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Renal cell cancer (RCC) has become a prototype example of the extensive intratumor heterogeneity and clonal evolution of human cancers. However, there is little direct evidence on how the genetic heterogeneity impacts on drug response profiles of the cancer cells. Our goal was to determine how genomic clonal evolution impacts drug responses. Finding from our study could help to define the challenge that clonal evolution poses on cancer therapy. We established multiple patient-derived cells (PDCs) from different tumor regions of four RCC patients, verified their clonal relationship to each other and to the uncultured tumor tissue by genome sequencing. Furthermore, comprehensive drug-sensitivity testing with 460 oncological drugs was performed on all PDC clones. The PDCs retained many cancer-specific copy number alterations and mutations in driver genes such as VHL, PBRM1, PIK3C2A, KMD5C and TSC2 genes. The drug testing highlighted vulnerability in the PDCs toward approved RCC drugs, such as the mTOR-inhibitor temsirolimus, but also novel sensitivities were uncovered. The individual PDC clones from different tumor regions in a patient showed distinct drug-response profiles, suggesting that genomic heterogeneity contributes to the variability in drug responses. Studies of multiple PDCs from a patient with cancer are informative for elucidating cancer heterogeneity and for the determination on how the genomic evolution is manifested in cancer drug responsiveness. This approach could facilitate tailoring of drugs and drug combinations to individual patients.

Notch signaling is frequently hyperactivated in breast cancer, but how the enhanced signaling contributes to the tumor process is less well understood. In this report, we identify the proinflammatory cytokine interleukin-6 (IL-6) as a novel Notch target in breast tumor cells. Enhanced Notch signaling upregulated IL-6 expression, leading to activation of autocrine and paracrine Janus kinase/signal transducers and activators of transcription signaling. IL-6 upregulation was mediated by non-canonical Notch signaling, as it could be effectuated by a cytoplasmically localized Notch intracellular domain and was independent of the DNA-binding protein CSL. Instead, Notch-mediated IL-6 upregulation was controlled by two proteins in the nuclear factor (NF)-κB signaling cascade, IKKα and IKKβ (inhibitor of nuclear factor kappa-B kinase subunit alpha and beta, respectively), as well as by p53. Activation of IL-6 by Notch required IKKα/IKKβ function, but interestingly, did not engage canonical NF-κB signaling, in contrast to IL-6 activation by inflammatory agents such as lipopolysaccharide. With regard to p53 status, IL-6 expression was upregulated by Notch when p53 was mutated or lost, and restoring wild-type p53 into p53-mutated or -deficient cells abrogated the IL-6 upregulation. Furthermore, Notch-induced transcriptomes from p53 wild-type and -mutated breast tumor cell lines differed extensively, and for a subset of genes upregulated by Notch in a p53-mutant cell line, this upregulation was reduced by wild-type p53. In conclusion, we identify IL-6 as a novel non-canonical Notch target gene, and reveal roles for p53 and IKKα/IKKβ in non-canonical Notch signaling in breast cancer and in the generation of cell context-dependent diversity in the Notch signaling output.

The receptor-tyrosine kinase ErbB4 was identified as a direct regulator of hypoxia-inducible factor-1α (HIF-1α) signaling. Cleaved intracellular domain of ErbB4 directly interacted with HIF-1α in the nucleus, and stabilized HIF-1α protein in both normoxic and hypoxic conditions by blocking its proteasomal degradation. The mechanism of HIF stabilization was independent of VHL and proline hydroxylation but dependent on RACK1. ErbB4 activity was necessary for efficient HRE-driven promoter activity, transcription of known HIF-1α target genes, and survival of mammary carcinoma cells in vitro. In addition, mammary epithelial specific targeting of Erbb4 in the mouse significantly reduced the amount of HIF-1α protein in vivo. ERBB4 expression also correlated with the expression of HIF-regulated genes in a series of 4552 human normal and cancer tissue samples. These data demonstrate that soluble ErbB4 intracellular domain promotes HIF-1α stability and signaling via a novel mechanism.

The special AT-rich sequence-binding protein 2 (SATB2), a nuclear matrix-associated transcription factor and epigenetic regulator, was identified as a tissue type-specific protein when screening protein expression patterns in human normal and cancer tissues using an antibody-based proteomics approach. In this respect, the SATB2 protein shows a selective pattern of expression and, within cells of epithelial lineages, SATB2 expression is restricted to glandular cells lining the lower gastrointestinal tract. The expression of SATB2 protein is primarily preserved in cancer cells of colorectal origin, indicating that SATB2 could function as a clinically useful diagnostic marker to distinguish colorectal cancer (CRC) from other types of cancer. The aim of this study was to further explore and validate the specific expression pattern of SATB2 as a clinical biomarker and to compare SATB2 with the well-known cytokeratin 20 (CK20). Immunohistochemistry was used to analyze the extent of SATB2 expression in tissue microarrays with tumors from 9 independent cohorts of patients with primary and metastatic CRCs (n=1882). Our results show that SATB2 is a sensitive and highly specific marker for CRC with distinct positivity in 85% of all CRCs, and that SATB2 and/or CK20 was positive in 97% of CRCs. In conclusion, the specific expression of SATB2 in a large majority of CRCs suggests that SATB2 can be used as an important complementary tool for the differential diagnosis of carcinoma of unknown primary origin.

