Helga M. Ögmundsdóttir

Linkage to BRCA2 region in hereditary male breast cancer

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.

Nordic biological specimen banks as basis for studies of cancer causes and control - More than 2 million sample donors, 25 million person years and 100 000 prospective cancers

The Nordic countries have a long tradition of large-scale biobanking and comprehensive, population-based health data registries linkable on unique personal identifiers, enabling follow-up studies spanning many decades. Joint Nordic biobank-based studies provide unique opportunities for longitudinal molecular epidemiological research. The purpose of the present paper is to describe the possibilities for such joint studies, by describing some of the major Nordic biobank cohorts with a standardised calculation of the cancer incidence in these cohorts. Altogether two million donors have since 1966 donated more than four million biological samples, stored at −20°C to −135°C, to 17 biobank cohorts in Finland, Iceland, Norway and Sweden. As a result of joint database handling principles, the accuracy of personal identifiers and completeness of follow-up for vital status in all participating biobanks was improved. Thereafter, the cancer incidence was determined using follow-up through the national cancer registries. Biobanks based on random samples of population typically showed slightly lower cancer incidence rates than the general population, presumably due to better participation rates among health-conscious subjects. On the other hand, biobanks including samples for viral screening or clinical testing showed 1.5 to 2.1 fold increased incidence of cancer. This excess was very high immediately after sampling, but for some cancer sites remained elevated for years after clinical sampling. So far, more than 100 000 malignant neoplasms have occurred after sample donation, and the annual increase of the cancer cases in these cohorts is about 10 000. The estimates on the population-representativity of the biobanks will assist in interpretation of generalizability of results of future studies based on these samples, and the systematic tabulations of numbers of cancer cases will serve in study power estimations. The present paper summarizes optimal study designs of biobank-based studies of cancer.

Altered expression of E-cadherin in breast cancer patterns, mechanisms and clinical significance

Abstract Reduced cell adhesion brought about by altered surface expression of E-cadherin has been implicated in invasive and metastatic malignant growth. We investigated the patterns of immunohistochemical E-cadherin expression in 120 breast carcinomas. Furthermore, we analysed DNA from the same samples for loss of heterozygosity (LOH) using three separate microsatellite markers on chromosome 16q22.1. Finally, the clinical outcome was ascertained for 108 patients. 19% (18/97) of infiltrating ductal carcinomas showed complete loss of E-cadherin expression compared with 64% (9/14) of infiltrating lobular carcinomas. LOH was detected in 46% (24/52) of infiltrating ductal carcinomas and 89% (8/9) of infiltrating lobular carcinomas. In the infiltrating lobular carcinomas, LOH was associated with complete loss of cell membrane expression of E-cadherin, although a cytoplasmic expression pattern was evident. In contrast, this association was not seen in the infiltrating ductal carcinomas. In a multivariate analysis, loss of E-cadherin expression was shown to be a significant independent risk factor for a poorer disease-free survival (P=0.019), in particular in the node-negative subset of patients (P=0.029). Significance was also approached for breast cancer corrected survival (P=0.056). We conclude that different mechanisms are involved in the altered E-cadherin expression seen in different subtypes of breast carcinomas. Furthermore, we implicate loss of E-cadherin, regardless of the genetic causes, as an independent prognostic marker for disease recurrence, especially in node-negative breast cancer patients, irrespective of the histological type.

The effect of a single BRCA2 mutation on cancer in Iceland.

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.

Altered expression of E-cadherin in breast cancer

Reduced cell adhesion brought about by altered surface expression of E-cadherin has been implicated in invasive and metastatic malignant growth. We investigated the patterns of immunohistochemical E-cadherin expression in 120 breast carcinomas. Furthermore, we analysed DNA from the same samples for loss of heterozygosity (LOH) using three separate microsatellite markers on chromosome 16q22.1. Finally, the clinical outcome was ascertained for 108 patients. 19% (18/97) of infiltrating ductal carcinomas showed complete loss of E-cadherin expression compared with 64% (9/14) of infiltrating lobular carcinomas. LOH was detected in 46% (24/52) of infiltrating ductal carcinomas and 89% (8/9) of infiltrating lobular carcinomas. In the infiltrating lobular carcinomas, LOH was associated with complete loss of cell membrane expression of E-cadherin, although a cytoplasmic expression pattern was evident. In contrast, this association was not seen in the infiltrating ductal carcinomas. In a multivariate analysis, loss of E-cadherin expression was shown to be a significant independent risk factor for a poorer disease-free survival (P=0.019), in particular in the node-negative subset of patients (P=0.029). Significance was also approached for breast cancer corrected survival (P=0.056). We conclude that different mechanisms are involved in the altered E-cadherin expression seen in different subtypes of breast carcinomas. Furthermore, we implicate loss of E-cadherin, regardless of the genetic causes, as an independent prognostic marker for disease recurrence, especially in node-negative breast cancer patients, irrespective of the histological type.

