High frequency of LOH, MSI and abnormal expression of FHIT in gastric cancer.

The FHIT gene is a putative tumour suppressor gene. In this study, we analysed a set of 50 gastric tumours for alterations of FHIT, and found 38 of 45 tumours (84%) exhibiting loss of heterozygosity (LOH) within the FHIT gene. We used both nested Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and single step RT-PCR to analyse the FHIT transcripts and found 34 of 39 (87%) tumours and seven of the 11 (64%) corresponding non-cancerous tissues showed low or aberrant expression of FHIT mRNA and the appearance of the aberrant FHIT transcripts depended on the conditions of the RT-PCR. In these aberrant transcripts, frequent deletions and/or insertions were detected by direct sequencing. All breakpoints for deletions and insertions were at splicing sites. All insertions came from the adjacent introns, whose appearance was completely in accordance with the 'GU-AG' rule for pre-mRNA splicing. It may be suggested that an alternative splicing mechanism functions in the formation of these aberrant transcripts. The fragile nature of FRA3B within the FHIT gene could be responsible for the formation of the aberrant mRNA. Negative or reduced Fhit expression was detected in 39 of 50 tumours (78%). Moreover, an association was found between abnormal Fhit expression and positive node status (P=0.012). Thirteen of 48 tumours (27%) displayed microsatellite instability (MSI), among which 10 tumours also showed MSI within the FHIT gene. Furthermore, we detected an association between MSI and negative node status (P=0.02). We conclude that the abnormalities of FHIT, presumably associated with the unstable nature of FRA3B within the FHIT gene, are involved in the carcinogenesis of gastric cancer, and lack of mismatch repair (MMR) could possibly promote its alteration in a subset of gastric tumours.

Characterization of specific IgE response in vitro against protein and drug allergens using atopic and normal donors.

BACKGROUND: As the incidence of allergy to different compounds increases in society, the need to understand and characterize specific IgE responses becomes obvious. Different cell culture systems have been evaluated for their ability to support such IgE secretion. METHODS: One system employed human peripheral lymphocytes (PBL) from normal donors stimulated with anti-CD3 activated T cells with or without the presence of allergens like benzylpenicillin (BP) and Phlenum pratense (PP). Secretion of IgE was analyzed in ELISA and compared to the IgG response to the nonallergenic antigen tetanus toxoid (TT). Another system employed stimulation of T and B cells with a heterotope, consisting of a T helper cell epitope derived from TT, and a B cell allergen epitope derived from BP. The specific IgE secretion was compared, using lymphocytes from normal as well as BP-allergic donors. RESULTS: Anti-CD3 stimulated T cells supported BP-specific IgE secretion in six of 11 normal donors. This response was inhibited in four donors and enhanced in two donors by the addition of the BP-allergen to the culture. In contrast, addition of the protein allergen (PP) or antigen (TT) to the same culture system inhibited both IgE and IgG synthesis in all experiments. Cells from the majority (10/16) of the BP-allergic donors failed to produce BP-specific IgE in vitro, when cultured in the presence of allergen. CONCLUSIONS: An allergen specific immune response is readily generated in vitro. The differential response against benzylpenicillin between different donor categories most probably reflects the level of pre-exposure to this allergen in vivo.

Alterations of E-cadherin and beta-catenin in gastric cancer.

BACKGROUND: The E-cadherin-catenin complex plays a crucial role in epithelial cell-cell adhesion and in the maintenance of tissue architecture. Perturbation in the expression or function of this complex results in loss of intercellular adhesion, with possible consequent cell transformation and tumour progression. METHODS: We studied the alterations of E-cadherin and beta-catenin in a set of 50 primary gastric tumours by using loss of heterozygosity (LOH) analysis, gene mutation screening, detection of aberrant transcripts and immunohistochemistry (IHC). RESULTS: A high frequency (75%) of LOH was detected at 16q22.1 containing E-cadherin locus. Three cases (6%) showed the identical missense mutation, A592T. This mutation is not likely to contribute strongly to the carcinogenesis of gastric cancer, because a low frequency (1.6%) of this mutation was also found in 187 normal individuals. We also detected a low frequency (0.36%, 0%) of this mutation in 280 breast tumours and 444 other tumours, including colon and rectum, lung, endometrium, ovary, testis, kidney, thyroid carcinomas and sarcomas, respectively. We also analyzed the aberrant E-cadherin mRNAs in the gastric tumours and found that 7 tumours (18%) had aberrant mRNAs in addition to the normal mRNA. These aberrant mRNAs may produce abnormal E-cadherin molecules, resulting in weak cell-cell adhesion and invasive behaviour of carcinoma cells. Reduced expression of E-cadherin and beta-catenin was identified at the frequency of 42% and 28%, respectively. Specially, 11 tumours (22%) exhibited positive cytoplasmic staining for beta-catenin IHC. An association was found between reduced expression of E-cadherin and beta-catenin. Moreover, an association was detected between reduced expression of E-cadherin and diffuse histotype. CONCLUSION: Our results support the hypothesis that alterations of E-cadherin and beta-catenin play a role in the initiation and progression of gastric cancer.