Background During hematopoiesis, multipotent stem cells and pluripotent precursors undergo a complex differentiation program to generate a diverse set of blood cell types with wide-ranging phenotypes and functions that may shape their initial response to therapies. Therefore, innate drug sensitivity in healthy cells provides insight into cell specific responses, aids identification of lineage specific anticancer therapies and reveals off-target effects. To characterize the diversity in drug response and intracellular signal transduction patterns in the major hematopoietic cell types, we simultaneously assessed their sensitivity to 71 small molecules utilizing a multi-parametric flow cytometry assay and mapped onto their proteomic and basal signaling profiles. Methods Human BM aspirates and PB samples were collected after written informed consent in compliance with the Declaration of Helsinki. Mononuclear cells from 331 BM aspirates or PB samples were isolated by density gradient centrifugation (Ficoll-Paque Premium; GE Healthcare). Cytometric analyses of drug responses were performed assay using a high throughput flow cytometer (iQue®Screener PLUS) in both 384 well (in 3 healthy samples, 71 drugs) and 96 well plate formats (in 26 samples, 6 drugs) to study drug effects in 5 concentrations (1-10,000 nM). Effect of bortezomib, clofarabine, dexamethasone, omipalisib, venetoclax and navitoclax were assessed on 10 cell populations, namely hematopoietic stem cells (HSCs) (CD34+CD38-), common progenitor cells (CPCs) (CD34+CD38+), monocytes (CD14+), B cells (CD45+CD19+), cytotoxic T cells (CD45+CD3+CD8+), T helper cells (CD45+CD3+CD4+), NK-T cells (CD45+CD3+CD56+), NK cells (CD45+CD56+CD3-), plasma cells (CD138+CD38) and granulocytes (CD45+, SSC++). Mass cytometry (CyTOF) was applied to investigate basal signaling activity of 9 proteins involved in MAPK, JAK-STAT, NF-κB and PI3K-mTOR signaling. Protein abundances in CD3+, CD14+ and CD19+ cells were investigated in six samples using mass spectrometry. To evaluate whether the distinct drug sensitivities detected in the cells of origin could be exploited in the malignant cell counterpart cell, ex vivo drug responses detected in healthy cells were compared to a cohort of 281 primary samples derived from multiple hematological malignancies. Results Unsupervised hierarchical clustering of drug response to 71 small molecules identified distinct drug responses in healthy cell subsets based on their cellular lineage. Compared to other cell types, B and NK cells were more sensitive to dexamethasone, venetoclax and midostaurin. Monocytes were more sensitive to trametinib which did not correlate to resting ERK1/2 phosphorylation. Venetoclax exhibited dose dependent cell selectivity to lymphocytes that inversely correlated to STAT3 phosphorylation. Elevated expression of catalase (CAT) and calprotectin (S100A8/S100A9) in monocytes corresponded to their intrinsic resistance to dexamethasone and venetoclax. Comparison of drug responses for six aforementioned drugs in healthy and neoplastic cells across 281 patient samples showed that healthy cell responses are predictive of the corresponding malignant cell response. Conclusion Applying a high content, multi-parametric single-cell assay, we could assess the diversity in drug effects on 10 different cell populations in individual donor samples. Our results demonstrate that cell subtypes are drastically different from each other with respect to protein abundance, signaling profiles and drug-response patterns against a diverse collection of anticancer drugs. Importantly, cell subset specific sensitivity and resistance mechanisms were clearly reflected in their malignant state. Taken together, understanding drug sensitivity in the healthy cell-of-origin provides opportunities to obtain a new level of therapy precision and avoid off-target toxicity. Disclosures Mustjoki: Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding. Wennerberg:Novartis: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Gjertsen:Seattle Genetics: Consultancy; BerGenBio: Consultancy; Kinn Therapeutics: Membership on an entity's Board of Directors or advisory committees; Alden Cancer Therapy 2: Equity Ownership; Boehringer Ingelheim: Research Funding; Alden Cancer Therapy 2: Membership on an entity's Board of Directors or advisory committees; Alden Cancer Therapy 2: Patents & Royalties: Alden Cancer Therapy II patent application in relation to CryoIT trial.; Kinn Therapeutics: Equity Ownership; Novartis: Consultancy. Heckman:Novartis: Research Funding; Orion Pharma: Research Funding; Celgene: Research Funding.

