 18q (100%), 9p (91%), 15q(73%), 21q (64%), 3p (55%), and 13q (55%). High-levels gains (tumor to normal ratio over 1.5) were detected at 3q, 6p, 7q, 8q, 12p, 19q, and 20q. Among the tumor biopsies, overrepresentations of 7p and 8q were most common (31%), followed by 5p, 5q, 11p, 11q, 12p, and 18q (23%), whereas the most frequent losses involved 18p and 18q (31%) and 6q and 17p (23%). The genetic changes in nine samples obtained from metastatic lesions did not differ significantly from those in 15 primary carcinomas. Most of the gains and losses detected in this CGH study correspond well to those identified in previous cytogenetic and molecular genetic investigations of pancreatic carcinomas. However, frequent gain of 12p and loss of 15q have not been previously reported. Molecular genetic analyses of these chromosome arms are warranted, and may lead to the discovery of novel genes important in pancreatic carcinogenesis.

Aged↗

PubMed1997

Genetic aberrations in pediatric acute lymphoblastic leukemia by comparative genomic hybridization.

Classical cytogenetic analysis plays an important role in the diagnosis and classification of childhood acute lymphoblastic leukemia (ALL). However, poor in vitro growth of the malignant cells and suboptimal quality of metaphase spreads may sometimes cause false-negative findings (normal karyotype). We used comparative genomic hybridization (CGH) to study whether this new method is able to detect and characterize genetic aberrations not detected by karyotyping. CGH showed clonal genetic aberrations in 8 of 13 cases, most of which showed gains of several chromosomes, indicating hyperdiploidy. The sensitivity of CGH was sufficient to detect a small interstitial deletion of 6q. One karyotypically complex case was resolved by CGH showing a high-level amplification of DNA sequences originating from the 12p12-13. Interphase fluorescence in situ hybridization (FISH) analyses confirmed the CGH findings in 2 cases, validating the accuracy of CGH. In conclusion, CGH experiments established the known fact that hyperdiploidy is the most common finding in pediatric ALLs and that CGH may detect aberrations that are not seen in the G-banded karyotype. CGH was also able to further characterize genetic aberrations such as gene amplification, which is occasionally involved in pediatric ALL as well as in other leukemias.

Child↗

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↗

PubMed1997

Genetic changes associated with the acquisition of androgen-independent growth, tumorigenicity and metastatic potential in a prostate cancer model.

Genetic changes underlying the progression of human prostate cancer are incompletely understood. Recently, an experimental model system that resembles human prostate cancer progression was developed based on the serial passage of an androgen-responsive, non-tumorigenic LNCaP prostate cancer cell line into athymic castrated mice. Six different sublines, derived after one, two or three rounds of in vivo passage, sequentially acquired androgen independence and tumorigenicity as well as metastatic capacity. Here, we used comparative genomic hybridization (CGH) and locus-specific fluorescence in situ hybridization (FISH) analysis to search for genetic changes that may underlie the phenotypic progression events in this model system. Six genetic aberrations were seen by CGH in the parental LNCaP cell line. The derivative sublines shared virtually all these changes, indicating a common clonal origin, but also contained 3-7 additional genetic changes. Gain of the 13q12-q13 chromosomal region as well as losses of 4, 6q24-qter, 20p and 21q were associated with androgen independence and tumorigenicity with additional changes correlating with metastasis. In conclusion, an accumulation of genetic changes correlates with tumour progression in this experimental in vivo model of prostate cancer progression. It is possible that the specific chromosomal aberrations involved in this model system may provide clues to the location of genes involved in human prostate cancer progression and metastasis.

Androgens↗

PubMed1997

Genetic changes in intraductal breast cancer detected by comparative genomic hybridization.

Ductal carcinoma in situ (DCIS) is considered a direct precursor of invasive ductal breast cancer (IDC). We combined tissue microdissection and comparative genomic hybridization to identify genetic changes in five DCIS lesions with no invasion and in two that were adjacent to IDC. Extensive genetic changes characterized pure DCIS cases with gains of 1q, 6q, 8q, and Xq as well as losses of 17p and chromosome 22 being most often involved. Except for the Xq gain, these changes are also common to IDC. Separate analysis of DCIS and IDC components in the same tumor revealed an almost identical pattern of genetic changes in one case, whereas substantial differences were found in another. We conclude that many of the common genetic changes in IDC may take place before development of invasive growth. However, a simple linear progression model may not always account for the DCIS-IDC transition.

Breast Neoplasms↗

PubMed1997

Optimizing DOP-PCR for universal amplification of small DNA samples in comparative genomic hybridization.

