Search

After identifying an association between maternal Epstein-Barr virus (EBV) reactivation and acute lymphoblastic leukemia (ALL), the authors analyzed a nested case-control study within Finnish and Icelandic maternity cohorts with 7 million years of follow-up to confirm EBV's role in ALL. Offspring of 550,000 mothers were followed up to age 15 years during 1975-1997 by national cancer registries to identify leukemia cases. Mothers of cases and three quarters of matched mothers of controls were identified by national population registers. First-trimester sera from mothers of 304 ALL cases and 39 non-ALL cases and from 943 mothers of controls were analyzed for antibodies to viral capsid antigen, early antigen, and EBV transactivator protein ZEBRA. Relative risk, estimated as odds ratio (95% confidence interval), was adjusted for birth order and sibship size. Combining early antigen and/or ZEBRA immunoglobulin G antibodies with the presence of viral capsid antigen immunoglobulin M antibodies did not increase the estimate for ALL risk for viral capsid antigen immunoglobulin M alone (odds ratio = 1.9, 95% confidence interval: 1.2, 3.0). Both ZEBRA immunoglobulin G antibodies and viral capsid antigen immunoglobulin M antibodies were associated with an increased risk of non-ALL in the offspring (odds ratio = 4.5, 95% confidence interval: 1.3, 16; odds ratio = 5.6, 95% confidence interval: 1.1, 29, respectively), suggesting EBV reactivation in the mothers of non-ALL cases. EBV reactivation may be associated with a proportion of childhood leukemia. [ABSTRACT FROM AUTHOR]

Some large ecological studies have noted a significant association of testicular cancer (TC) with maternal smoking during pregnancy, while several more controlled studies have been negative. It has been difficult to obtain reliable data on exposure because of the long lag time to cancer diagnosis. We performed a case-control study nested within Finnish, Swedish and Icelandic maternity cohorts exploiting early pregnancy serum samples to evaluate the role of maternal smoking in the risk of TC in the offspring. After reviewing the literature, we also performed a meta-analysis of published studies. For each index mother of the TC patient, three to nine matched control mothers with a cancer-free son born at the same time as the TC case were identified within each cohort. First trimester sera were retrieved from the 70 index mothers and 519 control mothers and were tested for cotinine level by a novel HPLC-MS-MS method developed. No statistically significant association between maternal cotinine level and risk of TC in the offspring was found (OR 0.68; 95% CI 0.35, 1.34). This is the first study based on individual exposure measurements. Its results agree with our meta-analysis of seven previous epidemiological studies (total number of 2149 cases, 2762 controls) using indirect exposure assessment (OR 1.0; 95% CI 0.88, 1.12). [ABSTRACT FROM AUTHOR]

Breast cancer is rare in men, and family history of the disease is a risk factor. The recently discovered BRCA2 gene on chromosome 13q is thought to account for some families with increased risk of breast cancer, including male breast cancer. We describe a family with multiple cases of male breast cancer but, interestingly, no increase in female breast cancer. Linkage to the BRCA2 region is demonstrated and all the affected men share the same haplotype for the BCRA2 markers and loss of the other alleles in their tumours. [ABSTRACT FROM AUTHOR]

Proton pump inhibitors (PPIs) are commonly used drugs among cancer patients. Due to conflicting reports on their safety, we aimed to determine whether PPI use is associated with mortality among prostate cancer patients. In this population-based cohort study, we identified incident diagnoses of prostate cancer between 2007 and 2012 (n = 1058). Follow-up was from 12 months after diagnosis until death, emigration or end the of study. Post-diagnosis use was defined as ≥2 filled prescriptions following diagnosis. We used time-dependent Cox proportional hazard regression models to compute hazard ratios (HRs) and 95% confidence intervals (CIs) for prostate cancer-specific and all-cause mortality associated with post-diagnosis use of PPIs. We identified 347 (32.8%) post-diagnosis PPI users and 711 (67.2%) non-users after diagnosis. Of the 347 patients using PPIs after diagnosis, 59 (17.0%) died due to any cause and 22 (6.3%) due to prostate cancer, compared with 144 (20.3%) and 76 (10.7%) among non-users after diagnosis, respectively. Post-diagnosis PPI use was not associated with prostate cancer-specific mortality (HR 0.88; 95% CI: 0.52-1.48) or all-cause mortality (HR 1.02; 95% CI: 0.73-1.43). Contrary to a previous report, this study did not find evidence of an association between post-diagnosis PPI use and mortality among prostate cancer patients., (© 2019 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Purpose: Increased expression of Vacuolar-type H + ATPases (V-ATPases), in the plasma membrane of cancer cells has been suggested to contribute to the development of aggressive cancer phenotypes by promoting acidic tumor microenvironments. Accumulating data suggest that proton pump inhibitors (PPIs) may elicit a chemopreventive effect via V-ATPase inhibition in some cancers, but evidence is still limited. Therefore, we aimed to explore a potential preventive role of PPIs in this study., Methods: In this population-based case-control study, we identified incident cases of breast cancer (n = 1739), prostate cancer (n = 1897), and malignant melanoma (n = 385) in Iceland between 2005 and 2014 from the Icelandic Cancer Registry. We assessed varying levels of PPI use through record linkages to the Icelandic Medicines Registry. For each case, we selected up to 10 age-matched, sex-matched, and calendar-matched population controls using risk-set sampling. Using conditional logistic regression, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) controlling for NSAID use., Results: Adjusted ORs associated with ever use of PPIs were 1.03 (95% CI: 0.92-1.16) for breast cancer, 1.12 (95% CI: 1.00-1.25) for prostate cancer, and 0.84 (95% CI: 0.69-1.12) for malignant melanoma. Analyses of high use of PPIs (≥1000 DDDs) yielded ORs of 0.97 (95% CI: 0.78-1.19), 1.20 (0.99-1.47), and 0.59 (0.40-1.13) for breast cancer, prostate cancer, and malignant melanoma, respectively. Analyses of cumulative exposure to PPIs did not support a dose-response relationship for any of the three cancer types., Conclusions: Our findings do not support a chemopreventive effect of PPI use on breast cancer, prostate cancer, or malignant melanoma., (© 2018 John Wiley & Sons, Ltd.)