The CHEK2 kinase is a tumor suppressor whose activation in response to DNA double-strand breaks contributes to cell cycle arrest or apoptosis. The c.1100delC mutation is associated with familial breast cancer, and tumors from mutation carriers show reduced or absent CHEK2 protein expression. We have here studied CHEK2 protein expression by immunohistochemistry on a tissue microarray of 611 unselected breast tumors and also evaluated the tumor characteristics among 1,297 unselected breast cancer patients defined for the c.1100delC germ line mutation status (2.5% carrier frequency). CHEK2 protein expression was reduced in 21.1% of the unselected breast cancers studied. Tumors with reduced CHEK2 expression had more often larger primary tumor size (pT3-4; nominal significance p = 0.002) compared to tumors with normal staining. A similar trend for larger tumor size was seen among the 37 breast tumors from c.1100delC germ line mutation carriers. Tumors from c.1100delC mutation carriers were of higher grade than those of noncarriers (nominal significance p = 0.02). The c.1100delC germ line mutation also associated strongly with bilateral breast cancer. No significant correlation was seen between CHEK2 status and hormone receptor status, histology, lymph node status, or overall survival.

Cell cycle checkpoint kinase 2 (CHEK2) is a transducer of cellular responses to DNA damage. The CHEK2 1100delC has previously been shown to be a low-penetrance breast cancer susceptibility allele. We have evaluated the role of another CHEK2 variant, I157T in the FHA domain of the gene, for association with breast cancer. I157T was found at a significantly higher frequency in the population-based series of breast cancer patients (77/1035, 7.4%, odds ratio [OR] = 1.43, 95% confidence interval [CI] = 1.06–1.95, p = 0.021) than among population controls (100/1885, 5.3%). The frequency in the familial breast cancer patients was not elevated (28/507, 5.5%, OR = 1.04, 95% CI = 0.68–1.61). The I157T protein, that undermines cellular responses to ionizing radiation and shows deficiency in substrate recognition in vivo, was expressed at normal level in tumor tissues as well as in cultured cells. The I157T protein was stable and it dimerized with the wild-type CHEK2 co-expressed in human cells. These functional properties of the I157T protein suggest that this variant may have negative effect on the pool of normal CHEK2 protein in heterozygous carrier cells by formation of heterodimers with wild-type CHEK2. The I157T variant may be associated with breast cancer risk, but the risk is lower than for 1100delC. © 2004 Wiley-Liss, Inc.

Disclosed are new methods comprising the use of in situ hybridization to detect abnormal nucleic acid sequence copy numbers in one or more genomes wherein repetitive sequences that bind to multiple loci in a reference chromosome spread are either substantially removed and/or their hybridization signals suppressed. The invention termed Comparative Genomic Hybridization (CGH) provides for methods of determining the relative number of copies of nucleic acid sequences in one or more subject genomes or portions thereof (for example, a tumor cell) as a function of the location of those sequences in a reference genome (for example, a normal human genome). The intensity(ies) of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences in the one or more subject genomes as a function of position along the reference chromosome spread. Amplifications, duplications and/or deletions in the subject genome(s) can be detected. Also provided is a method of determining the absolute copy numbers of substantially all RNA or DNA sequences in subject cell(s) or cell population(s).

To identify chromosomal regions that may contain loci for tumor suppressor genes involved in adrenocortical tumor development, a panel of 60 tumors (39 carcinomas and 21 adenomas) were screened for loss of heterozygosity. Although the vast majority of loss of heterozygosity (LOH) were detected in the carcinomas and involved chromosomes 2, 4, 11, and 18, only few were found in the adenomas. Therefore, 2 loci that harbor the familial cancer syndromes Carney complex in 2p16 and the multiple endocrine neoplasia type 1 gene in 11q13 were further studied in 27 (13 carcinomas and 14 adenomas) of the 60 tumors. Detailed analysis of the 2p16 region mapped a minimal area of overlapping deletions to a 1-centimorgan region, which is separate from the Carney complex locus. LOH for a microsatellite marker (PYGM), very close to the MEN1 gene, was detected in all 8 informative carcinomas (100%) and in 2 of 14 adenomas. Of the 27 cases analyzed in detail, 13 cases (11 carcinomas and 2 adenomas) showed LOH on chromosome 11 and was therefore selected for MEN1 gene mutation analysis. In 6 cases a common polymorphism (Asp418Asp) was found, but no mutation was detected. In conclusion, our data indicate the existence of tumor suppressor genes at multiple chromosomal locations, whose inactivations are involved in the development of adrenocortical carcinomas. Loss of genetic material from 2p16 was strongly associated with the malignant phenotype, as it was seen in almost all carcinomas but not in any of the adenomas. LOH in 11q13 also occurred frequently in the carcinomas, but was not associated with a MEN1 mutation, suggesting the involvement of a different tumor suppressor gene on this chromosome.

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.

All cases of lung cancer diagnosed in the Tampere University Hospital catchment area in 1983–1987 were identified, analyzed for DNA flow cytometry and followed up to 1992. The patients were classified into 3 groups: screen-detected, symptom-detected, and detected by chance. The biological aggressiveness as indicated by DNA flow cytometry was not related to the survival of the symptom-detected patients. Also the screen-detected patients with an aggressive tumour (aneuploid or high S-phase fraction, SPF) had the same survival as the symptom-detected patients. The survival of screen-detected patients with a diploid or low SPF tumour was significantly better than that in the other groups. It is concluded that some of the previously known discrepancy of no effect on mortality and effect on survival of lung-cancer screening may be due to over-diagnosis, i.e., detection of morphologically malignant but biologically indolent lesions by screening. © 1996 Wiley-Liss, Inc.