CD40 employs p38 MAP kinase in IgE isotype switching.

IgE switching requires the prior induction of C epsilon germline transcripts which is mediated by the concerted binding of STAT-6 and NF kappa B to the C epsilon promoter. These transcription factors are regulated by IL-4 and CD40, respectively. However the latter can effect other signaling pathways and the present study explores the role of p38 MAPK in induction of C epsilon germline transcripts. CD40 and IL-4, both alone and in synergy, were initially shown to activate the C epsilon promoter in a B cell lymphoma cell line. Under the same conditions CD40 caused activation of p38 MAPK, whereas IL-4 was ineffective. The p38 MAPK inhibitor, SB203580, and a dominant negative form of p38 MAPK decreased the CD40 activation of the C epsilon promoter by reducing the ability of CD40 to increase the transactivation potential of NF kappa B. This study suggests that p38 MAPK is crucially important in mediating CD40 activation of NF kappa B which acts to induce C epsilon germline transcripts, ultimately facilitating IgE switching.

FHIT alterations in breast cancer.

The FHIT gene encodes a diadenosine hydrolase and may be involved in growth control pathways of the cell. Studies on protein-protein interactions, cell lines, including tumourigenicity tests, and knockout mice suggest that the Fhit protein is involved in cell proliferation and apoptosis, and might act as a tumour suppressor. In several different cancers, including breast cancer, alterations in the FHIT gene have been detected in high frequency. The most common alterations are: deletions, DNA hypermethylation, abnormal transcripts and reduced expression at RNA and protein level. The FHIT gene is located at the FRA 3B fragile site at chromosome 3p 14.2, and alterations in the FHIT gene and Fhit protein have been found associated with genome instability, particularly in BRCA 2 mutated breast tumours. This paper will focus on some of the functional aspects of the Fhit protein with respect to tumour pathogenesis and on aberrations detected in breast cancer.

Alterations of the FHIT gene in breast cancer: association with tumor progression and patient survival.

Our previous results on breast tumors show that LOH (loss of heterozygosity) at the FHIT locus is associated with reduced Fhit protein expression. We have also shown that LOH at this locus is significantly higher in tumors from patients carrying the BRCA2 999de15 mutation than in tumors without this mutation, presumably because of lack of DNA repair. Here, our aim was to determine the relationship of FHIT LOH with breast tumor progression. Five microsatellite markers located within the FHIT gene were typed in 239 breast tumors and corresponding normal tissue, and the LOH results were compared with clinicopathologic factors and LOH at other chromosome regions. LOH at FHIT is associated with estrogen- and progesterone-negative breast tumors, high S-phase fraction, reduced patient survival, and LOH at chromosome regions 6q, 7q, 8p, 9p, 11p, 11q, 13q, 16q, 17p, 17q, 18q, and 20q. A multivariate analysis shows that LOH at FHIT results in a 60% increased relative risk of dying. We conclude that the loss of FHIT results in growth advantage of breast tumor cells, is associated with unstable genome, and may be of prognostic value.

Similar regions of human chromosome 3 are eliminated from or retained in human/human and human/mouse microcell hybrids during tumor growth in severe combined immunodeficient (SCID) mice.

