Olli-Pekka Kallioniemi

Comparative genomic hybridization

Altering DNA copy number is one of the many ways that gene expression and function may be modified. Some variations are found among normal individuals ( 14, 35, 103 ), others occur in the course of normal processes in some species ( 33 ), and still others participate in causing various disease states. For example, many defects in human development are due to gains and losses of chromosomes and chromosomal segments that occur prior to or shortly after fertilization, whereas DNA dosage alterations that occur in somatic cells are frequent contributors to cancer. Detecting these aberrations, and interpreting them within the context of broader knowledge, facilitates identification of critical genes and pathways involved in biological processes and diseases, and provides clinically relevant information. Over the past several years array comparative genomic hybridization (array CGH) has demonstrated its value for analyzing DNA copy number variations. In this review we discuss the state of the art of array CGH and its applications in medical genetics and cancer, emphasizing general concepts rather than specific results.

Improving the prognostic value of DNA flow cytometry in breast cancer by combining DNA index and S‐phase fraction: A proposed classification of DNA histograms in breast cancer

To optimize the prognostic value of DNA flow cytometry in breast cancer the authors calculated several parameters from the DNA histogram, including the DNA index, the size and number of aneuploid peaks as well as S‐phase and G2/M‐phase cell cycle fractions. Of these, DNA index and S‐phase fraction (SPF) proved to be the most valuable prognostic indices. DNA aneuploidy was associated with a threefold risk of death as compared to DNA diploidy (P < 0.0001). The highest risk of death was associated with hypertetraploid (>2.20) DNA index, whereas a tetraploid DNA index (1.80–2.20) was associated with a relatively low risk. The SPF had significant additional prognostic value in both DNA diploid (P = 0.0002) and DNA aneuploid (P = 0.02) tumors. By combining DNA index and SPF the authors defined three types of DNA histograms, which were associated with favorable, intermediate, and poor prognosis of the patients. DNA diploidy together with low (<7%) SPF (type I DNA histogram) was associated with very favorable prognosis, whereas DNA aneuploidy with high DNA index (>2.20) or high (>12%) SPF (type III DNA histogram) was related to the worst prognosis with approximately eight‐fold relative risk of death. In a Cox multivariate regression analysis the type of DNA histogram was an independent and most powerful prognostic indicator in breast cancer. The other independent factors in the Cox analysis were primary tumor size, nodal status, and progesterone receptor status.

Prognostic significance of DNA index, multiploidy, and S-phase fraction in ovarian cancer

Paraffin‐embedded tumor samples from 157 ovarian cancer patients were analyzed by DNA flow cytometry. Tumor ploidy had prognostic significance in both early and advanced ovarian cancer. After adjusting for stage, residual tumor mass, histopathologic type, treatment, and age of patient in a Cox regression analysis, the relative risk of death was two‐fold higher in single DNA‐aneuploid and sixfold higher in DNA‐multiploid tumors as compared to DNA‐diploid tumors (P < 0.0001). A tetraploid DNA index was associated with a relatively low risk ratio, whereas hypertetraploid tumors were highly malignant. S‐phase fraction (SPF) had prognostic value especially in DNA‐diploid tumors. The simultaneous evaluation of DNA index, the number of aneuploid cell clones, and SPF gave more prognostic information than the determination of tumor ploidy alone. On the basis of these parameters we have developed a classification of tumor DNA histograms for better prognostic assessment of ovarian cancer.

Evaluation of cell proliferation in breast carcinoma. Comparison of Ki-67 immunohistochemical study, DNA flow cytometric analysis, and mitotic count

Growth kinetics of 102 breast carcinomas were studied by immunohistochemical analysis with the monoclonal antibody Ki‐67, which reacts with a nuclear antigen in proliferating cells. The results were correlated with ploidy and S‐phase fraction (SPF) analyzed by DNA flow cytometric study and with mitotic count analyzed by light microscopic study. The proportion of Ki‐67‐positive cells (Ki‐67 score) in breast carcinomas varied from 0.6% to 80% (median, 6.3%). Ki‐67 scores were significantly higher in the DNA aneuploid than in the DNA diploid tumors. Ki‐67 scores correlated significantly with tumor SPF in DNA aneuploid tumors. In DNA diploid tumors SPF showed no correlation with Ki‐67 score. High Ki‐67 scores were associated with high mitotic counts (P < 0.0001) and histologic grade (P < 0.0001). Nuclear pleomorphism, tubule formation, or lymph node status were not correlated with Ki‐67 score. In conclusion, Ki‐67 immunostaining correlates with other measures of cell proliferation (SPF, mitotic count) supporting its clinical significance.

Aneuploid DNA Content and High S-Phase Fraction of Tumour Cells are Related to Poor Prognosis in Patients with Primary Breast Cancer

The prognostic impact of DNA content and S-phase fraction (SPF) of tumour cells was studied in 93 patients with primary breast cancer. Aneuploid DNA content and high SPF were clearly associated with poor differentiation state of tumours and absence of steroid, especially progesterone receptors. Aneuploidy and high SPF tended to become more common with increasing primary tumour size, with more extensive nodal involvement and with more advanced stage of the cancer. Patients with diploid tumours had a slightly longer disease-free interval and survival than those with aneuploid tumours, whereas below median SPF as compared to above median SPF was associated with significantly longer (P less than 0.01) relapse-free interval and survival in patients with stage II-III cancer. We conclude that the DNA analysis of tumour cells is a promising method for the estimation of prognosis in breast cancer patients.