FISH techniques have opened new possibilities for high-resolution genome mapping. Effective utilization of these techniques for the rapid orientation and ordering of adjacent and overlapping probes as well as for the characterization of long-range genomic contigs would facilitate physical mapping and positional cloning efforts. Here, we have evaluated our recently developed improved fiber-FISH technique for the physical mapping of a 500-kb region at 1p32 as well as for the detection of genomic rearrangement affecting this region. Our fiber-FISH technique is based on the hybridization of probes to unfixed linearized DNA fibers on a microscope slide. Preparation of the target DNA from cells embedded in pulsed-field gel electrophoresis (PFGE) blocks makes it possible to obtain long intact DNA fibers that give an excellent signal-to-noise ratio in FISH. The linear range of the method reached from 2 to 500 kb with a measuring accuracy approaching that of PFGE. Fiber-FISH was used to establish the order, orientation, and distances for several probes for this region, including six large insert phage, cosmid, and P1 clones and seven genomic subclones. This has significantly facilitated our efforts to develop a genomic contig for this region, recently discovered to contain the gene for inherited neuronal ceroid lipofuscinosismore » (INCL). Finally, we also demonstrated how rearrangements affecting the L-myc gene at this locus in small-cell lung cancer can be visualized with fiber-FISH. In conclusion, fiber-FISH is very useful for high-resolution physical mapping and contig evaluation as well as for detecting genetic rearrangements. 22 refs., 2 figs., 1 tab.« less

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.

Our understanding of key epigenetic regulators involved in specific biological processes and cancers is still incomplete, despite great progress in genome-wide studies of the epigenome. Here, we carried out a systematic, genome-wide analysis of the functional significance of 615 epigenetic proteins in prostate cancer (PrCa) cells. We used the high-content cell-spot microarray technology and siRNA silencing of PrCa cell lines for functional screening of cell proliferation, survival, androgen receptor (AR) expression, histone methylation and acetylation. Our study highlights subsets of epigenetic enzymes influencing different cancer cell phenotypes. Plant homeo domain (PHD) finger proteins have a key role in cell survival and histone methylation, whereas histone deacetylases were primarily involved in regulating AR expression. In contrast, JumonjiC-domain (JmjC) containing histone lysine demethylases (KDMs) mainly had an impact on cell proliferation. Our results show that the KDMs JARID1B, PHF8, KDM3A, KDM3B and KDM4A were highly expressed in clinical PrCa samples. The PHD-finger protein 8 (PHF8), a transcriptional coactivator with both PHD- and JmjC-domains, was moderately to strongly expressed in 80% of clinical PrCa samples, whereas 76% of normal and benign samples were negative or only showed weak PHF8 expression. Strong PHF8 expression correlated significantly with high Gleason grade and was borderline significant for poor prognosis. The results of functional PHF8 knockdown implicate a role in cell migration and invasion, as shown by cell motility and 3-D invasion assays. Our study suggests that various cellular phenotypes are regulated by distinct subsets of epigenetic enzymes. Proteins interpreting and modifying histone methylation, such as JmjC-domain and particularly PHD-finger proteins like PHF8, are activated in subsets of PrCa's and promote cancer relevant phenotypes.

We report the effects of monitored smoking cessation on adrenergic regulation in chronic smokers. The beta 2 adrenoceptor density of mononuclear leukocytes (MNLs) and plasma catecholamines was analyzed before cessation and 2, 3, and 8 weeks after cessation. We found a progressive increase in beta-adrenoceptor density after smoking cessation. During smoking the beta-adrenoceptor density was 1.456 +/- 83 (mean +/- SEM) binding sites per cell (n = 10), whereas 3 weeks after cessation the density was 1,774 +/- 157 sites per cell (n = 10; p less than 0.05), and at 8 weeks, 1,900 +/- 227 sites per cell (n = 8; p less than 0.05), representing an overall increase of 23%. Smoking cessation had no effect on binding affinity nor on lymphocyte subgroup distribution. The density of MNL cell beta-adrenoceptors in age-matched nonsmoking men was higher, at 1,896 +/- 271 sites per cell, than that of the chronic smokers before cessation, 1,419 +/- 117 sites per cell (n = 14; p less than 0.01). Plasma epinephrine decreased as a result of cessation from 0.36 pmol/ml (0.26-0.44, 95% confidence interval; baseline) to 0.26 pmol/ml (0.20-0.32) at 8 weeks (p less than 0.05), and norepinephrine decreased from 2.09 pmol/ml (1.38-2.80) to 1.69 pmol/ml (1.14-2.24; p = 0.06). We conclude that stopping smoking progressively increases beta 2-adrenoceptor density on MNL cells. Eight weeks after cessation the adrenoceptor density reaches the corresponding level of nonsmokers. These reversible changes in adrenergic regulation after smoking cessation may be associated with the relatively rapid reduction in cardiovascular disease risk among ex-smokers.