By passaging microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background through severe combined immunodeficient (SCID) mice (elimination test), we have previously defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a second eliminated region (ER2) at 3p21.1-p14 and a common retained region (CRR) at 3q26-qter. In the present work, chr3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chr3. Four MCHs were generated. Compared with KH39, they developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analyzed in comparison with corresponding MCHs by chr3 arm-specific painting, 19 fluorescent in situ hybridization (FISH) probes, and 27 polymorphic markers. Three MCHs that maintained the intact exogenous chr3 in vitro lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four tumors contained mixed cell populations that lacked either the exogenous or one endogenous KH39 derived 3p. In one MCH the exogenous chr3 showed deletions within CER1 and ER2 already in vitro. It remained essentially unchanged in 8/9 derived tumors. The third, exogenous copy of the 3q26-q27 region (part of CRR) was retained in 16/20 tumors. It can be concluded that the human/human MCH-based elimination test identifies similar eliminated and retained regions on chr3 as the human/murine MCH-based test.

Low-frequency magnetic noise in micron-scale magnetic tunnel junctions.

We have observed low-frequency noise due to quasiequilibrium thermal magnetization fluctuations in micron-scale magnetic tunnel junctions (MTJs). This strongly field-dependent magnetic noise occurs within the magnetic hysteresis loops, either as 1/f or Lorentzian (random telegraph) noise. We attribute it to the thermally excited hopping of magnetic domain walls between pinning sites. Our results show that magnetic stability is a crucial factor in reducing the low-frequency noise in small MTJs.

Rapid polarization of Th2 cells during induction of antigen-specific IgE antibodies in vitro.

BACKGROUND: Type 2 T-helper cells (Th2) are involved in the regulation of the humoral immune response against antigens and allergens and directly affect which isotype will be produced. The mechanism that regulates antigen-specific IgE secretion and immune deviation is still not known. OBJECTIVES: To delineate mechanisms behind antigen-specific IgE secretion we have used in vitro immunization and focused on T-cell phenotype and the activation status of the transcription factor NFkappaB. METHODS: Peripheral blood lymphocytes (PBMC) from seronegative donors were immunized in vitro with a peptide consisting of both a T-cell and a B-cell epitope. RESULTS: Antigen-specific IgE antibodies could be detected after a primary immunization, during which T-helper cells secreted type 2 cytokines. Specific IgE was also detected in the secondary immunization, but due to a rapid polarization from Th2 to Th1 phenotype, exogenous IL-4 was required for the specific IgE secretion. Analysis of NFkappaB activation in B and T cells during primary and secondary immunization showed that NFkappaB could be detected in both B and T cells during primary immunization, but was dependent on exogenous IL-4 in the secondary immunization. CONCLUSION: This is the first evidence of antigen-specific IgE induction in vitro using naive B cells, demonstrating the involvement of T-helper cell phenotype and NFkappaB and demonstrates the usefulness of in vitro cultures to study the effect of antigens on human immunocytes.

Analysis of the fragile histidine triad (FHIT) gene in lobular breast cancer.

The fragile histidine triad (FHIT) gene is a candidate tumour suppressor gene in breast and other cancers. We investigated deletions within the FHIT gene in lobular breast cancer and found that 16% of cases showed loss of heterozygosity (LOH) within the gene. We compared LOH within FHIT in lobular and ductal breast tumours and found a significant association between LOH at FHIT and the ductal histological type (P<0.001). To determine whether genomic alteration of the FHIT gene in lobular breast cancer leads to Fhit inactivation we have assessed the level of Fhit expression by immunohistochemical detection and determined that 27% (15 of 55) consecutive sporadic lobular tumours showed negative or reduced Fhit expression. A significant association was found between LOH at the FHIT gene and reduced Fhit expression in lobular and ductal tumours (P=0.025 and P=0.001, respectively). Thus, genetic alterations within the FHIT gene, leading to loss of Fhit protein, may play an important role in the carcinogenesis of a significant number of sporadic lobular breast cancers, even though the apparent frequency of genomic alterations within the gene is lower than in ductal breast cancer.

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

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.

Population studies and validation of paternity determinations by six microsatellite loci.

A single locus system of 6 microsatellite markers was evaluated for paternity testing. A nonradioactive method based on peroxidase labeling of a DNA probe was used to estimate the allele frequency of markers D1S216, D3S1217, D7S480, D9S157, D13S153, and D16S422 by genotyping 1134-1698 chromosomes. The number of detected alleles were 22, 15, 23, 10, 16, and 19, respectively, and the allele frequency varied from 0.001 to 0.317. The genotype of 87 families, consisting of mother, father, and child was determined. The probability that a random individual will give a positive paternity was evaluated. We conclude that the markers can be reliably typed and give sufficient and reliable information for paternity testing.

