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Biomedical subjects

O P Kallioniemi

Publications and source records attributed to O P Kallioniemi.

At least 109 records · Page 6Linked to original sources
PubMed1994

Automated peak detection and cell cycle analysis of flow cytometric DNA histograms.

We describe an algorithm for fully automated flow cytometric DNA histogram classification and analysis that provides rapid, reproducible determination of DNA index and S-phase fraction (SPF). Automated classification agreed with subjective assessment of DNA ploidy in 96-98% of DNA histograms. Automated and conventional analyses of DNA index (r = 0.95) and SPF (r = 0.89) were also highly correlated with one another. In a series of 86 node-negative breast carcinomas, SPF calculated with the fully automated method was a significant predictor of 10 year survival (p = 0.009). Automation greatly increased the speed of DNA histogram analysis, allowing evaluation of the same set of histograms with different methods. In a preliminary study exploring the optimization of DNA histogram analysis, the best association between SPF and prognosis of breast cancer patients was achieved using sliced nuclei debris modeling, reporting only the aneuploid SPF (in aneuploid histograms), while excluding small aneuploid clones (< 15% of total cell count) from evaluation. In conclusion, automated DNA histogram analysis does not replace the need for close human supervision but provides a useful guideline for less experienced users, facilitates interlaboratory comparisons, and makes possible extensive reanalyses of large data sets.

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

Elevated erbB-2 oncoprotein levels in preoperative and follow-up serum samples define an aggressive disease course in patients with breast cancer.

BACKGROUND: Recent evidence indicates that a soluble fragment of the erbB-2 oncogene product may be released from cell surface and become detectable in the serum of patients with breast cancer. METHODS: To study the diagnostic utility of this phenomenon, the authors measured serum erbB-2 levels with a quantitative enzyme-linked immunosorbent assay in 227 preoperative samples from women who underwent breast surgery and in 339 samples from 225 patients with breast cancer during follow-up. RESULTS: Eleven (9%) of 114 preoperative samples from patients with a histologically verified breast cancer and 2 of 113 (1.8%) from patients with benign breast tumors had elevated (greater than 20 U/ml) serum erbB-2 antigen levels. Ten (91%) of the 11 carcinomas and one of the benign tumors from patients with elevated serum erbB-2 levels also showed overexpression of the erbB-2 protein in immunohistochemical analysis of tissue sections. Elevated preoperative serum erbB-2 levels were predominantly found in patients with large tumors, and those with axillary lymph node or distant metastases. Sixty-three of the 339 (19%) follow-up samples had elevated serum erbB-2 antigen levels. Approximately one-third (30.9%) of the samples taken during recurrent disease were serum erbB-2 positive, which is close to the overall overexpression rate of this oncogene. Elevated erbB-2 levels were more common in patients whose disease was not responsive to treatment. Patients with distant metastases had elevate erbB-2 levels more often (40%) than did those with locoregional recurrence (20%). Elevated erbB-2 levels predicted the appearance of metastases within the next 6 months in 10 of 27 (37%) patients. CONCLUSION: The study's results suggest that assay serum erbB-2 levels may be valuable in the follow-up and monitoring of patients with breast cancer whose primary tumors show erbB-2 overexpression by immunohistochemistry.

Adult↗
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↗
PubMed1994

Molecular cytogenetics of human breast cancer.

Our studies of human breast cancer using CGH and FISH with precisely mapped probes have revealed a surprising amount of intratumor and intertumor heterogeneity in human breast cancers. However, some regions of abnormal copy number are found frequently. We believe that the regions of frequent abnormality harbor novel cancer genes, and that identification of these is important for diagnosis, prognostication, and the development of new therapies.

Breast Neoplasms↗
PubMed1994

High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agarose-embedded cells.

Fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) are essential techniques in physical mapping and in positional cloning. We present a technique that utilizes agarose-embedded high molecular weight DNA prepared for PFGE as a target for FISH. The agarose blocks are melted, and the DNA is extended on a poly-L-lysine-coated microscope slide. The resulting DNA fibers appear on the slide as long straight strands and are a suitable target for high resolution FISH mapping as demonstrated here with cosmid and plasmid hybridizations.

Chromosome Mapping↗
PubMed1994

Sensitive detection of chromosome copy number aberrations in prostate cancer by fluorescence in situ hybridization.

The pattern of chromosomal aberrations and their significance in prostate cancer are poorly understood. We studied 23 prostate cancer and 10 benign prostatic hyperplasia (BPH) specimens by fluorescence in situ hybridization (FISH) using pericentromeric repeat-specific probes for 10 different chromosomes. The aims of the study were: 1) to compare the sensitivity of FISH and DNA flow cytometry in aneuploidy detection, 2) to determine which chromosome copy number changes are most common, and 3) which probe combinations would be most effective in aneuploidy diagnosis. Disaggregated tumor cells from formalin-fixed, paraffin-embedded tissues were pretreated with our newly developed method based on hot glycerol solution to improve probe penetration. All BPH specimens were diploid by DNA flow cytometry and showed no numerical chromosome aberrations by FISH. In prostate cancer, flow cytometry showed abnormal DNA content in 35% of cases, whereas 74% were abnormal by FISH. Aberrant copy number of chromosomes 8 (48% of cases), X (43% of cases), and 7 (39% of cases) were most common. Ninety-four percent of all aneuploid cases would have been detected with these three probes alone. Simple chromosome losses were uncommon but in DNA tetraploid tumors relative losses (trisomy or disomy) of several chromosomes were often found, suggesting progression of prostate cancer through tetraploidization followed by losses of selected chromosomes. In conclusion, our results indicate that FISH using three selected chromosome-specific probes is two to three times more sensitive than flow cytometric DNA content analysis in aneuploidy detection.