Immunohistochemical determination of estrogen and progesterone receptors in human breast carcinoma. Correlation with histopathology and dna flow cytometry

Human breast carcinomas (n = 232) were evaluated for estrogen and progesterone receptors (ER, PR) by immunohistochemical study and by cytosol steroid‐binding assay (n = 185). The staining was scored (histoscore) by estimates of relative nuclear staining intensity and the percentage of positively stained carcinoma cells. Of the invasive ductal carcinomas 72% were ER‐positive and 55% were PR‐positive. The invasive lobular, intraductal, tubular, and mucinous carcinomas were the most frequent ER‐positive tumor types, whereas comedo and medullary carcinomas only rarely contained ER. Progesterone receptor was most frequently present in intraductal, tubular, and mucinous carcinomas. Better differentiated tumors with lower histologic grade were significantly associated with high prevalence of immunohistochemically determined ER and PR (P < 0.0001). Proliferative cell fraction, determined by DNA flow cytometric study (n = 63), was inversely related to ER (P = 0.03) and PR (P = 0.05) status. Aneuploidy was independent of ER or PR content.

Novel findings in gene expression detected in human osteosarcoma by cDNA microarray.

cDNA microarray analysis was used to screen for gene expression alterations in human osteosarcoma cell lines. The analysis using three cell lines revealed changes in the expression of several genes in comparison with normal human osteoblasts. Among the 5,184 sequences that were analyzed, 35 showed aberrant expression in all the cell lines. Eight of these showed overexpression and 27 underexpression compared to their expression levels in osteoblasts. The most highly up-regulated genes included heat shock protein 90beta and polyadenylate-binding protein-like 1. Commonly down-regulated genes included fibronectin 1 and thrombospondin 1. RT-PCR was used to verify these changes in the cell lines and in three primary osteosarcoma samples. This study shows that (1) gene expression pattern in osteosarcoma cell lines differs considerably from normal osteoblasts, (2) osteosarcoma cell lines can be used as a model system to detect novel gene expression alterations present in primary tumors, (3) the overexpression of heat shock protein 90beta and polyadenylate-binding protein-like 1, and (4) the down-regulation of fibronectin 1 and thrombospondin 1 may play a role in the development and/or progression of osteosarcoma. This study indicates that microarray-based expression surveys may be used to establish the molecular fingerprint of osteosarcoma, however, larger cDNA chips and more tumor specimens are required to define the clinically relevant gene expression patterns.

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.

BARD1 variants Cys557Ser and Val507Met in breast cancer predisposition

BARD1 (BRCA1-associated RING-domain 1) is a tumor suppressor whose protein product interacts with BRCA1, and in which rare somatic and germline mutations have been reported in breast, uterine, and endometrial cancers. We aimed to evaluate whether there are BARD1 genetic variants that contribute to breast cancer risk by screening the gene for germline alterations in 45 Finnish familial breast cancer patients and in seven patients with both breast and ovarian cancer. Two of the missense alterations identified (Cys557Ser and Val507Met) were recently suggested to associate with an increased breast cancer risk. We also analyzed these variants in large and independent series of familial and unselected breast cancer patients and healthy controls. No clearly deleterious mutations were detected in the initial mutation screening. No association of the Cys557Ser and breast cancer risk was observed as the variant was found altogether in 1.4% (16/1181) of familial and 2.2% (34/1565) of unselected breast cancer patients, and in 2.5% (27/1083) of healthy controls. The frequency of the Val-allele of the Val507Met variant was modestly higher among breast cancer patients than among healthy controls, although the difference did not reach statistical significance. No statistically significant association of the Cys557Ser or Val507Met variants with any clinicopathologic parameters was observed. These results suggest that the contribution of the BARD1 germline variants to breast cancer predisposition is very limited, and that neither Cys557Ser nor Val507Met have an effect on familial breast cancer susceptibility.

Flow Cytometric Analysis of Tumour dna Profile Related to Response to Radiotherapy and Survival in Inoperable Lung Cancer

DNA flow cytometric analysis was done from 100 paraffin-embedded non-small cell (NSCLC) and 29 small cell lung carcinomas (SCLC). Most of the patients had locally advanced disease and all were considered inoperable and treated by radiotherapy. DNA aneuploidy was detected in 62 (62%) of the NSCLCs and in 20 (69%) of the SCLCs. Radiotherapy induced a slightly more extensive and prolonged tumour regression in DNA-aneuploid NSCLC with high S-phase fraction (SPF) than in those with low SPF and no evidence of aneuploidy. However, neither in NSCLC nor in SCLC were significant survival differences detected between DNA-diploid and DNA-aneuploid cases or between those with low ( 12%) S-phase fraction (SPF). Neither did the degree of differentiation have prognostic significance in this material.