The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis. The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma. Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells. The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding. In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3. Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux. Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma. Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy.

Epithelial to mesenchymal transition (EMT) is a developmental event characterized by phenotypic switching from a polarized epithelial phenotype to an unpolarized mesenchymal phenotype. Changes to plasma membrane function accompany EMT yet the differences in lipid composition of cells that have undergone EMT are relatively unexplored. To address this the lipidome of two cell models of EMT in breast epithelial tissue, D492 and HMLE, were analyzed by untargeted LC-MS. Detected masses were identified and their abundance was compared through multivariate statistical analysis. Considerable concordance was observed in eight lipid components between epithelial and mesenchymal cells in both cell models. Specifically, an increase in phosphatidylcholine and triacylglycerol were found to accompany EMT while phosphatidylcholine- and phosphatidylethanolamine plasmalogens, as well as diacylglycerols decreased. The most abundant fatty acid lengths were C16 and C18 but mesenchymal cells had on average shorter and more unsaturated fatty acids. The results are consistent with enhanced cell mobility post EMT and reflect a consequence of oxidative stress pre- and post EMT in breast epithelial tissue., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Autophagy is a degradation pathway important for cellular homeostasis. The E1-like enzyme ATG7 is a key component of the autophagy machinery, with the main function of mediating the lipidation of LC3/GABARAP during autophagosome formation. By analysing mRNA-sequencing data we found that in addition to the full-length ATG7 isoform, various tissues express a shorter isoform lacking an exon of 27 amino acids in the C-terminal part of the protein, termed ATG7(2). We further show that ATG7(2) does not bind LC3B and fails to mediate the lipidation of members of the LC3/GABARAP family. We have thus identified an isoform of ATG7 that is unable to carry out the best characterized function of the protein during the autophagic response. This short isoform will have to be taken into consideration when further studying the role of ATG7.

Background: The use of proton-pump inhibitors (PPIs) has grown worldwide, and there are concerns about increased unsubstantiated long-term use. The aim of the study was to describe the real-world use of PPIs over the past decade in an entire national population., Methods: This was a nationwide population-based drug-utilization study. Patterns of outpatient PPI use among adults in Iceland between 2003 and 2015 were investigated, including annual incidence and prevalence, duration of use, and dose of tablet used (lower versus higher), as well as the proportion of PPI use attributable to gastroprotection., Results: We observed 1,372,790 prescription fills over the entire study period, of which 95% were for higher-dose PPIs. Annual incidence remained stable across time (3.3-4.1 per 100 persons per year), while the annual prevalence increased from 8.5 per 100 persons to 15.5 per 100 persons. Prevalence increased with patient age and was higher among women than men. Duration of treatment increased with patients' age (36% of users over 80 years remained on treatment after 1 year compared with 13% of users aged 19-39 years), and was longer among those initiating on a higher dose compared with a lower dose. The proportion of PPI users concurrently using nonsteroidal anti-inflammatory drugs decreased over the study period, while the proportion concurrently using acetylsalicylic acid, oral anticoagulants, or platelet inhibitors increased., Conclusions: In this nationwide study, a considerable increase in overall outpatient use of PPIs over a 13-year period was observed, particularly among older adults. Patients were increasingly treated for longer durations than recommended by clinical guidelines and mainly with higher doses., Competing Interests: Conflict of interest statement: The authors declare no conflicts of interest in preparing this article.

Genetic variations in the hyaluronan synthase 1 gene (HAS1) influence HAS1 aberrant splicing. HAS1 is aberrantly spliced in malignant cells from multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM), but not in their counterparts from healthy donors. The presence of aberrant HAS1 splice variants predicts for poor survival in multiple myeloma (MM). We evaluated the influence of inherited HAS1 single nucleotide polymorphisms (SNP) on the risk of having a systemic B cell malignancy in 1414 individuals compromising 832 patients and 582 healthy controls, including familial analysis of an Icelandic kindred. We sequenced HAS1 gene segments from 181 patients with MM, 98 with monoclonal gammopathy of undetermined significance (MGUS), 72 with Waldenstrom macroglobulinemia (WM), 169 with chronic lymphocytic leukemia (CLL), as well as 34 members of a monoclonal gammopathy-prone Icelandic family, 212 age-matched healthy donors and a case-control cohort of 295 breast cancer patients with 353 healthy controls. Three linked single nucleotide polymorphisms (SNP) in HAS1 intron3 are significantly associated with B-cell malignancies (range p = 0.007 to p = 10(-5)), but not MGUS or breast cancer, and predict risk in a 34 member Icelandic family (p = 0.005, Odds Ratio = 5.8 (OR)), a relatively homogeneous cohort. In contrast, exon3 SNPs were not significantly different among the study groups. Pooled analyses showed a strong association between the linked HAS1 intron3 SNPs and B-cell malignancies (OR = 1.78), but not for sporadic MGUS or for breast cancer (OR<1.0). The minor allele genotypes of HAS1 SNPs are significantly more frequent in MM, WM, CLL and in affected members of a monoclonal gammopathy-prone family than they are in breast cancer, sporadic MGUS or healthy donors. These inherited changes may increase the risk for systemic B-cell malignancies but not for solid tumors.