Aneuploidy↗
PubMed1993

Extrachromosomal gene amplification in acute myeloid leukemia; characterization by metaphase analysis, comparative genomic hybridization, and semi-quantitative PCR.

A case of acute myeloid leukemia (M-3) with complex karyotypic aberrations and double minute (dmin) chromosomes is presented. The patient had no history of prior exposure to mutagenic or carcinogenic agents or of other malignancies. She died from CNS involvement six weeks after the initial diagnosis. We used comparative genomic hybridization to identify the amplified sequences presumed to represent the dmin of the leukemic cells; the tumor/normal ratios indicated increased signal intensity at 8q24. This localization prompted investigation by semi-quantitative PCR that revealed amplification of the MYC oncogene. The extent of chromosome aberrations and the oncogene amplification, both linked with poor prognosis, may relate to the rapid course of this patient's disease.

Base Sequence↗
PubMed1993

Prognostic value of cells with more than 5c DNA content in node-negative breast cancer as determined by image cytometry from tissue sections.

The aim of this investigation was to study the prognostic significance of 5c cells (presence of cancer cells with > 5c DNA content; ie, over 18 pg of DNA per nucleus) in axillary node-negative breast cancer. Tissue sections (3 microns) from 134 tumors were stained for DNA using the Feulgen method and screened for the percentage of 5c cells with the CAS 200 image analysis system (Cell Analysis System, Inc, Lombard, IL). Cancer cells with a DNA content exceeding the 5c level were found in 45% (60 of 134) of the cases, accounting for a median of 0.2% (range, 0.05% to 1.05%) of all cells. The presence of 5c cells was associated with a high histologic grade of the tumor (P = .0001), a large number of mitoses (P < .0001), flow cytometric DNA aneuploidy and high S-phase fraction (P = .0002 and P < .0001, respectively), and c-erbB-2 oncoprotein and p53 tumor suppressor gene product overexpression (P = .0002 and P = .0006, respectively). Patients with 5c cell-positive tumors had a significantly worse 8-year survival rate (P = .003) than those with 5c cell-negative tumors. Subgroup analysis showed that the presence of 5c cells had a prognostic impact in low malignancy tumors, ie, in well-differentiated (grade I or II) and slowly proliferating tumors. Our findings suggest that determination of 5c cells may be a useful additional prognostic factor in axillary node-negative breast cancer. It adds prognostic information, especially in cases that are otherwise thought to have a favorable course.

Adult↗
PubMed1993

Cathepsin D expression detected by immunohistochemistry has independent prognostic value in axillary node-negative breast cancer.

PURPOSE: Increased expression of the lysosomal protease cathepsin D (CD) has been implicated in the metastatic progression of breast cancer. This study was designed to determine the prognostic significance of CD expression in axillary node-negative (ANN) breast cancer. The relationship of CD expression and onset of soft tissue recurrences and visceral metastatases was also studied. PATIENTS AND METHODS: We analyzed a population-based group of 262 ANN breast cancer patients, none of whom had received any adjuvant chemotherapy or endocrine therapy. An immunohistochemical method based on a new monoclonal antibody (1C11) with a distinct epitope specificity made it possible to study CD expression from archival paraffin-embedded specimens and to distinguish staining in tumor cells from the high-level expression found in tumor-infiltrating macrophages. RESULTS: High-level CD expression, as defined by cytoplasmic immunoreactivity in greater than 10% of the cancer cells, was found in 95 cases (36%). High-level CD expression was associated with large primary tumor size (P = .014), but not with histologic grade, estrogen and progesterone receptors, DNA index, or S-phase fraction, or with c-erbB-2 and p53 overexpression. Patients with CD-positive tumors developed significantly more often both soft tissue recurrences and visceral metastases (P = .0007) and had a significantly shorter disease-free survival (P < .0001). Eight-year overall survival of patients with high-level CD expression was 64% as compared with 90% in those with low-level expression (relative risk, 2.97; 95% confidence interval [Cl], 1.6 to 4.4; P < .0001). According to a Cox multivariate model and a regression-tree analysis, high-level CD expression was an independent predictor of poor overall survival in conjunction with tumor size and S-phase fraction. CONCLUSION: These results indicate that CD expression determined by immunohistochemistry is a powerful prognostic factor in ANN breast cancer. The most significant prognostic information was obtained when CD expression (predicting metastatic activity) was combined with estimate of cell-proliferation rate (S-phase fraction).

