Circulating microRNAs as prognostic therapy biomarkers in head and neck cancer patients.

BACKGROUND: The prediction of therapy response in head and neck squamous cell cancer (HNSCC) requires biomarkers, which are also a prerequisite for personalised therapy concepts. The current study aimed to identify therapy-responsive microRNAs (miRNAs) in the circulation that can serve as minimally invasive prognostic markers for HNSCC patients undergoing radiotherapy. METHODS: We screened plasma miRNAs in a discovery cohort of HNSCC patients before therapy and after treatment. We further compared the plasma miRNAs of the patients to age- and sex-matched healthy controls. All miRNAs identified as biomarker candidates were then confirmed in an independent validation cohort of HNSCC patients and tested for correlation with the clinical outcome. RESULTS: We identified a signature of eight plasma miRNAs that differentiated significantly (P=0.003) between HNSCC patients and healthy donors. MiR-186-5p demonstrated the highest sensitivity and specificity to classify HNSCC patients and healthy individuals. All therapy-responsive and patient-specific miRNAs in plasma were also detectable in tumour tissues derived from the same patients. High expression of miR-142-3p, miR-186-5p, miR-195-5p, miR-374b-5p and miR-574-3p in the plasma correlated with worse prognosis. CONCLUSIONS: Circulating miR-142-3p, miR-186-5p, miR-195-5p, miR-374b-5p and miR-574-3p represent the most promising markers for prognosis and therapy monitoring in the plasma of HNSCC patients. We found strong evidence that the circulating therapy-responsive miRNAs are tumour related and were able to validate them in an independent cohort of HNSCC patients.

MiR-221/-222 differentiate prognostic groups in advanced breast cancers and influence cell invasion.

BACKGROUND: MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells. METHODS: MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT-PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222. RESULTS: In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed. CONCLUSION: This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.

In situ quantification of HER2-protein tyrosine kinase 6 (PTK6) protein-protein complexes in paraffin sections from breast cancer tissues.

BACKGROUND: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. METHOD AND RESULTS: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6-HER2 protein-protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). CONCLUSION: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2-PTK6 complexes are of prognostic relevance.

Prognostic value of protein tyrosine kinase 6 (PTK6) for long-term survival of breast cancer patients.

The cytoplasmic tyrosine kinase PTK6 (BRK) shows elevated expression in approximately two-thirds of primary breast tumours, and is implicated in EGF receptor-dependent signalling and epithelial tumorigenesis. Using immunohistochemistry, we performed a retrospective study on 426 archival breast cancer samples from patients with long-term follow-up and compared the protein expression levels of PTK6, the HER receptors, Sam68 (a substrate of PTK6), and signalling proteins including MAP kinase (MAPK), phosphorylated MAPK (P-MAPK), and PTEN. We show that PTK6 expression is of significant prognostic value in the outcome of breast carcinomas. In multivariate analysis, the disease-free survival of patients of >or=240 months was directly associated with the protein expression level of PTK6 (P

Accumulation of DSBs in gamma-H2AX domains fuel chromosomal aberrations.

DNA double strand breaks (DSBs) pose a severe hazard to the genome as erroneous rejoining of DSBs can lead to mutation and cancer. Here, we have investigated the correlation between X irradiation-induced gamma-H2AX foci, theoretically induced DSBs, and the minimal number of mis-rejoined DNA breaks (MNB) in irradiated lymphocytes obtained from two healthy humans by painting of the whole chromosome complement by spectral karyotyping. There were less gamma-H2AX foci/dose than theoretically expected, while misrepair, as expressed by MNB/gamma-H2AX focus, was similar at 0.5 and 1Gy but 3.6-fold up at 3Gy. Hence, our results suggest that X-ray-induced gamma-H2AX foci in G0 lymphocyte nuclei contain more than one DSB and that the increasing number of DSBs per gamma-H2AX repair factory lead to an increased rate of misrepair.

Array CGH demonstrates characteristic aberration signatures in human papillary thyroid carcinomas governed by RET/PTC.