BackgroundCurrent prognostic molecular markers for epithelial ovarian cancer (EOC) are insufficient. The aim of the current study was to investigate the role of Sox11 in EOC.MethodsUsing an in silico transcriptomic screen, Sox11 was identified as a potential EOC biomarker. Sox11 protein expression was evaluated using immunohistochemistry (IHC) in 76 EOC cases, which were analysed using automated algorithms to develop a quantitative scoring model.ResultsSox11 mRNA expression was upregulated in EOC compared to normal tissues. Automated analysis of Sox11 in the EOC cohort revealed high expression of Sox11, in 40% of tumours, who had an improved recurrence-free survival (RFS) (p = 0.002). Multivariate analysis confirmed that Sox11 was an independent predictor of improved RFS after controlling for stage and grade.ConclusionsThese data suggest that Sox11 is a new prognostic marker in EOC. Loss of Sox11 is associated with a decreased RFS and a more aggressive phenotype.

In this work, we calculated the pair wise chemical similarity for a subset of small molecules screened against the NCI60 cancer cell line panel. Four different compound similarity calculation methods were used: Brutus, GRIND, Daylight and UNITY. The chemical similarity scores of each method were related to the biological similarity data set. The same was done also for combinations of methods. In the end, we had an estimate of biological similarity for a given chemical similarity score or combinations thereof. The data from above was used to identify chemical similarity ranges where combining two or more methods (data fusion) led to synergy. The results were also applied in ligand-based virtual screening using the DUD data set. In respect to their ability to enrich biologically similar compound pairs, the ranking of the four methods in descending performance is UNITY, Daylight, Brutus and GRIND. Combining methods resulted always in positive synergy within a restricted range of chemical similarity scores. We observed no negative synergy. We also noted that combining three or four methods had only limited added advantage compared to combining just two. In the virtual screening, using the estimated biological similarity for ranking compounds produced more consistent results than using the methods in isolation.

Members of the ErbB subfamily of receptor tyrosine kinases are important regulators of normal mammary gland physiology, and aberrations in their signaling have been associated with breast tumorigenesis. Therapeutics targeting epidermal growth factor receptor (EGFR = ErbB1) or ErbB2 in breast cancer have been approved for clinical use. In contrast, relatively little is known about the biological significance of ErbB4 signaling in breast cancer. This review focuses on recent advances in our understanding about the role of ErbB4 in breast carcinogenesis, as well as in the potential clinical relevance of ErbB4 in breast cancer prognostics and therapy.

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 less than 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.

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 less than 0.0001) and histologic grade (P less than 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.

Biallelic mutations in the ataxia-telangiectasia mutated (ATM) gene result in ataxia-telangiectasia (A-T). Studies on A-T families have shown that obligate female carriers have increased risk of developing breast cancer. Here we have evaluated the role of known Finnish ATM germ line mutations as possible breast cancer predisposing alleles outside A-T families by analyzing their prevalence in large cohorts of familial and unselected breast cancer cases. Of seven different alterations, two were observed in the studied breast cancer material. ATM 6903insA (causing protein truncation) was seen in 3/541 familial and 5/1124 unselected cases, but not among healthy population controls (0/1107). 7570G>C (Ala2524Pro) occurred in 1/541 familial and 2/1124 unselected cases compared with 1/1107 in controls. Additionally, 8734A>G (Arg2912Gly) associated previously with breast cancer susceptibility and suggested to be causative also for A-T was detected in 2/541 of familial cases, but not in unselected cases (0/1124) or controls (0/1107). In total, heterozygous ATM mutation carriers were observed in 6/541 familial [P = 0.006, odds ratio (OR) 12.4, 95% confidence interval (CI) 1.5-103.3) and 7/1124 unselected cases (P = 0.07, OR 6.9, 95% CI 0.9-56.4), compared with 1/1107 in controls, suggesting an apparent yet overall limited contribution to predisposition to cancer. The current results also provided evidence for founder effects in the geographical distribution of these mutations. Interestingly, results from functional analysis of the breast cancer-associated ATM mutations indicated that cancer susceptibility is not restricted to mutations with dominant-negative effect on kinase activity, displayed only by 7570G>C, whereas 8734A>G showed only a partial defect in the phosphorylation of ATM substrates, and 6903insA seemed to be a null allele.

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.

The CHEK2 kinase is a tumor suppressor whose activation in response to DNA double-strand breaks contributes to cell cycle arrest or apoptosis. The c.1100delC mutation is associated with familial breast cancer, and tumors from mutation carriers show reduced or absent CHEK2 protein expression. We have here studied CHEK2 protein expression by immunohistochemistry on a tissue microarray of 611 unselected breast tumors and also evaluated the tumor characteristics among 1,297 unselected breast cancer patients defined for the c.1100delC germ line mutation status (2.5% carrier frequency). CHEK2 protein expression was reduced in 21.1% of the unselected breast cancers studied. Tumors with reduced CHEK2 expression had more often larger primary tumor size (pT3-4; nominal significance p = 0.002) compared to tumors with normal staining. A similar trend for larger tumor size was seen among the 37 breast tumors from c.1100delC germ line mutation carriers. Tumors from c.1100delC mutation carriers were of higher grade than those of noncarriers (nominal significance p = 0.02). The c.1100delC germ line mutation also associated strongly with bilateral breast cancer. No significant correlation was seen between CHEK2 status and hormone receptor status, histology, lymph node status, or overall survival.