Aurora kinases play a vital part in successful mitosis and cell division. Aberrant Aurora-A and -B expression is commonly seen in various types of tumors. Small molecule Aurora inhibitors have already entered clinical trials. Aurora-A amplification has been shown to be associated with breast tumors from BRCA2-mutation carriers and such patients might therefore be candidates for treatment with Aurora kinase inhibitors. There is a need to identify markers that can predict sensitivity to Aurora inhibition. In this study sensitivity to the inhibitor ZM447439 was tested on a panel of 15 non-malignant and malignant epithelial cell lines that differed with respect to BRCA2 and p53 status and related to level of Aurora kinase expression. The IC(50) value for cell survival ranged from 1.9-8.1 μM and was not related to presence or absence of BRCA2 mutation. The levels of Aurora-A and -B expression correlated with each other but sensitivity towards ZM447439 did not correlate with levels of Aurora-A and -B mRNA expression, alone. Cells treated with the Aurora kinase inhibitor completed mitosis but cytokinesis was inhibited resulting in polyploidy and multinucleation. Different levels of polyploidy could not be fully explained by defects in p53. Only cell lines with a combination of high Aurora-A and -B expression, BRCA2 mutation and p53 defects showed more sensitivity towards Aurora inhibition than other cell lines. In conclusion, BRCA2-mutated cells showed variable sensitivity towards Aurora kinase inhibition. The level of sensitivity could not be predicted by Aurora expression levels alone but BRCA2 mutated tumors with high Aurora expression and non-functional p53 are likely candidates for treatment with Aurora inhibitors.

Macrocyclic bisbibenzyls are a class of characteristic compounds, exclusively produced by liverworts. They are attracting increasing attention due to their wide range of biological activities, including antibacterial, antifungal, and antioxidative properties as well as cytotoxicity. Marchantin A is a cyclic bisbibenzyl that has previously been isolated from Marchantia polymorpha and other liverwort species and has been shown to exert cytotoxic effects. In the present study we found that the Icelandic M. polymorpha species produces marchantin A and through an in vitro cell growth inhibition assay, marchantin A was shown to induce a reduction in cell viability of breast cancer cell lines A256 (IC₅₀ = 5.5 µM), MCF7 (IC₅₀ = 11.5 µM), and T47D (IC₅₀ = 15.3 µM). The effect was considerably increased in all cell lines in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously. Fluorescence microscopy confirmed the antimicrotubular effect of marchantin A, and cell cycle analysis indicated enhanced cell division failure when combining this mitotic-spindle inhibitor with the checkpoint modulator., (© Georg Thieme Verlag KG Stuttgart · New York.)

Tetraploidy and aneuploidy can be caused by cell division errors and are frequently observed in many human carcinomas. We have recently reported delayed cytokinesis in primary human fibroblasts from BRCA2 mutation carriers, implying a function for the BRCA2 tumour suppressor in completion of cell division. Here, we address ploidy aberrations in breast tumours derived from BRCA2 germline mutation carriers. Ploidy aberrations were evaluated from flow cytometry histograms on selected breast tumour samples (n=236), previously screened for local BRCA mutations. The ploidy between BRCA2-mutated (n=71) and matched sporadic (n=165) cancers was compared. Differences in ploidy distribution were examined with respect to molecular tumour subtypes, previously defined by immunohistochemistry on tissue microarray sections. Tetraploidy was significantly 3 times more common in BRCA2 breast cancers than sporadic. However, no differences were found in the overall ploidy distribution between BRCA2-mutation carriers and non-carriers. In BRCA2 cancers, tetraploidy was associated with luminal characteristics. The increased frequency of tetraploidy in BRCA2 associated cancers may be linked to cell division errors, particularly cytokinesis. Additionally, tetraploidy emerges predominantly in BRCA2 breast cancers displaying luminal rather than triple-negative phenotypes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The lichen compound usnic acid (UA) is a lipophilic weak acid that acts as a proton shuttle and causes loss of mitochondrial inner membrane potential. In the current study we show that UA treatment induced the formation of autophagosomes in human cancer cells, but had minimal effects on normal human fibroblasts. However, autophagic flux was incomplete, degradation of autophagosomal content did not occur and acidification was defective. UA-treated cells showed reduced ATP levels and activation of AMP kinase as well as signs of cellular stress. UA is thus likely to trigger autophagosome formation both by energy depletion and stress conditions. Our findings indicate that the H(+)-shuttling effect of UA operates not only in mitochondria as previously shown, but also in lysosomes, and have implications for therapeutic manipulation of autophagy and pH-determined drug distribution.

Routinely used prognostic factors fail to predict clinical outcome in a significant proportion of breast cancer patients, implying that they can not detect some important biological characteristics. Chromosomal changes have been described in breast carcinomas for many years but their significance is not clear. We compared chromosomal changes with clinico-pathological characteristics and clinical outcome in 203 breast cancer patients with a follow-up of 9-18 years. Combining data from classical cytogenetics and flow cytometry revealed chromosomal abnormalities in 142 cases (70%). Of these, 51 (35.9%) contained two or more cytogenetically abnormal clones. Polyclonality was significantly associated with poor breast-cancer-specific survival (P = 0.03) within 5 years, independent of tumor size, lymph node metastases, and hormone receptors. Specific changes were similar to those previously described, but a new finding was a significant association between del 3p12p21 and poor survival. Polyclonality was significantly associated with TP53-mutations but not with a germline BRCA2 mutation. Less than one third of the polyclonal samples were identified by flow cytometry alone. Cytogenetic changes were detected in 17 out of 114 samples from non-tumorous tissue (15%), two of them identical with a clone in the corresponding tumor. Several samples contained clearly unrelated clones within the tumor and outside, implying either multifocal origin or early divergence. In conclusion, the common deletion on Chromosome 3p12p21 was associated with poor clinical outcome. Chromosomal polyclonality is common in breast carcinomas and predicts poor survival. Polyclonality was poorly detected by one-sample flow cytometry. Multiple sampling might improve the detection rate., (Copyright © 2011 Wiley-Liss, Inc.)