The aim of this study is to investigate additional genetic alterations in papillary thyroid carcinomas (PTCs) with known RET/PTC rearrangements. We applied array-based comparative genomic hybridization (array CGH) to 33 PTC (20 PTC from adults, 13 post-Chernobyl PTC from children) with known RET/PTC status. Principal component analysis and hierarchical cluster analysis identified cases with similar aberration patterns. Significant deviations between tumour-groups were obtained by statistical testing (Fisher's exact test in combination with Benjamini-Hochberg FDR-controlling procedure). FISH analysis on FFPE sections was applied to validate the array CGH data. Deletions were found more frequently in RET/PTC-positive and RET/PTC-negative tumours than amplifications. Specific aberration signatures were identified that discriminated between RET/PTC-positive and RET/PTC-negative cases (aberrations on chromosomes 1p, 3q, 4p, 7p, 9p/q, 10q, 12q, 13q and 21q). In addition, childhood and adult RET/PTC-positive cases differ significantly for a deletion on the distal part of chromosome 1p. There are additional alterations in RET/PTC-positive tumours, which may act as modifiers of RET activation. In contrast, alterations in RET/PTC-negative tumours indicate alternative routes of tumour development. The data presented serve as a starting point for further studies on gene expression and function of genes identified in this study.

Effects of modulated microwave radiation at cellular telephone frequency (1.95 GHz) on X-ray-induced chromosome aberrations in human lymphocytes in vitro.

The case for a DNA-damaging action produced by radiofrequency (RF) signals remains controversial despite extensive research. With the advent of the Universal Mobile Telecommunication System (UMTS) the number of RF-radiation-exposed individuals is likely to escalate. Since the epigenetic effects of RF radiation are poorly understood and since the potential modifications of repair efficiency after exposure to known cytotoxic agents such as ionizing radiation have been investigated infrequently thus far, we studied the influence of UMTS exposure on the yield of chromosome aberrations induced by X rays. Human peripheral blood lymphocytes were exposed in vitro to a UMTS signal (frequency carrier of 1.95 GHz) for 24 h at 0.5 and 2.0 W/kg specific absorption rate (SAR) using a previously characterized waveguide system. The frequency of chromosome aberrations was measured on metaphase spreads from cells given 4 Gy of X rays immediately before RF radiation or sham exposures by fluorescence in situ hybridization. Unirradiated controls were RF-radiation- or sham-exposed. No significant variations due to the UMTS exposure were found in the fraction of aberrant cells. However, the frequency of exchanges per cell was affected by the SAR, showing a small but statistically significant increase of 0.11 exchange per cell compared to 0 W/kg SAR. We conclude that, although the 1.95 GHz signal (UMTS modulated) does not exacerbate the yield of aberrant cells caused by ionizing radiation, the overall burden of X-ray-induced chromosomal damage per cell in first-mitosis lymphocytes may be enhanced at 2.0 W/kg SAR. Hence the SAR may either influence the repair of X-ray-induced DNA breaks or alter the cell death pathways of the damage response.

PTK (protein tyrosine kinase)-6 and HER2 and 4, but not HER1 and 3 predict long-term survival in breast carcinomas.

The HER receptors are of therapeutic and prognostic significance in breast cancer, and their function is modulated by cytoplasmic tyrosine kinases like PTK6 (brk). We performed a retrospective study on archival breast cancer samples from patients with long follow-up and compared the protein expression between individual HERs and between HERs and the PTK6. Univariate and multivariate analyses were used to study the prognostic value of parameters. Metastases-free survival of patients for longer than 240 months was inversely associated (P< or =0.05) with nodal status, tumour size, and oestrogen receptor status, but was also directly associated with high protein expression levels of HER4 and PTK6 in Kaplan-Meier analysis. In multivariate analysis for metastases-free survival of >240 months, the stepwise selected parameters were tumour size (relative risk 3.1), PTK6 expression (0.4), and number of positive lymph nodes (1.2). Furthermore, we demonstrated a timedependence of the prognostic value attributed to the parameters. The HER receptors (HER2,4), but not PTK6, were independent prognostic markers for metastases-free survival at 60 months, whereas at 240 months PTK6 is the strongest prognostic marker. We demonstrate that PTK6 is a prognostic marker of metastases-free survival in breast cancer, and is independent of the classical morphological and molecular markers of lymph node involvement, tumour size, and HER2 status.

Mutations that affect meiosis in male mice influence the dynamics of the mid-preleptotene and bouquet stages.