The standard comparative genomic hybridization (CGH) protocol relies on availability of macroscopic tumor samples, which do not contain too much interfering normal cells. Recently, CGH after universal amplification of genomic DNA with degenerate oligonucleotide primed PCR (DOP-PCR) has been used to detect genetic aberrations in microdissected tumor specimens. However, owing to the technical difficulties, CGH results of only few microdissected samples have so far been published. We have developed an improved protocol for DOP-PCR, which includes direct incorporation of fluorochrome-conjugated nucleotides into the PCR product. Among the four polymerase enzymes tested. ThermoSequenase gave the best yield, with PCR products ranging from 100-4,000 bp. A two-step PCR-procedure was used, consisting of a preamplification with low stringency conditions followed by amplification in more stringent conditions. The method was first validated by hybridizing DOP-PCR-amplified normal DNA against nick-translated reference DNA, which showed uniform amd even hybridization result for all chromosomes. Comparison of DOP-PCR CGH to conventional CGH in MCF-7 breast cancer cell line further indicated that genetic aberrations can be reliable detected after DOP-PCR amplification. The sensitivity of the DOP-PCR-CGH was tested by serial dilution of MCF-7 DNA. Fifty picograms of sample DNA (corresponding roughly to two MCF-7 cells) was sufficient for high quality CGH. Experiments with cells microdissected from intraductal breast cancer demonstrated that carcinoma cells from 1 to 2 ducts were sufficient for a successful DOP-PCR CGH analysis. We conclude that the improved DOP-PCR-CGH protocol provides a powerful tool to study genetic aberrations in different histological subpopulations of malignant as well as precancerous lesions. DOP-PCR also improves the success rate of conventional paraffin-block CGH, because a poor quality or a too low yield of extracted DNA can be compensated by universal DNA amplification by DOP-PCR.

DNA, Neoplasm↗

PubMed1997

Accumulation of genetic changes is associated with poor prognosis in grade II astrocytomas.

Unexpectedly aggressive clinical course of some grade II astrocytomas is a diagnostic dilemma for routine histopathology. Because increasing tumor malignancy is a consequence of progressive accumulation of chromosomal alterations, we investigated whether aggressive behavior of grade II astrocytomas could be predicted by the number and type of gross chromosomal aberrations. We used comparative genomic hybridization to analyze 11 grade II astrocytomas with typical (good, n = 7) or poor (n = 4) prognosis. The results were also compared with a reference material of 13 grade III-IV astrocytomas and nine established cell lines. We found a median of two aberrations (range 0 to 4) in tumors with good prognosis and of 15.5 changes (range 8 to 28) in tumors with poor prognosis. Chromosomal gains were present in both groups, whereas chromosomal losses were frequent in tumors with poor prognosis (median 9.5, range 3 to 14) but rare in tumors with good prognosis (range 0 to 2). All chromosomal gains were also found in the high-grade astrocytoma group and the majority of them in cell lines. Chromosomal losses in grade II astrocytomas with poor prognosis were very similar to those in grade III-IV astrocytomas and cell lines. We conclude that an early accumulation of genetic changes in grade II astrocytomas is closely associated with poor patient prognosis, suggesting diagnostic use for comparative genomic hybridization in characterization of grade II astrocytomas.

Adolescent↗

PubMed1996

Evaluation of camera requirements for comparative genomic hybridization.

Comparative genomic hybridization (CGH) is based on quantitative digital image analysis of fluorescence intensities from metaphase chromosomes. High-quality CCD cameras are commonly used for image acquisition, but the minimal requirements of CCD cameras have not been determined. We first evaluated minimal camera requirements by artificially reducing spatial and dynamic resolution of images produced by a scientific-grade CCD camera (Xillix MicroImager). The results showed that reduction of dynamic resolution from 4,096 to 256 gray levels (12-bit image transformed to an 8-bit image) had negligible effect on CGH profiles and no effect on their interpretation. Similarly, CGH profiles obtained from spatially reduced images (from 1,340 x 1,035 to 670 x 517 pixels) were virtually identical to those obtained from the original image. For a practical test, we compared two 8-bit frame integrating video-rated CCD cameras (Cohu 4910 and Photometrics ImagePoint) to the Xillix Micro-Imager in a real CGH setting. Images collected from the same metaphase cells with all three cameras resulted in the identification of the same genetic changes in the samples studied. We conclude that requirements for camera resolution in CGH analysis are not stringent, and therefore that low-priced video-rated cameras capable of frame integration are sufficient for comparative genomic hybridization.

DNA↗

PubMed1996

Gain of 17q24-qter detected by comparative genomic hybridization in malignant tumors from patients with von Recklinghausen's neurofibromatosis.