Cell cycle checkpoint kinase 2 (CHEK2) is a transducer of cellular responses to DNA damage. The CHEK2 1100delC has previously been shown to be a low-penetrance breast cancer susceptibility allele. We have evaluated the role of another CHEK2 variant, I157T in the FHA domain of the gene, for association with breast cancer. I157T was found at a significantly higher frequency in the population-based series of breast cancer patients (77/1035, 7.4%, odds ratio [OR] = 1.43, 95% confidence interval [CI] = 1.06-1.95, p = 0.021) than among population controls (100/1885, 5.3%). The frequency in the familial breast cancer patients was not elevated (28/507, 5.5%, OR = 1.04, 95% CI = 0.68-1.61). The I157T protein, that undermines cellular responses to ionizing radiation and shows deficiency in substrate recognition in vivo, was expressed at normal level in tumor tissues as well as in cultured cells. The I157T protein was stable and it dimerized with the wild-type CHEK2 co-expressed in human cells. These functional properties of the I157T protein suggest that this variant may have negative effect on the pool of normal CHEK2 protein in heterozygous carrier cells by formation of heterodimers with wild-type CHEK2. The I157T variant may be associated with breast cancer risk, but the risk is lower than for 1100delC.

The majority of microarray studies focus on analysis of gene expression differences between various specimens or conditions. However, the causes of this variability from one cancer to another, from one sample to another and from one gene to another often remain unknown. In this study, we present a systematic procedure for finding genes whose expression levels are altered due to an intrinsic or extrinsic explanatory phenomenon. The procedure consists of three stages: preprocessing, data integration and statistical analysis. We tested and verified the utility of this approach in a case study, where expression and copy number levels of 13,824 genes were determined in 14 breast cancer cell lines. The procedure resulted in identification of 92 genes whose expression levels could be explained by the variability of gene copy number. This set includes several genes that are known to be both overexpressed and amplified in breast cancer. Thus, these genes may represent an important set of primary, genetically altered genes that drive cancer progression.

Disclosed are new methods comprising the use of in situ hybridization to detect abnormal nucleic acid sequence copy numbers in one or more genomes wherein repetitive sequences that bind to multiple loci in a reference chromosome spread are either substantially removed and/or their hybridization signals suppressed. The invention termed Comparative Genomic Hybridization (CGH) provides for methods of determining the relative number of copies of nucleic acid sequences in one or more subject genomes or portions thereof (for example, a tumor cell) as a function of the location of those sequences in a reference genome (for example, a normal human genome). The intensity(ies) of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences in the one or more subject genomes as a function of position along the reference chromosome spread. Amplifications, duplications and/or deletions in the subject genome(s) can be detected. Also provided is a method of determining the absolute copy numbers of substantially all RNA or DNA sequences in subject cell(s) or cell population(s).

Background Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell proliferation, migration, and permeability during embryonic vasculogenesis as well as in physiological and pathological angiogenesis. The recently isolated VEGF-B and VEGF-C cDNAs encode novel growth factor genes of the VEGF family. Methods and Results Southern blotting and polymerase chain reaction analysis of somatic cell hybrids and fluorescence in situ hybridization (FISH) of metaphase chromosomes were used to assess the chromosomal localization of VEGF-B and VEGF-C genes. The VEGF-B gene was found on chromosome 11q13, proximal to the cyclin D1 gene, which is amplified in a number of human carcinomas. However, VEGF-B was not amplified in several mammary carcinoma cell lines containing amplified cyclin D1. The VEGF-C gene was located on chromosome 4q34, close to the human aspartylglucosaminidase gene previously mapped to 4q34-35. Conclusions The VEGF-B locus in 11q13 and the VEGF-C locus in 4q34 are candidate targets for mutations that lead to vascular malformations or cardiovascular diseases.

The aim of this investigation was to study the prognostic significance of 5c cells (presence of cancer cells with5c 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.

We present a clinicopathological and flow cytometric evaluation of eight primary small cell carcinomas of the esophagus representing 1.5% of all esophageal malignancies diagnosed during a 22-year period (1965-1987) in the Tampere University Central Hospital. The mean age of the patients (four male and four female) was 67 years (range 55-75 years). Five cases had distant metastases at the time of diagnosis. Three patients were treated by esophageal resection, one by laser vaporisation, and four by chemotherapy. The median survival time was 4 months (range 9 days to 8 months). A complete local response to chemotherapy in serial esophagogramms was detected in one patient. All four patients given chemotherapy survived longer than those treated with esophageal resection only. Four (67%) of the six carcinomas analyzed by DNA flow cytometry contained DNA-aneuploid stemlines. The median S-phase fraction of these small cell carcinomas was high (16.3%), reflecting rapid cell proliferation rate, which may be related to their responsiveness to chemotherapy.

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.