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of mid-preleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm(-/-), Spo11(-/-), Mei1(m1Jcs/m1Jcs), Mlh1(-/-), Terf1(+/-) and Hr6b(-/-) spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1(-/-), Gmcl1(-/-), Asm(-/-)) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11(-/-)Atm(-/-) spermatogenesis suggests that DSBs contribute to the Atm(-/-)-correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects in early stages of DSB repair (Dmc1(-/-), Hop2(-/-)) suggest that there is an ATM-specific influence on bouquet stage duration. Altogether, it appears that several pathways influence telomere dynamics in mammalian meiosis.

RET rearrangements in post-Chernobyl papillary thyroid carcinomas with a short latency analysed by interphase FISH.

Tissue samples from 13 post-Chernobyl childhood thyroid tumours that occurred within a short period of time (4-8 years) after the Chernobyl accident have been investigated by interphase FISH analysis for rearrangements of RET. In all, 77% of cases showed RET/PTC rearrangements and a distinct intratumoural genetic heterogeneity. The data were compared to findings on 32 post-Chernobyl PTCs that occurred after a longer period of time (9-12 years) after the accident. In none of the cases from either group were 100% of cells positive for RET rearrangement. In addition, the pattern of RET-positive cells was different in the two groups (short vs longer latency). A significant clustering of aberrant cells could be detected in the long-latency subgroup, whereas the aberrant cells were more homogeneously distributed among the short-latency tumours. The findings suggest that oligoclonal tumour development occurs in post-Chernobyl PTCs. This pattern of different clones within the tumour appears to become more discrete in cases with longer latencies, suggesting either outgrowth of individual clones or development of later subclones with time.

SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis.

For a retrospective dose estimation of human exposure to ionising radiation, a partial genome analysis is routinely used to quantify radiation-induced chromosome aberrations. For this purpose, fluorescence in situ hybridisation (FISH) with whole chromosome painting probes for selected chromosomes is usually applied covering about 20% of the whole genome. Since genome-wide screening techniques like spectral karyotyping (SKY) and multiplex FISH (mFISH) have been developed the detection of radiation-induced aberrations within the whole genome has now become feasible. To determine the correspondence between partial and whole genome analysis of radiation-induced chromosome aberrations, they were measured comprehensively in this study using in vitro irradiated blood samples from three donors. We were able to demonstrate that comparable results can be detected with both approaches. However, complex aberrations might be misinterpreted by partial genome analysis. We therefore conclude that whole genome analysis by SKY is useful especially in the high dose range to correct aberration data for complex exchange aberrations.

Cytogenetic analyses in peripheral lymphocytes of persons living in houses with increased levels of indoor radon concentrations.

Published data concerning the effects of indoor radon exposure on the frequency of chromosome aberrations in peripheral lymphocytes of residents are contradictory. Possible reasons for this may be the low radon concentration in dwellings and/or the limited number of investigated persons. We therefore studied the relationship of domestic radon exposure and the occurrence of chromosome aberrations in peripheral lymphocytes in 61 persons living in houses with radon concentrations from 80 up to 13,000 Bq/m3. We analyzed 60,000 cells from fluorescence plus Giemsa (FPG)-stained slides. It could be clearly demonstrated that in groups of persons living in dwellings with indoor radon concentrations >200 Bq/m3 the number of cells containing dicentrics and/or centric rings (C(dic + cr)) (2.45 +/- 0.50 x 10(-3)) was significantly increased (p < 0.05) in comparison to the control level (1.03 +/- 0.15 x 10(-3)). However, there was no difference in the mean frequency of C(dic + cr) between the groups living in dwellings with higher radon concentrations. Using the fluorescence in situ hybridization (FISH) technique for the detection of translocations, we analyzed 23,315 cells in 16 persons of the highest exposed group (>5,000 Bq/m3). The observed frequency of translocations was 3.9 +/- 0.64 x 10(-3). In comparison to the control group (2.02 +/- 0.18 x 10(-3)), there was a slight but not statistically significant increase in the exposed group (P = 0.055). If, however, the age of the examined persons is taken into account, the values are significantly increased (P < 0.05) in the exposed persons older than 40 years in comparison to the age-matched controls. Since most of the translocations were found in stable cells, it is concluded that translocations are also induced in blood-forming tissue and are transmitted to peripheral blood.

Distribution of radiation-induced exchange aberrations in all human chromosomes.