An epithelial cell line, referred to as A163, was established from breast carcinoma derived from a patient with a strong family history of breast cancer but no known breast cancer susceptibility mutation. A163 was propagated in a serum-free culture medium including the epidermal growth factor. Immunophenotypic characterization demonstrated a mixed luminal and basal-like phenotype. When epidermal growth factor was excluded from the culture medium, A163 entered a quiescent period followed by a period of increased cell proliferation in a subpopulation of the cells. The epidermal growth factor-independent subpopulation retained the basal-like phenotype of the parental cell line. Karyotype and fluorescent in situ hybridization analysis showed an amplification of epidermal growth factor receptor on 7q in A163-S1 only, resulting in high expression of total and phosphorylated epidermal growth factor receptor. The A163-S1 sub-line piles up in culture, indicating a loss of contact inhibition. When grown on transwell filters, A163 shows basal expression of P63 and cytokeratin 14, whereas A163-S1 expresses P63 ubiquitously, and has lost the basal specific expression of cytokeratin 14, indicating a loss of polarity. Furthermore, when cultured in reconstituted basement membrane matrix, A163 form polarized normal like acini. In contrast, A163-S1 form large disorganized structures with lack of polarity. These cell lines may prove useful to understand molecular changes in breast cancer progression, in particular basal-like breast cancer subtype with bad prognosis and no current treatment options.

Background: In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and alpha-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules., Results: Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1) along the nuclear envelope, 2) irregular, and 3) along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent., Conclusions: These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.

We performed a large nested case-control study within the Finnish and Icelandic maternity cohorts to verify/falsify the association of maternal EBV infection with an increased risk of acute lymphoblastic leukemia (ALL) in the offspring found in previous studies. All hematologic malignancies diagnosed among children born during 1983 to 2006 in Finland and 1997 to 2005 in Iceland were identified through national cancer registries. For each index mother of a leukemia case, three matched control mothers with cancer-free offspring were identified. First trimester sera from 561 ALL and 144 non-ALL index mothers and from 2,105 control mothers were analyzed for antibodies to EBV viral capsid antigen (IgG and IgM), early antigen (IgG) and ZEBRA protein (IgG). Conditional logistic regression-based estimates of odds ratios and 95% confidence intervals adjusted for birth order and sib-ship size were calculated. Overall, there was no evidence of increased risk of ALL associated to EBV viral capsid antigen IgM (odds ratio, 0.9; 95% confidence interval, 0.5-1.8). The early antigen and ZEBRA antibodies (EBV reactivation markers) were also not associated with risk. The data argue against a role of EBV in ALL.

According to the leading hypothesis on testicular cancer (TC) etiology exposure to a specific pattern of steroid hormones in utero, in particular, to high levels of estrogens and low levels of androgens is the major determinant of TC risk in the offspring. We performed a case-referent study nested within Finnish, Swedish and Icelandic maternity cohorts exploiting early pregnancy serum samples to evaluate the role of maternal endogenous steroid hormones with regard to the risk of TC. TC cases and referents were aged between 0 and 25 years. For each case-index mother pair, three or four matched referent-referent mother pairs were identified using national population registries. First trimester or early second trimester sera were retrieved from the index mothers of 73 TC cases and 286 matched referent mothers, and were tested for dehydroepiandrosterone sulfate (DHEAS), androstenedione, testosterone, estradiol, estrone, and sex hormone binding globulin (SHBG). Offspring of mothers with high DHEAS levels had a significantly decreased risk of TC (OR for highest vs. lowest DHEAS quartile, 0.18 (95% CI 0.06-0.58). In contrast, offspring of mothers with high androstenedione levels had an increased risk of TC (OR 4.1; 95% CI 1.2-12.0). High maternal total estradiol level also tended to be associated with an increased risk of TC in the offspring (OR 32; 95% CI 0.98-1,090). We report the first direct evidence that interplay of maternal steroid hormones in the early pregnancy is important in the etiology of TC in the offspring., (Copyright 2008 UICC.)

During open heart surgery in infants the thymus was usually removed, partly or completely. Our previous studies on 16 such children indicated reduced T-cell output later in life with signs of extrathymic maturation of the T cells, but no reduction in T regulatory cells (CD4+CD25+). The diversity of the T-cell repertoire in these children was examined to test if the extrathymic microenvironment could alter Vbeta usage. The expression of Foxp3 and CD127 in CD4+CD25(high) T cells was measured in order to determine whether the T regulatory cells had the phenotype of natural T regulatory cells. There was a wide distribution of Vbeta usage in both study and control groups. Significant variability was found in Vbeta usage for CD4+ and CD8+ T cells when the distribution of the percentage of T cells expressing each Vbeta family was analysed between individuals within each group (P < 0.001; Kruskal-Wallis). Significant difference was also found in average usage of Vbeta2, Vbeta5.1 and Vbeta14 chains within CD4+ T cells and Vbeta2, Vbeta8 and Vbeta21.3 chains within CD8+ cells between the groups (P < 0.05; Student's t-test). There was no difference between the two groups with regard to the proportion of CD4+CD25(high) T cells and no difference in the average expression of Foxp3 or CD127 within the CD4+CD25(high) population. Our data provide evidence that cardiothoracic surgery in infants and total or partial thymectomy alters Vbeta usage, suggesting more limited selection in such children than in the control group. The frequency of natural T regulatory cells seems to be unimpaired.

During recent decades the incidence of testicular cancer (TC) has increased rapidly around the world. Associated exogenous etiological factors might therefore be identifiable. We performed a case-control study nested within Finnish, Swedish and Icelandic maternity cohorts exploiting early pregnancy serum samples to evaluate the role of congenital or neonatal infections with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) as risk factors of TC in the offspring. For each case-index mother pair, three or four matched control-control mother pairs were identified using national population registries. First trimester sera were retrieved from the index mothers of 66 TC cases and 258 matched control mothers, and were tested for antibodies to EBV and CMV. High level of maternal EBV IgG antibodies was associated with significantly increased risk of TC in the offspring (odds ratio (OR), 2.50; 95% confidence interval (CI), 1.15, 5.40), especially with risk of non-seminoma TC (OR, 2.73; 950% CI, 1.25, 5.99) and non-seminoma TC diagnosed under 8 years of age (OR, 2.72; 95% CI, 1.05, 7.04). In contrast, offspring of CMV IgG-seropositive mothers had a decreased risk of TC diagnosed under 8 years of age (OR, 0.35; 95% CI, 0.14, 0.89). Our results suggest that EBV and CMV infections may be associated with TC.