The genetic changes leading to the development of malignant peripheral nerve sheath tumors (MPNSTs) are largely unknown. The few tumors that have been investigated cytogenetically had highly complex karyotypes and no consistent rearrangements, and the attempts to pinpoint consistent DNA-level changes have met with only limited success. We used comparative genomic hybridization to analyze seven MPNSTs and one dermatofibrosarcoma protuberans from eight patients with von Recklinghausen's disease (neurofibromatosis type 1), as well as three sporadic MPNSTs. Gains and losses of DNA sequences were found in all tumors, with an average of four losses (range, 0-14) and two gains (range, 0-5) per tumor. Two striking observations were made: (a) an increase in copy number of the distal part of the long arm of chromosome 17, with the smallest region of overlap 17q24-qter, was seen in five of seven MPNSTs and in the only dermatofibrosarcoma protuberans, all of which were from patients with neurofibromatosis, whereas none of the three sporadic MPNSTs had this alteration; and (b) loss of 13q, with the smallest region of overlap 13q14-q21, was found in 6 of 10 MPNSTs. The consistent involvement of these two chromosomal regions probably reflects two different pathogenetic mechanisms for MPNSTs.

Chromosome Deletion↗

PubMed1996

Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy.

The differentiation between malignant and benign adrenocortical tumors is often difficult, and better markers are required. Because the genetic background of adrenocortical tumors is poorly characterized, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes in 8 sporadic primary adrenocortical cancers and 14 adenomas. There was a strong relationship between the number of genetic aberrations detected using CGH and both tumor size and malignancy. No alterations were seen in the smaller adenomas (< 5 cm), whereas the two largest adenomas (5 cm each) and seven of the eight cancers (7-20 cm) showed an increased number of genetic alterations. The presence of genetic aberrations detected using CGH was associated with an aneuploid DNA pattern. In the cancers, losses most often involved the chromosomal regions 2, 11q, and 17p (four of eight tumors), whereas gains took place at chromosomes 4 and 5 (four of eight tumors). In conclusion, our data indicate that genetic changes may help to define the malignant potential of adrenocortical tumors. Furthermore, the CGH results implicate several chromosomal regions that may contain genes with an important role in the development of adrenocortical cancers.

Adenoma↗

PubMed1996

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

The molecular basis of malignant mesothelioma is poorly known. We examined genetic changes in 11 mesothelioma specimens by comparative genomic hybridization (CGH). Five DNA specimens originated from uncultured tumor tissues and six from cell lines established from the same patients. Findings from the classical karyotypic characterization of both primary tumors and cell lines have been reported previously. In the CGH analyses the most common genetic alterations in the 11 mesothelioma specimens were losses of chromosomal regions in 1p, 8p, 14q, and 22q and gains of 5p, 6p, 8q, 15q, 17q, and 20. The cell lines had on average a much higher total number of genetic changes than the uncultured tumor specimens. Clonal relationship between the cell lines and the uncultured tissue specimens could not usually be demonstrated even though they originated from the same patient. The observed differences may partly be due to high frequency of chromosomal rearrangements, which CGH cannot detect, partly due to contamination of tumor specimens with normal tissue, and partly due to genetic evolution in tumor cell lines.

Chromosome Aberrations↗

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↗

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

Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization.

Genetic changes leading to the development of prostate cancer and factors that underlie the clinical progression of the disease are poorly characterized. Here, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes along all chromosomes in 31 primary and 9 recurrent uncultured prostate carcinomas. The aim of the study was to identify those chromosome regions that contain genes important for the development of prostate cancer and to identify genetic markers of tumor progression. CGH analysis indicated that 74% of primary prostate carcinoma showed DNA sequence copy number changes. Losses were 5 times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). Allelic loss studies with 5 polymorphic microsatellite markers for 4 different chromosomes were done from 13 samples and showed a 76% concordance with CGH results. In local recurrences that developed during endocrine therapy, there were significantly more gains (P < 0.001) and losses (P < 0.05) of DNA sequences than in primary tumors, with gains of 8q (found in 89% of recurrences versus 6% of primary tumors), X (56% versus 0%), and 7 (56% versus 10%), as well as loss of 8p (78% versus 32%), being particularly often involved. In conclusion, our CGH results indicate that losses of several chromosomal regions are common genetic changes in primary tumors, suggesting that deletional inactivation of putative tumor suppressor genes in these chromosomal sites is likely to underlie development of prostate cancer. Furthermore, the pattern of genetic changes seen in recurrent tumors with the frequent gains of 7, 8q, and X suggests that the progression of prostate cancer and development of hormone-independent growth may have a distinct genetic basis. These chromosome aberrations may have diagnostic utility as markers of prostate cancer progression.

Chromosome Deletion↗