PURPOSE: To investigate the DNA-proportional distribution of radiation-induced chromosome aberrations for all chromosomes of a male and a female human karyotype. MATERIALS AND METHODS: Metaphases were prepared from whole blood cultures obtained from two healthy donors and set up after irradiation with 3 Gy 220 kV X-rays. Single whole-chromosome FISH painting simultaneously with pancentromeric DNA painting were performed separately for each chromosome of the human karyotype. One thousand exclusively first-division metaphases were analysed per chromosome and donor. After statistical analysis, the data obtained were compared with theoretically expected values. RESULTS: All aberration types (translocations, dicentrics) showed deviations from a DNA-proportional distribution. For both donors, chromosomes 2 and 3 exhibited significantly less and chromosome 4 more symmetrical translocations than expected. Chromosomes 15 and 22 showed more symmetrical translocations than predicted for one of the two donors. Less dicentrics than expected became apparent for chromosomes 2, 3 and 18, while more dicentrics were seen for chromosomes 15, 16 and 17. Moreover, chromosomes 4, 14 and 22 showed a significant deviation from the theoretically expected 1:1 ratio of the yields of symmetrical translocations to the yields of dicentrics. CONCLUSION: The results from the whole-chromosome complement in two different donors confirmed published data from the analysis of single chromosomes that some human chromosomes were not involved in radiation-induced dicentrics and symmetrical translocations proportional to their DNA content. This must be taken into account if chromosome subsets for dose reconstruction are selected or if whole genomic frequencies have to be calculated from partial genome analysis.

Instability of microsatellites in radiation-associated thyroid tumours with short latency periods.

PURPOSE: To determine the instability of microsatellite sequences in post-Chernobyl thyroid tumours from children and young adults, and to ascertain whether they correlated with the age of the patient at the time of the accident and the tumour latency period. MATERIALS AND METHODS: The stability of 26 microsatellite markers was investigated in 122 radiation-associated thyroid tumours (96 children, 26 adults) from Belarus and 39 spontaneous thyroid tumours (adults) from Munich without radiation history. RESULTS: A significant correlation between patient age at the time of the accident and the instability of microsatellite sequences was established. Also, a high instability of microsatellite sequences was found in 28 early thyroid tumours from Belarus with latency periods of 6-8 years, in contrast to a low instability of microsatellites in 94 tumours emerging 9-11 years after the accident. Microsatellite instability in the reference group from Munich proved similar to the early thyroid tumours from Belarus. CONCLUSION: Early, fast-growing and aggressive post-Chernobyl thyroid tumours are characterized by an increase in microsatellite instability.

Her-2/neu gene amplification, elevated mRNA expression, and protein overexpression in the metaplasia-dysplasia-adenocarcinoma sequence of Barrett's esophagus.

SUMMARY: The importance of alterations of the Her-2/neu oncogene in the tumorigenesis of Barrett's adenocarcinoma (BCA) is discussed controversially. In the present study, we evaluated for the first time the Her-2/neu status in the metaplasia-dysplasia-adenocarcinoma sequence of BCA simultaneously at the DNA, mRNA, and protein level using resection specimens of 25 patients. The locus-specific Her-2/neu gene status was quantified by performing fluorescence in situ hybridization, and information about the ploidy status of chromosome 17 was obtained. Tissue sections from the same areas were used for quantitative RT-PCR (TaqMan RT-PCR) of laser-microdissected tumor cells and for immunohistochemistry to quantify Her-2/neu mRNA and oncoprotein expression. Her-2/neu gene amplification was observed in 35% of BCA, and all of these samples showed strong overexpression of both mRNA and oncoprotein. A polysomy 17 without Her-2/neu gene amplification was observed in 52% of BCA, showing a normal or moderately elevated mRNA expression and no or weak immunopositivity. From 13 areas of high-grade dysplasia (HGD) we found four to be amplified for the Her-2/neu locus, whereas five showed a polysomy 17. All four samples of HGD areas with Her-2/neu gene amplification displayed mRNA and strong oncoprotein overexpression; however, lower mRNA levels were seen than in the amplified BCA areas. None of the samples with low-grade dysplasia (LGD) showed a locus-specific Her-2/neu amplification, but polysomy 17 was present in four of eight cases. No changes were detected in BCA-associated intestinal metaplasia and squamous epithelium. In summary, only a locus-specific Her-2/neu gene amplification was associated with strong mRNA overexpression and strong membranous Her-2/neu immunostaining in BCA and HGD. A chromosome 17 polysomy, as found in the majority of BCA, led to no or weak mRNA overexpression and no or weak immunopositivity. In the metaplasia-dysplasia-adenocarcinoma sequence, a chromosome 17 polysomy without Her-2/neu gene amplification was already present in LGD. This may be a result of an early polyploidization, preceding the later genetic events, such as Her-2/neu gene amplification in HGD and BCA.