The aim of this study was to examine the natural history of monoclonal gammopathy using a retrospective approach and a long observation period. Protein electrophoresis (PE) and immunofixation (IF) was performed on frozen prediagnosis serum samples from 65 multiple myeloma (MM) and 10 Waldenström's macroglobulinemia (WM) cases. Paraprotein was found in 28% and 46% of the samples from cases using PE and IF respectively. The type of paraprotein was IgA in 33.4% of cases, IgG in 57%, and IgM in 8.5%. Excluding light chain or non-secretory disease, 72 % of MM cases had a prodromal MGUS phase within 10 years of diagnosis MM and WM were preceded by MGUS in at least half of the cases, confirming the premalignant nature of this condition.

Lipid peroxidation of polyunsaturated fatty acids (PUFA) generates reactive products that may cause DNA damage. To examine the possible relationship between DNA damage in peripheral blood mononuclear cells (PBMC) and the concentration of PUFA in red blood cells (RBC), endogenous DNA strand breaks, formamidopyrimidine DNA glycosylase (FPG) sites, and hydrogen peroxide (H2O2) sensitive sites were evaluated by the comet assay in blood samples from 98 Icelandic women. Fatty acid composition of RBC was analyzed by gas chromatography. Endogenous DNA strand breaks in PBMC correlated positively with the concentration of total PUFA, total n-3 PUFA, docosahexaenoic acid, linoleic acid, oleic acid, and palmitic acid in RBC. However, there was no association between FPG sites or H(2)O(2) sensitive sites in DNA in PBMC and the concentration of total PUFA or total saturated fatty acid in RBC. As there was no association between oxidative DNA damage or sensitivity of DNA to oxidative stress and the concentration of PUFA in RBC, the positive association between endogenous DNA strand breaks in PBMC and the concentration of total PUFA in RBC is probably not related to oxidative stress.

Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits with the tissue of origin. Microvasculature was localized in situ by immunohistochemistry in breast samples. From this tissue, collagen-rich stroma and adipose tissue were dissected mechanically and further disaggregated to release microvessel organoids. BRENCs were cultured from these organoids in endothelial specific medium and characterized by staining for endothelial markers. Microvessels were a prominent feature of intralobular tissue as evidenced by immunostaining against endothelial specific markers such as CD31, VE-cadherin, and von Willebrand factor (VWF). Double staining against VE-cadherin and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) showed that blood and lymphatic vessels could be distinguished. An antibody against CD31 was used to refine protocols for isolation of microvasculature from reduction mammoplasties. BRENCs retained critical traits even at high passage, including uptake of low-density lipoprotein, and had E-selectin induced upon treatment with tumor necrosis factor-alpha. The first signs of senescence in passage 14 were accompanied by gain of trisomy 11. At passage 18 cells showed chromosomal aberrations and growth arrest as revealed by beta-galactosidase staining. We demonstrate here that breast microvasculature may serve as a large-scale source for expansion of BRENCs with molecular and functional traits preserved. These cells will form the basis for studies on the role of endothelial cells in breast morphogenesis.

Our previous study showed that children who had been partially or completely thymectomized during heart surgery as infants had lower proportions and numbers of total lymphocytes and reduced proportions of T cells (CD3(+)), helper T cells (CD4(+)) and naive T cells (CD3(+) CD4(+) CD45RA(+)), but normal proportion of cytotoxic T cells (CD8(+)). In this study T lymphocytes from a selected group of eight of these children and age- and gender-matched controls were characterized further using flow cytometry to determine phenotypes of T cells and T cell subsets related to T cell regulation and phenotypes suggestive of extrathymic maturation. Immune function was assessed by measuring autoantibodies and antibodies against vaccines. The study group had significantly lower numbers of all the main subsets of T lymphocytes and the composition was different. Thus, the proportions of lymphocytes with the following phenotypes: CD3(+), CD2(+), CD7(+), CD4(+), CD62L(+), CD4(+) CD62L(+) and CD4(+) CD69(-) were significantly reduced in the study group compared with the control group, but significantly higher proportions were seen of lymphocytes expressing CD8alpha(+) CD8beta(-) and TCRgammadelta(+) CD8alpha(+) CD8beta(-). The absolute number and proportion of CD4(+) CD25(+) cells were reduced but the proportions of the subgroup of naive regulatory T cells (CD4(+) CD25(+) CD62L(+)) and non-activated regulatory T cells (CD4(+) CD25(+) CD69(-)) were not reduced in the thymectomized children. We conclude that the phenotypic characteristics of T lymphocytes of children who have lost their thymus in infancy are indicative of extrathymic maturation. T regulatory cells appear to be less affected than other subsets by the general reduction in T cell numbers.

PUFA are susceptible to oxidation. However, the chain-reaction of lipid peroxidation can be interrupted by antioxidants. Whether an increased concentration of PUFA in the body leads to decreased antioxidant capacity and/or increased consumption of antioxidants is not known. To elucidate the relationship between plasma total antioxidant capacity (TAC), the concentration of antioxidant vitamins, and the proportion of PUFA in red blood cells (RBC), plasma TAC was measured by a Trolox equivalent antioxidant capacity assay in blood samples from 99 Icelandic women. Concentrations of tocopherols and carotenoids in the plasma were determined by HPLC, and the FA composition of RBC total lipids was analyzed by GC. Plasma TAC and the plasma concentration of alpha-tocopherol correlated positively with the proportion of total n-3 PUFA, 20:5n-3, and 22:6n-3 in RBC, whereas the plasma lycopene concentration correlated negatively with the proportion of total n-3 PUFA and 20:5n-3. On the other hand, plasma TAC correlated negatively with the proportion of n-6 PUFA in RBC. Plasma TAC also correlated positively with the plasma concentration of alpha-tocopherol, alcohol consumption, and age. Both the plasma concentration of alpha-tocopherol and age correlated positively with the proportion of n-3 PUFA in RBC; however, n-3 PUFA contributed independently to the correlation with plasma TAC. Because the proportion of n-3 PUFA in RBC reflects the consumption of n-3 PUFA, these results suggest that dietary n-3 PUFA do not have adverse effects on plasma TAC or the plasma concentration of most antioxidant vitamins.