Flow cytometric DNA ploidy determination has been regarded as an objective prognostic parameter in several types of human cancer. To test whether DNA histograms are similarly interpreted, a series of flow cytometric DNA histograms was posted to six investigators working in the field for independent classification. The histograms were produced from paraffin-embedded adrenal adenomas or non-neoplastic tissue and had several different patterns. Only 44% of the histograms were similarly classified by all investigators, and 85% by five of the six participants, when DNA ploidy was evaluated. Different criteria for tetraploidy existed, and also some uncertainty in classifying peridiploid and small aneuploid peaks. It is concluded that lack of consensus on histogram classification may result in widely varying percentages of DNA aneuploid tumors found even if the data are similar. Until general agreement is reached on the definition of DNA aneuploidy and its subclasses, classification of DNA histograms is variable and subjective.

To characterize the lipoprotein metabolism of lipid-filled cells of atherosclerotic lesions, uptake of 3,3'-dioctadecylindocarbocyanine (DiI)-labelled low density lipoprotein (LDL), acetylated LDL (Ac-LDL) and beta-very low density lipoprotein (beta-VLDL) was studied by fluorescence microscopy and flow cytometry in primary cultures of enzymatically dispersed aortic cells from cholesterol-fed rabbits. Most of the foam cells were identified as macrophages on the basis of Fc-receptors and high activities of nonspecific esterase and acid lipase, although cholesteryl ester (CE) inclusions were found by filipin staining also in smooth muscle cells (SMCs). During the culture only SMCs proliferated and were confluent in about 1 week. After incubation with DiI-Ac-LDL most macrophage foam cells were brightly fluorescent, but also many SMCs accumulated fluorescence. In SMCs, an excess of LDL inhibited the uptake of DiI-beta-VLDL and DiI-LDL, indicating that these lipoproteins were taken up by the apoB,E receptor; the activity of this receptor was low 2 days after cell isolation but increased considerably during SMC proliferation. DiI-beta-VLDL was not taken up by the macrophage foam cells until after 7 days' culture, when their CE content had decreased, reflecting a feed-back regulation of these receptors as well. Our results indicate that, in primary cultures of enzyme-dispersed cells from rabbit atherosclerotic lesions, most of the foam cells have lipoprotein receptors resembling those described in macrophages and that also many SMCs accumulate Ac-LDL.

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 three-fold risk of death as compared to DNA diploidy (P less than 0.0001). The highest risk of death was associated with hypertetraploid (greater than 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 (less than 7%) SPF (type I DNA histogram) was associated with very favorable prognosis, whereas DNA aneuploidy with high DNA index (greater than 2.20) or high (greater than 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.

The DNA incorporation of a thymidine analogue bromodeoxyuridine (BrdUrd) in herpes simplex virus type 2 (HSV-2) infected cervical cancer cell lines (CaSki and C-33 A) was studied by dual parameter flow cytometry. HSV-2 infection resulted in an exponential increase in DNA synthesis in the CaSki cells. In the less permissive C-33 A cells the proportion of DNA-synthesizing cells cycled during HSV-2 infection. The inhibition of viral DNA synthesis by phosphonoformate (PFA) was able to inhibit the virus induced changes in the CaSki but not in the C-33 A cells. In the C-33 A cells a part of the virus induced cellular DNA synthesis represents repair replication of cellular DNA.

Flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) from paraffin-embedded tumors has become an important diagnostic and prognostic tool in clinical pathology and investigative oncology. The present study aimed at elucidating the reliability of the method. About 90% of the 1,400 paraffin-embedded tumors analyzed were evaluable for DNA index and about 70% for SPF, although considerable differences between various tumor types were detected. The within-assay coefficients of variation for determination of tumor DNA index and SPF were 2% and 6%, respectively. Intratumor variation in DNA index was observed in 24% of breast and in 21% of ovarian carcinomas and variation in SPF in 36% and 29% of the respective tumors. Intratumor variation in SPF was greatest in DNA-diploid tumors, which may indicate that SPF values in these tumors may be less reliable owing to variations in the proportions of tumor and nontumor cells. If the methodological variation and the intratumor variation were taken into account, there was a good correlation between DNA indices (r = 0.980) and between SPF values (r = 0.794) obtained from fresh and paraffin-embedded tumors. It is concluded that accurate determination of DNA index and SPF from paraffin-embedded tumors is possible in the majority of cases. Regardless of the type of starting material used for DNA flow cytometry it is advantageous to study several samples from each tumor to account for the intratumor heterogeneity.

We have studied the nuclear DNA content of histologically favourable (n = 82) or unfavourable (n = 117) non-Hodgkin lymphomas (NHLs) diagnosed between 1957 and 1978 in the Tampere University Central Hospital. The DNA analysis was done by applying a trypsin digestion method to archival tumour samples. DNA aneuploidy was seen in 40 per cent of the unfavourable cases and in 10 per cent of the favourable cases, but varied considerably between different histological subtypes. The unfavourable cases showed high proliferative activity (S-phase fraction, SPF), while considerable variation in the SPF among the favourable NHLs was noted. Among the unfavourable NHLs, cases with DNA-aneuploid tumours had significantly (P less than 0.01) worse prognosis than stage and treatment matched cases with DNA-diploid tumours. In general, survival of the patients who had high SPF tumours was significantly lower compared with patients with low SPF tumours (P less than 0.01). However, SPF was not related to the prognosis in the unfavourable NHLs. We conclude that the flow cytometric DNA analysis revealed characteristic features in the favourable and unfavourable NHLs and may be useful in predicting the clinical outcome of patients.