Time-course of translocation and dicentric frequencies in a radiation accident case.

PURPOSE: To assess the persistence of exchange aberrations measured by FISH chromosome painting after accidental radiation exposure. MATERIALS AND METHODS: Chromosome analyses were carried out in peripheral lymphocytes of a 13-year-old boy exposed to protracted low dose-rate whole-body and short-time partial-body irradiation from a radiation accident in Estonia in 1994. Up to November 1998, the frequencies of translocations and dicentrics were periodically measured using FISH chromosome painting of the target chromosomes 1, 4 and 12, with a simultaneous pancentromeric probe. RESULTS: For the yields of dicentrics, an expected rapid temporal decline was found with a half-time of 14.2+/-1.9 months. The yields of reciprocal translocations also revealed a gradual but significant reduction with a half-time of 51.7+/-12.7 months. CONCLUSION: An unchanged temporal persistence of so-called stable translocations cannot be assumed. Any significant reduction of this aberration type with time obviously limits the application of FISH-based translocation measurements for reliable long-term biodosimetry after combined protracted whole-body and partial-body radiation exposure.

Technical report: application of the Metafer2 fluorescence scanning system for the analysis of radiation-induced chromosome aberrations measured by FISH-chromosome painting.

The Metafer2 fluorescence scanning system was used for routine analysis of radiation-induced exchange aberrations measured by fluorescence in situ hybridisation (FISH) chromosome painting in human peripheral lymphocytes. The system enables a rapid and unbiased fully-automated finding and image acquisition of fluorescently stained metaphase spreads. The chromosome aberration analysis is performed interactively from stored digitised processed gallery images, presented on a screen. Appropriate software image filters are available to further improve these pictures by background correction, noise reduction and fluorescence signal enhancement. Data sets generated by computer-assisted and manual scoring of radiation-induced reciprocal translocations (2B) and total 2B (2B+related 'one-way' types) or complete dicentrics (2A) and total 2A (2A+related 'one-ways') involving painted target chromosomes 2, 3 or 4 were compared and no significant differences were found.A linear-quadratic dose-response curve for total translocations (2B+'one-ways'+complex-derived types) based on computer-assisted analysis of 27,741 metaphases with chromosome 4 painting was compared to a curve obtained earlier for manually scored translocations in a set of target chromosomes 1, 4 and 12. After extrapolation to the whole genome, no significant difference between both curves was found. From our results it can be derived that computer-assisted aberration analysis using the Metafer2 system is a reliable alternative to manual analysis. Since time saving for computer-assisted translocation analysis is about 50% compared to manual scoring, this system is highly promising for a practical application in retrospective biodosimetry of human radiation exposure.

Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.

PURPOSE: To investigate within the framework of a multilaboratory study the suitability of FISH chromosome painting to measure so-called stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers (from the Southern Urals) and their use for retrospective biodosimetry. MATERIALS AND METHODS: Chromosime analyses were carried out from 69 workers who had received protracted occupational radiation exposures (0.012-6.065 Gy) up to approximately 40 years before blood sampling. Twenty-one unexposed people living in the same area were controls. A multicolour FISH-painting protocol with the target chromosomes 1, 4 and 8 simultaneously with a pancentromeric probe was used to score potentially transmissible chromosome-type aberrations (reciprocal translocations 2B and related 'one-way' patterns I-III according to the S&S classification). RESULTS: Individual biodosimetry estimates were obtained in terms of these potentially long-term surviving aberration types based on the linear component of a low dose-rate gamma-ray calibration curve produced using identical staining and scoring protocols. For comparison, the workers personal and total background doses were converted to red bone marrow doses. The estimated doses were mainly lower than would be predicted by the calibration curve, particularly at accumulated higher dose levels. CONCLUSIONS: Owing to the limited life-time of circulating T-lymphocytes, the long-term persistence of translocations in vivo requires the assumption of a clonal repopulation of these naturally senescing cells from the haemopoietic stem cell compartments. Obviously such a replacement cannot be fully achieved, leading to a temporal decline even of the yield of transmissible aberrations types. Assuming further a highly selective capacity of stem cells against any type of chromosomal damage and the fact that one must rely on partial genome findings, the potential of FISH chromosome painting for retrospective dose reconstruction is probably limited to a decade or so after high-level protracted radiation exposure.