Germ line mutations in BRCA1 and BRCA2 account for a large proportion of inherited breast and ovarian cancer. Both genes are involved in DNA repair by homologous recombination and are thought to play a vital role in maintaining genomic stability. A major drawback for long-term functional studies of BRCA in general and BRCA2 in particular has been a lack of representative human breast epithelial cell lines. In the present study, we have established three cell lines from two patients harboring the 999del5 germ line founder mutation in the BRCA2 gene. Primary cultures were established from cellular outgrowth of explanted tissue and subsequently transfected with a retroviral construct containing the HPV-16 E6 and E7 oncogenes. Paired cancer-derived and normal-derived cell lines were established from one patient referred to as BRCA2-999del5-2T and BRCA2-999del5-2N, respectively. In addition, one cell line was derived from cancer-associated normal tissue from another patient referred to as BRCA2-999del5-1N. All three cell lines showed characteristics of breast epithelial cells as evidenced by expression of breast epithelial specific cytokeratins. Cytogenetic analysis showed marked chromosomal instability with tetraploidy and frequent telomeric associations. In conclusion, we have established three breast epithelial cell lines from two patients carrying the BRCA2 Icelandic 999del5 founder mutation. These cell lines form the basis for further studies on carcinogenesis and malignant progression of breast cancer on a defined genetic background.

A case-control study was nested within two maternity cohorts with a total of 7 million years of follow-up for assessment of the role of bacterial infections in childhood leukemia. Offspring of 550,000 mothers in Finland and Iceland were combined to form a joint cohort that was followed for cancer up to age 15 years during 1975-1997 through national cancer registries. For each index mother-case pair, three or four matched control mother-control pairs were identified from population registers. First-trimester serum samples were retrieved from mothers of 341 acute lymphoblastic leukemia cases and 61 other leukemia cases and from 1,212 control mothers. Sera were tested for antibodies to the genus Chlamydia, Helicobacter pylori, and Mycoplasma pneumoniae. Odds ratios and 95% confidence intervals, adjusted for sibship size, were calculated as estimates of relative risk. M. pneumoniae immunoglobulin M appeared to be associated with increased risk (odds ratio (OR) = 1.6), but the association lost statistical significance when the specificity of the immunoglobulin M was considered (OR = 1.5, 95% confidence interval: 0.9, 2.4). In Iceland, H. pylori immunoglobulin G was associated with increased risk of childhood leukemia in offspring (OR = 2.8, 95% confidence interval: 1.1, 6.9). Since H. pylori immunoglobulin G indicates chronic carriage of the microorganism, early colonization of the offspring probably differs between Iceland and Finland, two affluent countries.

Background: The aim of this work was to study the constituents and cytotoxicity of the essential oils from the fruits of Angelica archangelica growing in Iceland., Materials and Methods: Three samples of essential oils were prepared by steam distillation. Their composition was established with GC/MS. The effects of the oils were examined in PANC-1 human pancreas cancer cells and Crl mouse breast cancer cells in concentrations ranging from 10-400 microg/ml, measuring the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl)-2- (4-sulphophenyl) -2H-tetrazolium (MTS) by mitochondrial enzymes., Results: Two types of essential oils were found, differing mainly in the absence or presence of beta-phellandrene. The ED50 of the oils ranged from 48.6 microg/ml to 108.3 microg/ml for PANC-1 and 48.0 microg/ml to 91.8 microg/ml for Crl cells., Conclusion: The cytotoxic activity of the essential oils was independent of the quantity of their main components.

Background: The purpose of this study was to examine the effect of a leaf extract from A. archangelica on the growth of Crl mouse breast cancer cells in vitro and in vivo., Materials and Methods: The antiproliferative activity of the extract was measured by 3H-thymidine uptake in the Crl cells in vitro. Twenty mice were injected with the Crl cells, and 11 of them were fed A. archangelica leaf extract, and the progress of the tumours was followed., Results: The leaf extract was mildly antiproliferative on the Crl cells with an EC50 of 87.6 microg/ml The antitumour activity of the extract was expressed in the mice by marked reduction in tumour growth. In the experimental animals, 9 out of 11 mice developed no or very small tumours, whereas control animals, not receiving the extract, developed significantly larger tumours (p<0.01), as estimated by Mann-Whitney U-test. The antitumour activity of the leaf extract could not be explained by the antiproliferative activity of furanocoumarins present in the extract., Conclusion: The results demonstrate the antiproliferative activity in vitro and antitumour activity in vivo of a leaf extract from A. archangelica

The aim of this work was to study the antiproliferative effect of a tincture from fruits of Angelica archangelica and the active components using the human pancreas cancer cell line PANC-1 as a model. Significant dose-dependent antiproliferative activity was observed in the tincture with an EC50 value of 28.6 microg/ml. Strong antiproliferative activity resulted from the two most abundant furanocoumarins in the tincture, imperatorin and xanthotoxin. The contribution of terpenes to this activity was insignificant. Imperatorin and xanthotoxin proved to be highly antiproliferative, with EC50 values of 2.7 microg/ml and 3.7 microg/ml, respectively, equivalent to 10 and 17 microM. The results indicate that furanocoumarins account for most of the antiproliferative activity of the tincture.

Infants undergoing open heart surgery often have all or part of their thymus removed. The activity of the immune system has not been investigated thoroughly in these children, and only shortly after the operation. Therefore, it was decided to investigate the activity of the immune system in more detail in children several years after their operation. Peripheral blood samples from 19 children who had undergone open heart surgery during their first months of life was collected (study group) and from 19 age- and gender-matched children (control group). The activity of the immune system was evaluated by measuring the number of different cell types in peripheral blood, the phenotype of lymphocytes and the response of T cells following in vitro stimulation by mitogen, tetanus toxoid and measles antigen. The study group had significantly lower counts of total lymphocytes, which was reflected in a lower number of T cells but not B cells. Furthermore, the study group had significantly lower proportion of T cells (CD3(+)) and helper T cells (CD4(+)), but not cytotoxic T cells (CD8(+)). The level of neutrophils in peripheral blood was significantly higher in the study group. This may indicate enhanced innate immunity when the acquired immunity is defective. The results indicate a shift to extrathymic T cell maturation, which is less efficient for CD4(+) helper cells than for CD8(+) cytotoxic cells.