We have previously defined three types of tumor DNA histograms, which are associated with favourable, intermediate and poor prognosis of patients with ovarian cancer. In the present study we evaluated the value of DNA histogram type combined with clinicopathological data in predicting long-term clinical outcome in stage I ovarian cancer. A stepwise discriminant analysis was done to find out the best combination of prognostic parameters in the distinction of two groups of stage I ovarian cancer patients; those with less than 5-year overall survival (22 cases) and those with longer than 5-year recurrence-free survival (47 cases). DNA histogram and histological type as well as FIGO stage in that order proved to be the most discriminating parameters and their combination allowed the correct prediction of clinical outcome in 78% of stage I ovarian cancer patients. In stage la the proportion of correctly classified cases based on DNA histogram and histological type was 82%. If DNA histogram was omitted from the discriminant analysis, the combination stage and histological type correctly classified a significantly lower percentage (68%) of patients. DNA flow cytometry thus improved the prognostic evaluation in stage I ovarian cancer, but even when combined with conventional clinicopathological factors failed to give correct prognostic assessment in about 20 % of patients with stage I ovarian cancer.

Eleven invasive and five non-invasive primary vaginal carcinomas were studied by DNA flow cytometry using archival paraffin-embedded tissue as starting material. Overall frequency of DNA aneuploidy in the invasive carcinomas was 8 11 (73%). DNA aneuploidy occurred in all four advanced stage (III–IV) and in 4 7 (57%) of the early stage (I–II) carcinomas. Among the squamous cell carcinomas aneuploid DNA content was also associated with non-keratinizing tumor type. Invasive vaginal carcinomas showed a high median S-phase fraction (SPF) (18.4%, range 6.9–31.8%). High SPF values were associated with advanced stage and nonkeratinizing tumors. Corrected 5-year survival rate in invasive vaginal cancer was 44%, with no significant relation to DNA ploidy or SPF. In situ carcinomas were almost as often DNA-aneuploid ( 3 5 , 60%) as the invasive carcinomas and had comparable median SPF value (13.4%, range 5.5–24.6). One in situ carcinoma with a high DNA-index and SPF relapsed, but overall 5-year survival rate was 100%. In conclusion, both invasive and in situ vaginal carcinomas frequently contain DNA-aneuploid stemlines and show a high SPF. Although DNA aneuploidy and high SPF correlate with advanced stage and non-keratinizing tumor type, they do not have much prognostic relevance in vaginal neoplasia.

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.

The majority of microarray studies evaluate gene ex- pression differences between various specimens or con- ditions. However, the causes of this variability often re- main unknown. Our aim is to identify underlying causes of these patterns, a process that would eventually enable a mechanistic understanding of the deregulation of gene expression in cancer. The procedure consists of three phases: pre-processing, data integration and statistical analysis. We have applied the strategy to identify genes that are overexpressed due to amplification in breast cancer. The data were obtained from 14 breast cancer cell lines, which were subjected to cDNA microarray based copy number and expression experiments. The re- sult of the analysis was a list that consisted of 92 genes. This set includes several genes that are known to be both overexpressed and amplified in breast cancer. The com- plete study was published in Journal of the Franklin In- stitute 2004, and in this paper we focus on the main issues of the study.

The serum concentrations of CA 125 and placental alkaline phosphatase were analyzed in 16 patients with ovarian cancer. Increased serum levels of CA 125 and placental alkaline phosphatase were observed in 75% and 50% of the cancer patients, respectively. The serum levels of these tumor markers were not correlated, supporting their distinct antigenic nature. CA 125 seems to be a more promising tumor marker for ovarian cancer than placental alkaline phosphatase.

Summary High-throughput screening (HTS) enables systematic testing of thousands of chemical compounds for potential use as investigational and therapeutic agents. HTS experiments are often conducted in multi-well plates that inherently bear technical and experimental sources of error. Thus, HTS data processing requires the use of robust quality control procedures before analysis and interpretation. Here, we have implemented an open-source analysis application, Breeze, an integrated quality control and data analysis application for HTS data. Furthermore, Breeze enables a reliable way to identify individual drug sensitivity and resistance patterns in cell lines or patient-derived samples for functional precision medicine applications. The Breeze application provides a complete solution for data quality assessment, dose–response curve fitting and quantification of the drug responses along with interactive visualization of the results. Availability and implementation The Breeze application with video tutorial and technical documentation is accessible at https://breeze.fimm.fi; the R source code is publicly available at https://github.com/potdarswapnil/Breeze under GNU General Public License v3.0. Contact swapnil.potdar@helsinki.fi Supplementary information Supplementary data are available at Bioinformatics online.