A prospective evaluation of plasma phospholipid fatty acids and breast cancer risk in the EPIC study.

BACKGROUND: Intakes of specific fatty acids have been postulated to impact breast cancer risk but epidemiological data based on dietary questionnaires remain conflicting. MATERIALS AND METHODS: We assessed the association between plasma phospholipid fatty acids and breast cancer risk in a case-control study nested within the European Prospective Investigation into Cancer and Nutrition study. Sixty fatty acids were measured by gas chromatography in pre-diagnostic plasma phospholipids from 2982 incident breast cancer cases matched to 2982 controls. Conditional logistic regression models were used to estimate relative risk of breast cancer by fatty acid level. The false discovery rate (q values) was computed to control for multiple comparisons. Subgroup analyses were carried out by estrogen receptor (ER) and progesterone receptor expression in the tumours. RESULTS: A high level of palmitoleic acid [odds ratio (OR) for the highest quartile compared with the lowest OR (Q4-Q1) 1.37; 95% confidence interval (CI), 1.14-1.64; P for trend = 0.0001, q value = 0.004] as well as a high desaturation index (DI16) (16:1n-7/16:0) [OR (Q4-Q1), 1.28; 95% C, 1.07-1.54; P for trend = 0.002, q value = 0.037], as biomarkers of de novo lipogenesis, were significantly associated with increased risk of breast cancer. Levels of industrial trans-fatty acids were positively associated with ER-negative tumours [OR for the highest tertile compared with the lowest (T3-T1)=2.01; 95% CI, 1.03-3.90; P for trend = 0.047], whereas no association was found for ER-positive tumours (P-heterogeneity =0.01). No significant association was found between n-3 polyunsaturated fatty acids and breast cancer risk, overall or by hormonal receptor. CONCLUSION: These findings suggest that increased de novo lipogenesis, acting through increased synthesis of palmitoleic acid, could be a relevant metabolic pathway for breast tumourigenesis. Dietary trans-fatty acids derived from industrial processes may specifically increase ER-negative breast cancer risk.

Epidermolytic palmoplantar keratoderma cosegregates with a keratin 9 mutation in a pedigree with breast and ovarian cancer.

Epidermolytic palmoplantar keratosis (EPPK) cosegregates with breast and ovarian cancers in a large French pedigree, raising the possibility that a single genetic mutation might cause these conditions and offering a potential lead to the identification of a hereditary breast/ovarian cancer gene. We have performed linkage analysis and show that the EPPK locus lies on the long arm of chromosome 17 near the type I keratin gene cluster and the proposed breast cancer gene (BRCA1). The type I keratin 9 gene has been partially sequenced in four affected individuals. A single base mutation within the rod domain of the protein cosegregates with EPPK in all affected individuals tested. Although inheritance of this mutation is likely responsible for EPPK, it is unlikely to be the cause of the breast and ovarian cancer.

Ovarian cancer risk in BRCA1 carriers is modified by the HRAS1 variable number of tandem repeat (VNTR) locus.

Women who carry a mutation in the BRCA1 gene (on chromosome 17q21), have an 80% risk of breast cancer and a 40% risk of ovarian cancer by the age of 70 (ref. 1). The variable penetrance of BRCA1 suggests that other genetic and non-genetic factors play a role in tumourigenesis in these individuals. The HRAS1 variable number of tandem repeats (VNTR) polymorphism, located 1 kilobase (kb) downstream of the HRAS1 proto-oncogene (chromosome 11p15.5) is one possible genetic modifier of cancer penetrance. Individuals who have rare alleles of the VNTR have an increased risk of certain types of cancers, including breast cancer (2-4). To investigate whether the presence of rare HRAS1 alleles increases susceptibility to hereditary breast and ovarian cancer, we have typed a panel of 307 female BRCA1 carriers at this locus using a PCR-based technique. The risk for ovarian cancer was 2.11 times greater for BRCA1 carriers harbouring one or two rare HRAS1 alleles, compared to carriers with only common alleles (P = 0.015). The magnitude of the relative risk associated with a rare HRAS1 allele was not altered by adjusting for the other known risk factors for hereditary ovarian cancer (5). Susceptibility to breast cancer did not appear to be affected by the presence of rare HRAS1 alleles. This study is the first to show the effect of a modifying gene on the penetrance of an inherited cancer syndrome.

The contribution of large genomic deletions at the CDKN2A locus to the burden of familial melanoma.