Adult↗
PubMed1993

Proliferating cell nuclear antigen immunohistochemistry using monoclonal antibody 19A2 and a new antigen retrieval technique has prognostic impact in archival paraffin-embedded node-negative breast cancer.

We evaluated whether proliferating cell nuclear antigen (PCNA) immunohistochemistry with antigen retrieval could be used as a measure of cell proliferation in archival, formalin-fixed, paraffin-embedded tissues and whether the staining results have long-term prognostic significance in axillary node-negative breast cancer. Primary tumor samples obtained from 109 axillary-node-negative breast cancer cases were used for the study. The best staining results were obtained with the 19A2 antibody after microwave heating in a solution of saturated lead thiocyanate. Using this method, there was a significant correlation (linear regression, r = 0.580, P < 0.001) between the proportion of PCNA19A2-positive carcinoma cells (PCNA19A2 score) and DNA flow cytometric S phase fraction. A high PCNA19A2 score was associated with high mitotic count, DNA aneuploidy, and absence of estrogen receptors. Axillary-node-negative patients with a high PCNA19A2 score (cut-point 8%) had significantly worse prognosis than those with a low PCNA19A2 score (P = 0.008). According to a Cox multivariate analysis, PCNA19A2 score had independent prognostic value but only if S phase fraction was excluded from the analysis. In our study, the PCNAPC10 score correlated weakly only with primary tumor size (analysis of variance) and prognosis (5-year univariate survival analysis), but the significance of these findings needs further evaluation. In conclusion, PCNA immunohistochemistry with the 19A2 antibody after an appropriate antigen retrieval treatment may offer a useful alternative to DNA flow cytometry for the analysis of cell proliferation activity from formalin-fixed, paraffin-embedded breast carcinomas.

Adult↗
PubMed1993

Comparative genomic hybridization: a rapid new method for detecting and mapping DNA amplification in tumors.

Recent evidence indicates that many more genes than the currently known oncogenes may undergo amplification in tumors. We have developed a new technique, Comparative Genomic Hybridization (CGH), which allows rapid detection of DNA amplification anywhere in the tumor genome and maps the amplified sequences on normal chromosomes. CGH is based on a competitive in situ hybridization of differentially labeled tumor DNA and normal DNA to a normal human metaphase spread. Regions of gain of DNA sequences are seen as an increased color ratio of two fluorochromes used to detect the labeled DNAs. Over 20 different regions of amplification have been identified using CGH.

Chromosome Mapping↗
PubMed1992

Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.

Comparative genomic hybridization produces a map of DNA sequence copy number as a function of chromosomal location throughout the entire genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes. Analysis of tumor cell lines and primary bladder tumors identified 16 different regions of amplification, many in loci not previously known to be amplified.

Chromosome Mapping↗
PubMed1992

Association of overexpression of tumor suppressor protein p53 with rapid cell proliferation and poor prognosis in node-negative breast cancer patients.

BACKGROUND: Recent evidence indicates that a subset of axillary node-negative (ANN) breast cancer patients can benefit from adjuvant therapy. Reliable prognostic markers are needed, however, to help clinicians identify these patients and arrive at more rational treatment decisions. PURPOSE: Mutations of the p53 tumor suppressor gene often result in overexpression of the p53 protein. In this study, we evaluated the prognostic significance of p53 protein overexpression in patients with ANN breast cancer. We also studied the association between the tumor cell proliferation rate and overexpression of the p53 and c-erbB-2 proteins, both of which have been implicated in cell cycle control. The c-erbB-2 protein is the product of the ERBB2 gene. METHODS: Two hundred eighty-nine ANN cases were randomly selected from a population-based cohort of patients who had not received any kind of adjuvant chemotherapy or endocrine therapy. Overexpression of the p53 and c-erbB-2 proteins was studied immunohistochemically in archival paraffin-embedded tumor samples, using the CM-1 polyclonal and the TAb 250 monoclonal antibodies, respectively. The tumor cell proliferation rate was measured as the S-phase fraction by DNA flow cytometry. Statistical analyses were performed using BMDP software. RESULTS: High-level p53 protein overexpression, found in 41 of the 289 tumors, was most common in tumors with high histologic grade, negative estrogen receptor status, c-erbB-2 protein overexpression, DNA index greater than 1.3, or high S-phase fraction. The lowest S-phase levels were found in tumors with neither p53 nor c-erbB-2 protein overexpression; the highest levels were seen in tumors showing overexpression of both proteins (P less than .0001). Both p53 and c-erbB-2 overexpression, as well as tumor size, had independent prognostic value in multivariate analysis. Eight-year survival of patients with p53 protein overexpression was 56% compared with 81% in patients with no overexpression (relative risk, 3.7; P less than .0001). If the S-phase fraction was included in a Cox regression analysis, however, only the tumor size and the S-phase fraction emerged as independent predictors of survival. CONCLUSIONS: Overexpression of the p53 and c-erbB-2 proteins indicates a high malignant potential in ANN breast cancer, but it is not a significant prognostic factor independent of the cell proliferation rate. The correlation between overexpression of these proteins and an increased S-phase fraction suggests that they may confer a proliferative advantage to cancer cells in vivo.

Breast Neoplasms↗