Cellular Mechanisms of the Anticancer Effects of the Lichen Compound Usnic Acid

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from CLADONIA ARBUSCULA and (-)-usnic acid from ALECTORIA OCHROLEUCA were shown to be equally effective inhibitors of DNA synthesis, with IC (50) 4.2 microg/mL and 4.0 microg/mL for (+) and (-)-usnic acid against T-47D, and 5.3 microg/mL and 5.0 microg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

Natural Products: Anti-proliferative Effects of Lichen-derived Inhibitors of 5-Lipoxygenase on Malignant Cell-lines and Mitogen-stimulated Lymphocytes

Several lichen species have been used traditionally as medicinal plants. It has previously been shown that two low‐molecular‐weight lichen metabolites, lobaric acid isolated from Stereocaulon alpinum Laur. and protolichesterinic acid isolated from Cetraria islandica L. (Ach.), have in‐vitro inhibitory effects on arachidonate 5‐lipoxygenase. We have studied the effects of these compounds on cultured cells from man, including three malignant cell‐lines (T‐47D and ZR‐75‐1 from breast carcinomas and K‐562 from erythro‐leukaemia), as well as normal skin fibroblasts and peripheral blood lymphocytes.

Solubilization of the lichen metabolite (+)-usnic acid for testing in tissue culture.

The pharmacological testing of natural products can often be hampered by the poor solubility of such compounds in non‐toxic solvents. There is thus a need for a suitable agent for solubilization of natural substances to allow testing on a variety of cell lines in‐vitro. Such an agent should ideally have no direct effects on any of the commonly used cell lines from a variety of tissues and mammalian species to allow proper comparison. In this study, the lichen metabolite (+)‐usnic acid, a dibenzofuran derivative, was used as a prototype for an insoluble natural product with the aim of finding a solvent that was both capable of solubilizing usnic acid and was free of direct activity against a test cell line. Solubilization was measured at different pH values in various concentrations of co‐solvents (glycofurol 75, propylene glycol, polyethylene glycol 400), surfactants (polysorbate 20 and Cremophor RH40), and the complexing agent 2‐hydroxypropyl‐β‐cyclodextrin. The solubility achieved in a 20% aqueous solution was 0.11 mg mL−1 for propylene glycol, 0.19 for PEG 400, 0.27 for glycofurol 75, 0.57 for Cremophor RH40, 0.68 for 2‐hydroxypropyl‐β‐cyclodextrin and 0.84 for polysorbate 20. The direct effects of the various solvent systems were tested on the human leukaemia cell line K‐562 in a standard proliferation assay. Most of the solvents proved toxic with the exception of propylene glycol, PEG 400 and 2‐hydroxypropyl‐β‐cyclodextrin. Anti‐proliferative activity of usnic acid could be demonstrated with an ED50 (amount of substance required to reduce thymidine uptake to 50% of uptake by untreated control culture) of 4.7μg mL−1 using PEG 400 and 2‐hydroxypropyl‐β‐cyclodextrin but only the latter gave satisfactory solubility. 2‐Hydroxypropyl‐β‐cyclodextrin was thus identified as a solubilizing agent that fulfilled both set criteria of solubility and lack of toxicity against the test cells.

Monoclonal gammopathy in Iceland: a population-based registry and follow-up.

Summary. The term monoclonal gammopathy (MG) signifies the benign or malignant clonal growth of B lymphocytes. In the present study, monoclonal gammopathy of unknown significance (MGUS) was defined as those patients with no identified haematological malignancy. A database was constructed of all 713 MG patients in Iceland between 1976 and 1997 and compared with the Icelandic Cancer Registry. The age‐standardized incidence per 100 000 of MG was 10·3 for males and 8·6 for females, calculated for the whole period, rising steadily from 5·8 (men) and 4·9 (women) during the 5‐year period 1976–80 to 14·7 (men) and 12·5 (women) during the last 5 year period. Age‐standardized incidence rates were very low for subjects under 50 years of age, then increased with age from 11 and 17 per 100 000 at 50‐54, to 169 and 119 per 100 000 at age 80–84, for men and women respectively. No association was detected between MG and non‐haematological malignancies, neither retrospectively nor prospectively. Haematological malignancy was diagnosed in 209 (29·3%) cases before the recorded finding of MG or within the same calendar year, leaving 504 (70·7%) patients diagnosed with MGUS. Of these, 51 (10%) progressed to multiple myeloma or Waldenströms macroglobulinaemia after a mean interval of 3·8 years; mean follow‐up was 7·4 years, median 6 years. The most common immunoglobulin (Ig) class was IgG (55%), followed by IgM (32%) and IgA (13%). MGUS was a highly significant risk factor for developing haematological malignancies and the risk was significantly greater for MG of the IgA class compared with either IgG or IgM.

TP53 mutations and abnormal p53 protein staining in breast carcinomas related to prognosis

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).