Mutations in two genes encoding cell cycle regulatory proteins have been shown to cause familial cutaneous malignant melanoma (CMM). About 20% of melanoma-prone families bear a point mutation in the CDKN2A locus at 9p21, which encodes two unrelated proteins, p16(INK4a) and p14(ARF). Rare mutations in CDK4 have also been linked to the disease. Although the CDKN2A gene has been shown to be the major melanoma predisposing gene, there remains a significant proportion of melanoma kindreds linked to 9p21 in which germline mutations of CDKN2A have not been identified through direct exon sequencing. The purpose of this study was to assess the contribution of large rearrangements in CDKN2A to the disease in melanoma-prone families using multiplex ligation-dependent probe amplification. We examined 214 patients from independent pedigrees with at least two CMM cases. All had been tested for CDKN2A and CDK4 point mutation, and 47 were found positive. Among the remaining 167 negative patients, one carried a novel genomic deletion of CDKN2A exon 2. Overall, genomic deletions represented 2.1% of total mutations in this series (1 of 48), confirming that they explain a very small proportion of CMM susceptibility. In addition, we excluded a new gene on 9p21, KLHL9, as being a major CMM gene.

Comprehensive analysis of CDKN2A (p16INK4A/p14ARF) and CDKN2B genes in 53 melanoma index cases considered to be at heightened risk of melanoma.

OBJECTIVE: Comprehensive analysis of the 9p21 locus including the CDKN2A, ARF, and CDKN2B genes in 53 individuals from melanoma index cases considered to be at heightened risk of melanoma. METHODS AND RESULTS: Using a combination of DNA sequencing, gene copy number by real time quantitative PCR, linkage analysis, and transcript analysis in haploid somatic cell hybrids, we found no evidence for germline alteration in either coding or non-coding domains of CDKN2A and CDKN2B. However, we identified a p14ARF exon 1beta missense germline mutation (G16D) in a melanoma-neural system tumour syndrome (CMM+NST) family and a 8474 bp germline deletion from 196 bp upstream of p14ARF exon 1beta initiation codon to 11233 bp upstream of exon 1alpha of p16(INK4A) in a family with five melanoma cases. For three out of 10 families with at least three melanoma cases, the disease gene was unlinked to the 9p21 region, while linkage analysis was not fully conclusive for seven families. CONCLUSIONS: These data reinforce the hypothesis that ARF is a melanoma susceptibility gene and suggest that germline deletions specifically affecting p14ARF may not be solely responsible for NST susceptibility. Predisposition to CMM+NST could either be due to complete disruption of the CDKN2A locus or be the result of more complex genetic inheritance. In addition, the absence of any genetic alteration in 50 melanoma prone families or patients suggests the presence of additional tumour suppressor genes possibly in the 9p21 region, and on other chromosomes.

Abnormal expression of vimentin intermediate filaments in human lymphoid cell lines with deletion or translocation of the distal end of chromosome 8.

The expression of vimentin, the major polypeptide of the intermediate filament (IFM) cytoskeleton of lymphoid cells, was studied in normal and malignant human lymphoid cell lines. Cells from 24 of 27 Burkitt's lymphoma cell lines (BLCL) were found to have an absent (16 lines) or decreased (8 lines) expression of vimentin IFM. In contrast, non-Burkitt's malignant lymphoid cell lines (5 lines) and lymphoblastoid cell lines (LCL) derived from normal B-cells (45 lines) exhibited a well-developed vimentin IFM network. However, low expression of vimentin was also found in 3 LCL derived from patients with the Langer-Giedion syndrome, which is characterized by a deletion of the distal end of chromosome 8. Treatment of vimentin-negative BLCL and Langer-Giedion LCL with azacytidine led to a transient reexpression of vimentin.

Establishment of lymphomatous cell lines from bone marrow samples from patients with Burkitt's lymphoma.

A total of 233 bone marrow aspirates were obtained from 43 patients with Burkitt's lymphoma (BL). Lymphoma cells were absent and lymphoblastoid cell lines could not be established from 197 samples, which were characterized by limited initial cell proliferation and development of an adherent population, followed by cell death after 2-4 weeks. In 14 aspirates, after a similar pattern of growth, cell proliferation began again after about 6 weeks, with a rapid appearance and growth of cell clumps from the feeder layer--a type of growth typical of spontaneous lymphoblastoid cell lines. In 22 aspirates, growth of malignant cells was observed in culture and cytocentrifuged, stained smears, including marrow samples from 9 patients in whom the presence of BL cells had not been ascertained or even suspected by cytology. Karyotypic anomalies characteristic of BL were found in these cells: t(8;14) in the majority, two t(8;22), two t(2;8), and one t(2;8;9).

Distinct reactivity of Burkitt's lymphoma cell lines with eight monoclonal antibodies correlated with the ethnic origin.