[Oncogene amplification and genetic heterogeneity in the metaplasia-dysplasia-adenocarcinoma sequence of Barrett esophagus]. / Onkogen-Amplifikation und genetische Heterogenität in der Metaplasie-Dysplasie-Adenokarzinom-Sequenz beim Barrett-Karzinom.

AIMS: Information about numerical genomic alterations in the tumorigenesis of Barrett's adenocarcinoma (BCA) is still limited. In order to search for gene amplification and ploidy status, a series of locus-specific DNA probes and associated centromere probes was analysed in the metaplasia-dysplasia-adenocarcinoma-sequence. METHODS: Fluorescence in situ hybridisation (FISH) was performed on paraffin sections with locus-specific DNA probes for D7S486, c-myc, cyclin D1, Her-2/neu, 20q13.2 and associated chromosomes 7, 8, 11, 17 and 20. Corresponding areas of intestinal metaplasia (IM, n = 5), low grade dysplasia (LGD, n = 9), high grade dysplasia (HGD, n = 15) and BCA (n = 16) were analysed. RESULTS: Gene amplification of c-myc, Cyclin D1, Her-2/neu and 20q13.2 was observed in 15-35% of BCA. Coincident amplification of genes was also present. Polysomies for all investigated centromere probes were highly prevalent (up to 85%). Gene amplification was also demonstrated in HGD lesions. Polysomies were observed in HGD in high frequency (up to 80%). Extensive genetic heterogeneity was observed in both, BCA and HGD displaying different levels of amplification. None of the samples with LGD showed a locus-specific amplification, but polysomies for all investigated chromosomes were present in 18-48% of LGD. No changes were detected in BCA associated IM and squamous epithelium. CONCLUSIONS: Our data indicate that oncogene amplification of c-myc, cyclin D1, Her-2/neu, and 20q13.2 occurring in BCA and less frequently in HGD is a late event in the tumorigenesis. Polysomies of chromosomes 7, 8, 11, 17 and 20, which were highly prevalent in BCA and HGD occur already at the stage of LGD. This may be a result of an early polyploidization, preceding the later genetic events, such as gene amplification in HGD and BCA. The detection of shared numerical genomic changes and the detected extensive genetic heterogeneity in the metaplasia-dysplasia-carcinoma-sequence in Barrett's esophagus supports the hypothesis of a process of multiclonal expansion underlying this progression.

Molecular genetic changes in metastatic primary Barrett's adenocarcinoma and related lymph node metastases: comparison with nonmetastatic Barrett's adenocarcinoma.

Lymph node metastasis is one of the strongest negative prognostic factors for patients with Barrett's adenocarcinoma (BCA). However, despite the importance of the metastatic process in BCA, the molecular basis of it remains poorly understood. To search for cytogenetic events associated with metastasis in regional or distant lymph nodes in BCA, we investigated 8 primary BCA and their lymph node metastases and compared them with 18 nonmetastatic BCA. In metastatic primary BCA, we observed significantly more DNA gains on 3q (P = .013), 17q (P = .019), and 22q (P = .021) compared with nonmetastatic primary BCA. No statistically significant correlation could be observed between DNA copy number changes and the histopathologic stage, grade, or survival (P > .05). The most frequent alteration observed only in lymph node metastases but not in the related primary tumor was loss of 2q (5 of 8). Coamplification of 7p and chromosome 17 was found in 6 of 8 lymph node metastases. A comparison of DNA copy number changes between primary tumors and their corresponding metastases indicated a high degree of genetic heterogeneity. Fluorescence in situ hybridization analysis demonstrated the involvement of the Her-2/neu gene in primary BCA and its related lymph node metastases. Each of the investigated primary tumors and related lymph node metastases also showed striking heterogeneity with respect to Her-2/neu, with several areas displaying different levels of amplification. In summary, our data indicate that DNA copy number changes on 2q, 3q, 7p, 17q, and 22q may be involved in the metastatic process in BCA. Furthermore, the striking genetic heterogeneity that we found between primary BCA and its lymph node metastases may underlie BCA's poor responsiveness to therapy and could help explain why prognostic biomarkers measured exclusively in primary tumors give an incomplete view of the biologic potential of BCA.