Chromosome 8p alterations in sporadic and BRCA2 999del5 linked breast cancer.

Chromosomal losses involving the short arm of chromosome 8 are frequent in a variety of tumour types, including breast cancer, suggesting the presence of one or more tumour suppressor genes in this region. In this study, we have used 11 microsatellite markers to analyse loss of heterozygosity (LOH) at chromosome 8p in 151 sporadic breast tumours and 50 tumours from subjects carrying the BRCA2 999del5 mutation. Fifty percent of sporadic tumours compared to 78% of BRCA2 linked tumours exhibit LOH at one or more markers at 8p showing that chromosome 8p alterations in breast tumours from BRCA2 999del5 carriers are more pronounced than in sporadic breast tumours. The pattern of LOH is different in the two groups and a higher proportion of BRCA2 tumours have LOH in a large region of chromosome 8p. In the total patient material, LOH of 8p is associated with LOH at other chromosome regions, for example, 1p, 3p, 6q, 7q, 9p, 11p, 13q, 17p, and 20q, but no association is found between LOH at 8p and chromosome regions 11q, 16q, 17q, and 18q. Furthermore, an association is detected between LOH at 8p and positive node status, large tumour size, aneuploidy, and high S phase fraction. Breast cancer patients with LOH at chromosome 8p have a worse prognosis than patients without this defect. Multivariate analysis suggests that LOH at 8p is an independent prognostic factor. We conclude that chromosome 8p carries a tumour suppressor gene or genes, the loss of which results in growth advantage of breast tumour cells, especially in carriers of the BRCA2 999del5 mutation.

Immature B cells in bone marrow express Fas/FasL.

Negative selection is a process by which autoreactive lymphocytes are eliminated from the developing antigen receptor repertoire. The mechanisms regulating negative selection of immature B lymphocytes in the bone marrow are poorly elucidated. Human bone marrow cells were examined in order to investigate the presence of the members of the Fas (APO-1/CD95) system. Here we demonstrate the expression of Fas in immature B lymphocytes (CD10/CD19+/CD40+/sIg+), and the presence of Fas natural ligand (FasL) in CD19+ bone marrow cells. The observed expression of apoptosis-related molecules might indicate how negative selection of autoreactive B cells can occur in human bone marrow.

Human interdigitating dendritic cells induce isotype switching and IL-13-dependent IgM production in CD40-activated naive B cells.

Interdigitating dendritic cells (IDC) represent a mature progeny of dendritic cells (DC) in vivo and are exhibiting a strong lymphocyte stimulatory potential. Because of the restricted localization to secondary lymphoid organs where decisive cellular interactions take place in the initial events of immunity, IDC regulatory function was addressed in relation to naive B cells. In this study, we demonstrate that human tonsillar IDC induce a dual response from CD40-activated IgD+/CD38- naive B lymphocytes. IDC direct naive B cells toward either isotype switching or an IL-13-dependent IgM secretion. IDC-dependent proliferation, isotype switching, and Ig production are all strictly mediated by soluble factors, suggesting that such skewing in B cell activation is the result of differential cytokine expression. Moreover, IDC-expressed IL-13 represents a novel source of a cytokine with recently established effects in Th2 induction as well as in immunological disorders resulting in allergic reactions.

The effect of mismatch repair deficiency on tumourigenesis; microsatellite instability affecting genes containing short repeated sequences.

We have investigated microsatellite instability (MSI) in colorectal, gastric, endometrial and ovarian cancer as a result of mismatch repair (MMR) deficiency. We detected frameshift mutations in several genes that carry short repeated sequences and are important in cell fidelity and growth control; hMSH3, hMSH6, BAX, IGFIIR, TGFbetaIIR, E2F4 and BRCA2. Accumulation of mutations was heterogeneous and mainly restricted to tumours showing MSI at several loci (MSI-H). Both insertions and deletions were evident and occasional intratumour heterogeneity was evident with more than one different additional allele in the tumour. Most MSI-H tumours had acquired mutations in more than one gene and longer repeated sequences were more frequently targets for mutations. The TGFbetaIIR gene was mutated in 62%, the hMSH3 gene in 43%, the E2F4 gene in 35%, the hMSH6 in 32%, the BAX gene in 32%, the IGFIIR gene in 26%, and the BRCA2 gene in 2% of the MSI-H tumours. Homozygous mutations or mutation of both alleles were evident in all genes except BRCA2, in total 23/105 mutated cases, varying from 7% for BAX to 50% for E2F4. E2F4 mutations were exclusively found in colon tumours and E2F4 polymorphisms was found in 8% of cases. No difference in mutation prevalence was noted between cancer types apart from TGFbetaIIR mutations, which were frequently found in colon and gastric tumours but not in endometrial tumours, suggesting that endometrial tumours progress by a different route where TGFbetaIIR mutations are less favourable.