Twenty-eight Burkitt's lymphoma(s) (BL) cell lines were analyzed with anti-human immunoglobulins and monoclonal antibodies: Y29/55, B1, and BA1 are slightly different pan-B-reagents; TU1 and BL13 are two discriminating markers of the follicle; RFT1 is a pan-T-reagent expressed on the follicle mantle; AL2 reacts with the common acute lymphoblastic leukemia antigen gp100; and 38:13 recognizes a BL-associated antigen. Those lines were classified into 3 groups according to their membrane phenotype. In the first 2 groups, cell lines were derived from BL of germinal center origin, whereas in the last group they were established from BL cells originating in the bone marrow. All cell lines in the last group were from Caucasian BL, whereas lines from African BL of a high-incidence area were in group 1. North African cases were in group 2. Those distinct subgroups were not related specifically to the reactivity with Epstein-Barr virus nuclear antigen, the type of chromosomal translocation, or the clinical features. The variations induced by growth culture as well as the clinical implications were discussed.

Effect of smoking on breast cancer in carriers of mutant BRCA1 or BRCA2 genes.

BACKGROUND: Smoking has carcinogenic effects, and possibly antiestrogenic effects as well, but it has not been found to be a risk factor for breast cancer in women in the general population. However, hereditary breast cancer is primarily a disease of premenopausal women, and interactions between genes and hormonal and environmental risk factors may be particularly important in this subgroup. METHODS: We conducted a matched case-control study of breast cancer among women who have been identified to be carriers of a deleterious mutation in either the BRCA1 or the BRCA2 gene. These women were assessed for genetic risk at one of several genetic counseling programs for cancer in North America. Information about lifetime smoking history was derived from a questionnaire routinely administered to women who were found to carry a mutation in either gene. Smoking histories of case subjects with breast cancer and age-matched healthy control subjects were compared. Odds ratios for developing breast cancer were determined for smokers versus nonsmokers by use of conditional logistic regression for matched sets after adjustment for other known risk factors. RESULTS: Subjects with BRCA1 or BRCA2 gene mutations and breast cancer were significantly more likely to have been nonsmokers than were subjects with mutations and without breast cancer (two-sided P = .007). In a multivariate analysis, subjects with BRCA1 or BRCA2 mutations who had smoked cigarettes for more than 4 pack-years (i.e., number of packs per day multiplied by the number of years of smoking) were found to have a lower breast cancer risk (odds ratio = 0.46, 95% confidence interval = 0.27-0.80; two-sided P = .006) than subjects with mutations who never smoked. CONCLUSIONS: This study raises the possibility that smoking reduces the risk of breast cancer in carriers of BRCA1 or BRCA2 gene mutations.

Characterization of intermediate filaments expressed by Ewing tumor cell lines.

The histogenesis of Ewing sarcoma is still controversial; we therefore studied the expression of intermediate filaments (IF) in cell lines derived from Ewing tumors since identification of IF in tumor cells is considered a reliable marker of tissue origin and differentiation. All nine lines studied expressed vimentin IF; in addition, a small number of Ewing cells from three lines expressed keratin filaments. After treatment with phorbol esters, a high percentage of cells from these three lines synthesize keratin IF identified by immunoblotting as keratin 8 and 18 polypeptides, which are expressed by single epithelia and epithelial cells in early embryonic development. Furthermore cells from a fourth line synthesize keratins after transplantation in nude mice. These data indicate that, under certain conditions, undifferentiated Ewing cells may acquire an IF phenotype related to that of epithelial cells.

Neuroectoderm-associated antigens on Ewing's sarcoma cell lines.

The histogenesis of Ewing's sarcoma, the second most frequent primary bone tumor in humans, remains controversial. Ten Ewing cell lines were analyzed by immunological methods. Surface antigens recognized on Ewing cells were found to be related to the neuroectoderm lineage. They included ganglioside GD2, a marker of neuroectodermal tissues and tumors, and an acidic glycolipid detected by monoclonal antibody HNK-1 in the nervous system. The P61 rat monoclonal antibody that reacts with a peptide moiety of neural cell adhesion molecule (N-CAM) and a rabbit antiserum raised to purified mouse N-CAM also stained Ewing cells. Flow cytometry analysis performed using these reagents allowed the definition of four distinct Ewing phenotypes: all reagents equally stained group 1 lines; group 2 lines were strongly reactive with anti-N-CAM reagents, by contrast with a fainter staining with HNK-1 and anti-GD2 antibodies; all reagents but P61 were strongly reactive with group 3 lines; in group 4, Ewing lines were stained by P61 but only poorly by the anti-N-CAM antiserum. Several antibodies to melanoma and neuroblastoma associated antigens including two monoclonal antibodies to the nerve growth factor receptor were also found to react with Ewing cells. By contrast, all antibodies detecting antigens specifically expressed in hematopoietic cell lineages were totally unreactive. HLA class II antigens were never detected while the level of expression of class I antigens varied to a large extent. Ewing cells are characterized by a specific t(11;22)(q23-24;q12) translocation also observed in neuroepithelioma, a neuroectodermal tumor. Thus, Ewing's sarcoma cells share antigenic and karyotypic features with derivatives of the neuroectoderm possibly indicating a related histogenesis.