A critical role for infection in the etiology of childhood leukemia has repeatedly been suggested. The authors undertook a case-control study nested within national maternity cohorts with altogether 7 million years of follow-up to assess the relative role of three maternal herpesvirus infections in childhood acute lymphoblastic leukemia (ALL). Offspring of 550,000 mothers in Finland and Iceland formed the joint study cohort that was followed up for cancer in the offspring before age 15 years during 1975-1997 through national cancer registries. For each index mother-case pair, three or four matched control mother-control pairs were identified from national population registers. First-trimester sera were retrieved from mothers of 342 ALL and 61 other leukemia cases and from 1,216 control mothers and were tested for antibodies to cytomegalovirus, Epstein-Barr virus (EBV), and human herpesvirus 6. Serum EBV DNA was also analyzed. Conditional logistic regression-based estimates of relative risk (odds ratio) adjusted for birth order and sibship size, and population attributable fractions, were calculated. Only EBV immunoglobulin M positivity in EBV-immunoglobulin-G-positive mothers was associated with a highly significant increased risk of ALL in the offspring (adjusted odds ratio = 2.9, 95% confidence interval: 1.5, 5.8). Results indicate that reactivation of maternal EBV infection is probably associated with childhood ALL.

Objective: Cervical cancer is a disease caused in part by an infection with an oncogenic subtype of human papillomavirus (HPV). In this study we analysed all cervical cancer samples diagnosed in Iceland during two periods, 1958-1960 and 1995-1996, and asked whether significant changes in viral or immunological parameters had occurred over a period that spanned both significant changes in sexual attitude and the implementation of organized screening for cervical cancer., Methods: Samples from 47 patients (46 squamous cell carcinomas (SCC) and 1 adenosquamous carcinoma (ASC)) in the first period and 30 patients (20 SCC, 4 ASC, and 6 adenocarcinomas (AC)) in the later period were analysed for viral subtype and expression of Fas, FasL, MHC class I, p53 and apoptosis., Results: AC and ASC are proportionately much more common today than 40 years ago (30% vs 2%). The distribution of HPV in cervical cancer is similar in both periods, with HPV16 found in 75% and HPV18 in 13% of cases. Other HPV types found were 31,33,45, and 59. No significant differences were found in the immunological profiles of tumors from the two periods except that a higher fraction of SCC in the later period stained positive for FasL. When SCC are compared with AC/ASC, the latter have less expression of MHC class I, less expression of Fas, and stronger FasL expression., Conclusions: AC/ASC tumors show some immunological features that suggest that they are more resistant to immune attack than SCC.

Objective: To estimate the risk of malignant diseases in families of probands with the same mutation in the BRCA2 gene., Design: A cohort study using record linkage of a breast cancer family resource and the Icelandic Cancer Registry., Setting: Iceland., Subjects: Families of 995 breast cancer patients, from which 887 were tested for a single founder 999del5 mutation; 90 had the mutation and 797 did not., Results: Relatives of probands with the mutation had significantly increased relative risk (RR) of breast cancer. For first degree relatives, the RR was 7.55 (95% CI 6.04 to 9.03) but was 1.72 (95% CI 1.49 to 1.96) in first degree relatives of probands without the mutation. For prostate and ovarian cancer, the first and second degree relatives of probands with the mutation had a significantly increased RR, but in families of probands without the mutation no significant familial risk was found., Conclusions: The 999del5 mutation in the BRCA2 gene explains a substantial proportion of familial risk of breast cancer in Iceland, but significant familial risk remains in relatives of probands without the mutation. For prostate and ovarian cancer, the mutation accounts for most of the familiality observed in families of breast cancer patients.

H. pylori infection is considered a causal agent of duodenal ulcer and a significant risk factor for gastric cancer. Retrospective cohort studies have demonstrated a significant association between presence of antibody to H. pylori and gastric cancer when using samples obtained years before the diagnosis but not at the time of diagnosis. The present study investigates, in a population-based cohort, whether a decline occurs in H. pylori antibody levels before the diagnosis of stomach cancer. Repeat samples (2 to 5) were available from 23 persons with gastric cancer taken up to 20 years before the diagnosis and 128 control subjects matched for gender, age, time and number of repeat samples. The odds ratio of developing stomach cancer was 1.16 (95% CI 1.05-1.28) for those showing decline in antibody levels of 1 relative antibody activity unit per year versus those with constant or rising levels. We conclude that this decline in antibody levels in cases, and not in controls, supports an active role of H. pylori in the pathogenesis of gastric cancer by causing atrophic gastritis, and provides a better risk assessment for gastric cancer compared to single measurements.

To clarify the important role of the tumor-suppressor gene p53 in maintaining genetic integrity, we estimated chromosome instability and staining of overexpressed p53 protein in the same cells of five primary breast carcinomas. The method included both fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH) on sections from formalin-fixed, paraffin-embedded breast cancer tissue. By using a centromeric FISH probe for chromosome 17 on interphase cells in these sections, we showed that cells with abnormal p53 protein expression had a statistically significant higher number of chromosome 17 than did cells with no p53 protein staining in the same samples as well as cells in four other tumor samples with no p53 protein staining. The samples identified positive for p53 abnormality by immunostaining were shown to have p53 mutation by constant denaturing gel electrophoresis analysis and DNA sequencing. These mutated samples were characterized by high DNA index, high S-phase, abnormal karyotype, and aneuploidy. The results strongly implicate p53 mutation as a cause for chromosomal instability and a crucial step in mammary carcinogenesis.