Chromosome alterations and E-cadherin gene mutations in human lobular breast cancer.

We have studied a set of 40 human lobular breast cancers for loss of heterozygosity (LOH) at various chromosome locations and for mutations in the coding region plus flanking intron sequences of the E-cadherin gene. We found a high frequency of LOH (100%, 31/31) at 16q21-q22.1. A significantly higher level of LOH was detected in ductal breast tumours at chromosome arms 1p, 3p, 9p, 11q, 13q and 18q compared to lobular breast tumours. Furthermore, we found a significant association between LOH at 16q containing the E-cadherin locus and lobular histological type. Six different somatic mutations were detected in the E-cadherin gene, of which three were insertions, two deletions and one splice site mutation. Mutations were found in combination with LOH of the wild type E-cadherin locus and loss of or reduced E-cadherin expression detected by immunohistochemistry. The mutations described here have not previously been reported. We compared LOH at different chromosome regions with E-cadherin gene mutations and found a significant association between LOH at 13q and E-cadherin gene mutations. A significant association was also detected between LOH at 13q and LOH at 7q and 11q. Moreover, we found a significant association between LOH at 3p and high S phase, LOH at 9p and low ER and PgR content, LOH at 17p and aneuploidy. We conclude that LOH at 16q is the most frequent chromosome alteration and E-cadherin is a typical tumour suppressor gene in lobular breast cancer.

Replication error in colorectal carcinoma: association with loss of heterozygosity at mismatch repair loci and clinicopathological variables.

Instability of microsatellite DNA or replication error (RER) is characteristic of tumours caused by mismatch repair (MMR) deficiency. Germline mutations in MMR genes are associated with Hereditary non-polyposis colorectal carcinoma (HNPCC) and somatic mutations in these genes are also found in a substantial fraction of colorectal cancers (CRC). In this study we concurrently screened colorectal tumours for the RER phenotype and loss of heterozygosity (LOH) at MMR gene loci. The RER phenotype was evident in 47/197 (24%) tumours. RER was more commonly detected in young patients (< 50 years) and in tumours located in the proximal colon. RER was positively associated with LOH at the hMSH2/hMSH6 loci on chromosome 2p, where LOH was observed in 46% of the RER+ tumours. LOH at hMLH1 and hPMS1 loci was more frequent in the younger patients (< 50 years). RER was not associated with clinicopathological parameters, such as Duke's stage and tumour differentiation (grade). The RER phenotype was associated with better overall survival, but there was a trend towards significance when multivariate analysis was used. This indicates that loss of MMR genes generate a less aggressive phenotype, and raises the question about RER being a useful indicator of prognosis for CRC patients.

Molecular genetics of breast cancer progression.

Somatic changes in the genome of breast cancer cells include amplifications, deletions and gene mutations. Several chromosome regions harboring known oncogenes are found amplified in breast tumors. Despite the high number of chromosome regions deleted in breast tumors the functional relationship to known genes at these locations and cancer growth is mainly undiscovered. Mutations in two tumor suppressor genes (TSG) have been described in a subset of breast carcinomas. These TSG are the TP53, encoding the p53 transcription factor, and the CDH1, encoding the cadherin cell adhesion molecule. Breast tumors of patients with a germ-line mutation in the BRCA1 or BRCA2 gene have an increase of additional genetic defects compared with sporadic breast tumors. This higher frequency of genetic aberrations could pinpoint genes that selectively promote tumor progression in individuals predisposed to breast cancer due to BRCA1 or BRCA2 germ-line mutations. Accumulation of somatic genetic changes during tumor progression may follow a specific and more aggressive pathway of chromosome damage in these individuals. Although the sequence of molecular events in the progression of breast tumor is poorly understood the detected genetic alterations fit the model of multistep carcinogenesis in both sporadic and hereditary breast cancer. This review will focus on the genetic lesions within the breast cancer cell.