A 1-kb Alu-mediated germ-line deletion removing BRCA1 exon 17.

Although more than 100 different BRCA1 germ-line mutations have already been identified in breast and/or ovarian cancer families, we report for the first time a deleterious genomic rearrangement in BRCA1. A 1-kb deletion comprising exon 17 was found in a large breast and ovarian cancer family, leading to a frameshift in the mutant mRNA due to the absence of exon 17. This deletion is probably the result of a recombination between two closely related Alu sequences. It was not detected by conventional PCR-based methods involving the genomic screening of the 22 coding exons or reverse transcription-PCR because the transcript without exon 17 is unstable in lymphoblastoid cell lines. Therefore, rearrangements in the BRCA1 gene should be sought in breast/ovarian cancer families in which no mutations have been found by PCR-based methods in the coding region or in the splice sites.

Identification of seven new BRCA1 germline mutations in Italian breast and breast/ovarian cancer families.

We analyzed 16 Italian breast and breast/ovarian cancer families for BRCA1 germline mutations using a combination of the protein truncation test (PTT) and the single-strand conformation polymorphism techniques. Genomic DNA from the affected proband of each family was analyzed by applying the PTT to exon 11 of the BRCA1 gene. This initial screening led to the identification of truncated protein products in three families that were shown to carry three different frameshift mutations. In the families that scored negative in the PTT, single-strand conformation polymorphism analysis of the entire coding sequence of the gene revealed four additional mutations consisting of one nonsense, one in-frame deletion, one frameshift, and one missense mutation (in a family with a case of male breast cancer). The four frameshift mutations resulted in a decreased expression of the mutant allele, whereas no loss of transcript was associated with the other three mutations. All mutant alleles were shown to cosegregate with the cancer phenotype within the families, and none have previously been reported.

Screening for germ-line rearrangements and regulatory mutations in BRCA1 led to the identification of four new deletions.

Most previous BRCA1 mutation screening studies conducted on breast cancer families were aimed at identifying mutations in the coding sequence and splice sites. Mutations in the promoter and untranslated regions, and large rearrangements are missed by standard mutation detection strategies. To look specifically for such germ-line mutations in the BRCA1 gene, we have analyzed a series of 27 American and 51 French breast cancer families in which no BRCA1 mutation was identified by classical techniques. No mutations were detected in either the promoter or untranslated regions, and we did not find any deletion of the whole gene. Four families were found to carry distinct deletions. Two of them, probably generated by Alu-mediated homologous recombination, were internal deletions of 3 and 23.8 kb, encompassing exon 15 and exons 8-13, respectively. These alterations both lead to a frameshift in the mutant mRNA and to premature stop codon-mediated mRNA decay. The other two deletions encompass exons 1 and 2. On the basis of previous and present analyses, rearrangements represent 8% (3/37) of all mutations in our set of BRCA1 American families. Consequently, the search for rearrangements appears mandatory in BRCA1 mutation screening studies.

BRCA1 expression during prenatal development of the human mammary gland.

Germ-line alterations of BRCA1 are associated with elevated risk of breast cancer. Evidence for the involvement of Brca1 in cellular differentiation and morphogenesis has been obtained in mouse models during embryogenesis. Although the presence of well-conserved functional domains might suggest a similar function for both human and mouse genes, very few data on BRCA1 expression in human fetal tissues are available. We have, therefore, investigated the expression of BRCA1 in the mammary gland from human female fetuses aged between 15 and 33 weeks. Quantification of BRCA1 transcripts, using a competitive reverse transcriptase PCR method, indicates a progressive decrease in BRCA1 expression with increasing fetal age between the 15th and 30th week of gestation. Subsequently, the amount of BRCA1 transcripts becomes similar to that found in adult mammary gland. Analysis of BRCA1 protein revealed, in fetal samples, a 220 kDa band corresponding to the 220 kDa BRCA1 protein described in human cell lines. These later experiments confirm that the relative level of the 220 kDa BRCA1 protein is highest in the early stages of mammary gland development. The temporal patterns of BRCA1 expression in human fetuses suggest a role for BRCA1 in the morphogenesis and differentiation of the human mammary gland.