Alterations of the TP53 gene were analyzed in samples from 87 primary breast cancer patients, using molecular and immunohistochemical approaches. Mutations were detected in 17% of the samples, using polymerase chain reaction (PCR) and constant denaturant gel electrophoresis (CDGE) on exons 5-8 of the TP53 gene, and were confirmed by sequencing. Abnormal TP53 protein staining was found in 55% of the primary samples, using the monoclonal TP53 antibody DO7. A statistically significant association was found between TP53 mutations and abnormal protein staining (p = 0.002). Our results suggest that dysfunction of the TP53 protein is associated with tumor progression, as we found an association between TP53 abnormalities and accumulation of genetic lesions, measured as overall allelic imbalance (AI), homogeneously staining regions (HSR) and strong ERBB2 overexpression. Furthermore, patients with TP53 mutation had a highly elevated risk of dying from breast cancer during the study period (p < 0.001, RR = 10.68) at a median follow-up time of 42 months. Abnormal TP53 staining was much more frequent than the mutations, but it was not of prognostic significance, whereas strong staining was an independent prognostic factor. We therefore conclude that loss of functional TP53 leads to genetic instability, resulting in poorer short-term prognosis, and that only strong staining of TP53, and not abnormal protein staining in general, is of prognostic significance.

Molecular genetics and cytogenetics are two different approaches to studying genetic changes in breast carcinoma. We have used karyotype analysis, fluorescence in situ hybridization, and molecular analysis of allelic imbalance on chromosomes 7q and 16q and on both arms of chromosome 17, to study 85 breast carcinomas. Twenty-five of these samples gave results that could be used to compare the two methods. Sixty-nine chromosome arms were compared, of which 48 (70%) gave concordant molecular and cytogenetical results. Samples were processed for karyotyping both by harvesting directly from the fresh tissue and after selective culture for a few days. Karyotypes among the direct harvest samples matched significantly better with the molecular genetics results than karyotypes among the cultured cell preparations.

The BRCA2 gene on chromosome 13 has been shown to be associated with familial male and female breast cancer. Here we describe a study on BRCA2 in 21 Icelandic families, including 9 with male breast cancer. We have previously reported linkage to the BRCA2 region in an Icelandic male breast cancer family and subsequently found a strong indication of linkage to BRCA2 and the same BRCA2 haplotype in breast cancer cases from 15 additional families, indicating a common origin. We describe a five base-pair deletion in exon 9 of BRCA2 in an affected male from the male breast cancer family. The same mutation occurs in all the families with the shared BRCA2 haplotype indicating a founder effect. Among mutation carriers there are 12 males with breast cancer, which accounts for 40% of all males diagnosed with breast cancer in Iceland over the past 40 years. Three of them have no family history of breast cancer indicating that this mutation may have variable penetrance. The same BRCA2 mutation appears to be associated with different cancer phenotypes in this population including male and female breast cancer, prostate cancer, pancreas cancer and ovarian cancer.

CA-125 is a high molecular weight glycoprotein that is best known as a tumour marker for ovarian carcinoma but has been found to be present on various epithelial surfaces including normal tissues. Elevated serum levels of CA-125 have been described in malignancies other than ovarian carcinoma as well as in inflammatory conditions. The expression of CA-125 was studied in paraffin-embedded tissue from 48 mammary carcinomas and 11 samples of normal mammary gland using two monoclonal antibodies, M2 and M11. CA-125 was detected in all normal tissue samples and 64% of the breast carcinomas. Eight of the thirty CA-125-positive carcinomas reacted with only one of the antibodies, indicating molecular change. In normal mammary tissue, CA-125 was seen on apical surfaces and in ductal contents, whilst the majority of the carcinomas (90%) expressed CA-125 in cytoplasmic granules, often showing membranous staining as well. In 16 samples of lymph node metastases CA-125 expression was similar to that seen in the primary tumour. Elevated serum levels of CA-125 were detected in only 3 out of 41 samples available from this patient group. No significant associations were detected with various clinical parameters. We conclude that CA-125 is normally expressed in the mammary gland and that the expression is frequently altered and sometimes absent in mammary carcinoma, possibly reflecting the loss of cellular polarity. Measuring serum levels of CA-125 is not relevant in breast carcinoma patients since one third of breast carcinomas were CA-125 negative and even patients with strongly CA-125-positive tumors had undetectable CA-125 serum levels.

Abnormalities in the TP53 tumour suppressor gene were evaluated in 106 unselected breast carcinomas and compared to clinical outcome of the disease. Tumours were screened for p53 abnormalities using immunohistochemical staining and polymerase chain reaction-constant denaturant gel electrophoresis (PCR-CDGE) analysis, followed by PCR and direct sequencing. Allelic loss at the TP53 locus was determined with polymorphic markers by comparing normal and tumour DNA. For approximately half of the patients, abnormal p53 protein expression in serum was determined by an ELISA assay. p53 abnormalities, detected as mutations and/or nuclear staining, were found in 37.6 (38/101) of cases. Nuclear staining for p53 protein could be identified in 33.7% of the tumours. Mutations in exons 5-8 were detected in 18.9% of the tumours, and an association was found between mutations and nuclear staining. Allelic loss in the TP53 region on 17p was more frequent in tumours showing changes in the TP53 gene (72.7%) compared to tumours with no mutation (45.8%). Serum levels of p53 antibodies showed no association with either TP53 mutations or nuclear staining. Women with TP53 mutations in their tumours had an elevated risk of dying during the study period (RR (relative risk) = 3.4, P = 0.014). The effects of p53 positive staining were similar (RR = 3.2, P = 0.013). Considering all abnormalities, mutation and/or staining, the relative risk of dying from breast cancer was 3.5 (P = 0.008).

Abnormalities in the p53 tumor suppressor gene have been shown to affect cell cycle control and lead to genetic instability in cell lines of murine and human origin. We have examined genetic instability in 183 primary human breast carcinomas with and without p53 abnormalities. Mutation analysis was performed by constant denaturant gel electrophoresis and DNA sequencing, and abnormal protein expression was examined by immunohistochemical staining methods. Genetic instability was studied by detection of gene amplification, allelic loss, karyotype analysis, and fluorescent in situ hybridization. We found a significant association between p53 abnormalities and genetic instability detected by these methods.