Down-regulation of BRCA1 in human sporadic breast cancer; analysis of DNA methylation patterns of the putative promoter region.

Germ-line alterations of BRCA1 are responsible for about 50% of familial breast cancers. Although its biological function(s) has not yet been fully determined, it has been suggested that it may act as a tumor suppressor gene in breast and ovarian cancers. In sporadic breast cancers alterations of BRCA1 have not been detected and in vitro experiments have indicated that BRCA1 negatively regulates cellular proliferation. The present study was designed to identify and quantify, the BRCA1 mRNA levels, in normal and neoplasic human breast tissue. BRCA1 mRNA molecules were quantified using competitive reverse transcriptase PCR assays. DNA methylation patterns of this gene have been analysed by Southern blot experiments using methylation sensitive restriction enzymes. We found that BRCA1 mRNA levels were significantly lower in sporadic breast cancers (37 cases analysed, 24 cases of invasive ductal carcinomas not otherwise specified (NOS), two lobular carcinomas in situ two medullary carcinomas, four invasive lobular carcinomas, two invasive mucinous carcinomas and three invasive ductal carcinomas with predominantly in situ component) compared with normal breast tissues (P=0.0003). This down-regulation of BRCA1 is observed in all histologic types analysed. In invasive ductal carcinomas NOS, this down-regulation does not correlate with any of the prognostic factors studied (tumor size, node status, histologic grade, hormone receptor status). In the samples analysed, alterations of DNA methylation patterns were not dectected in the vicinity of the major transcription start site. These data suggest the involvement of BRCA1 in the carcinogenesis of these histologic types.

Expression of P0- and P3-RNA from the normal and translocated c-myc allele in Burkitt's lymphoma cells.

We have studied the allele specific expression of c-myc P0- and P3-RNA in Burkitt's lymphoma (BL) cells. The steady state levels of P0-RNA show considerable variations in BL cells. Expression of P0-RNA was found to be restricted to the translocated allele, but could be induced by TPA from the normal allele. P0-transcription was particularly sensitive to inhibitors of protein synthesis compared to expression of P1-, P2- and P3-RNA. Transcription of P3-RNA is initiated in the first intron of the c-myc gene and has previously been described to be specific for translocated c-myc alleles in BL cells broken within exon 1 or intron 1. Here we show that P3-RNA is also expressed from an unrearranged c-myc gene. In the BL cell line Raji, substantial amounts of c-myc RNA are derived from the P3-promoter of the normal allele. This indicates that repression of the normal allele in BL cells does not include the P3-promoter. The potential coding capacity of P3-RNA is discussed.

Oncogenic activation of RET by two distinct FMTC mutations affecting the tyrosine kinase domain.

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are two dominantly inherited disorders caused by germline mutations of the RET proto-oncogene. The RET gene codes for a receptor tyrosine kinase. The majority of MEN2A and FMTC mutations are clustered in the extra-cellular cysteine-rich domain and result in constitutive activation of the tyrosine kinase through the formation of disulfide-bonded RET homodimers. Recently, two novel point mutations have been identified in the germline of five distinct FMTC families. Both mutations occur within the catalytic domain of the RET kinase and lead to the substitution of either glutamic acid 768 or valine 804 by an aspartic acid and a leucine respectively. We have introduced each FMTC mutation in two RET isoforms: RET51 the long isoform (1114 aa) and RET9 the short isoform (1072 aa) which differ in the C-terminal region of the protein. The RET51 isoform carrying either E768D or V804L mutation was autophosphorylated, displayed a transforming activity upon expression in Rat1 fibroblasts and induced neuronal differentiation of PC12 cells. However, the transforming capacity of these RET51-FMTC mutants was found to be severalfold less potent compared to the same isoform carrying either the MEN2A mutation (C634R) or the MEN2B mutation (M918T). In contrast, RET9 containing mutations E768D or V804L was not autophosphorylated, exhibited a poor oncogenic potential in fibroblasts and did not promote neuritic outgrowth upon expression in PC12 cells. Overall, these findings demonstrate that mutations E768D and V804L are gain-of-function mutations that confer to the long RET isoform the capacity to exert a biological effect, although these mutations are more weakly activating than the MEN2A and MEN2B mutations. These results may provide a biochemical basis as to why the phenotypic consequences of these mutations are restricted to thyroid C-cells.