The role of targeted BRCA1/BRCA2 mutation analysis in hereditary breast/ovarian cancer families of Portuguese ancestry.

We report the analysis of altogether 1050 suspected hereditary breast/ovarian cancer (HBOC) families, 524 fully screened for BRCA1/BRCA2 mutations and 526 tested only for the most common mutations. Of the 119 families with pathogenic mutations, 40 (33.6%) had the BRCA2 c.156_157insAlu rearrangement and 15 (12.6%) the BRCA1 c.3331_3334del mutation, the former being specific of Portuguese ancestry and the latter showing a founder effect in Portugal. Interestingly, the two most common mutations were found in a significant proportion of the HBOC families with an a priori BRCAPRO mutation probability <10%. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry, even those fulfilling moderately stringent clinical-criteria for genetic testing, should be specifically analyzed for the two most common BRCA1/BRCA2 founder mutations, and we here present a simple method for this first tier test. Screening of the entire coding regions of BRCA1 and BRCA2 should subsequently be offered to those families with a mutation probability ≥10% if none of those founder mutations are found.

Gene amplification of the histone methyltransferase SETDB1 contributes to human lung tumorigenesis.

Disruption of the histone modification patterns is one of the most common features of human tumors. However, few genetic alterations in the histone modifier genes have been described in tumorigenesis. Herein we show that the histone methyltransferase SETDB1 undergoes gene amplification in non-small and small lung cancer cell lines and primary tumors. The existence of additional copies of the SETDB1 gene in these transformed cells is associated with higher levels of the corresponding mRNA and protein. From a functional standpoint, the depletion of SETDB1 expression in amplified cells reduces cancer growth in cell culture and nude mice models, whereas its overexpression increases the tumor invasiveness. The increased gene dosage of SETDB1 is also associated with enhanced sensitivity to the growth inhibitory effect mediated by the SETDB1-interfering drug mithramycin. Overall, the findings identify SETDB1 as a bona fide oncogene undergoing gene amplification-associated activation in lung cancer and suggest its potential for new therapeutic strategies.

The MSH2 c.388_389del mutation shows a founder effect in Portuguese Lynch syndrome families.

The MSH2 c.388_389del mutation has occasionally been described in Lynch families worldwide. At the Portuguese Oncology Institute in Porto, Portugal, we have identified 16 seemingly unrelated families with this germline mutation. To evaluate if this alteration is a founder or a recurrent mutation we performed haplotype analysis in the 16 Portuguese index cases and 55 relatives, as well as in four index cases and 13 relatives reported from Germany, Scotland, England, and Argentina. In the Portuguese families we observed a shared haplotype of approximately 10 Mb and all were originated from the north of Portugal. These results suggest that this alteration is a founder mutation in Portugal with a relatively recent origin. In the reported families outside Portugal with this mutation different haplotype backgrounds were observed, supporting the hypothesis that it occurred de novo on multiple occasions. We also conclude that the high proportion of families with the MSH2 c.388_389del mutation indicates that screening for this alteration as a first step may be cost-effective in the genetic testing of Lynch syndrome suspects of Portuguese ancestry, especially those originating from the north of Portugal.

Environmental impacts on soil and groundwater at airports: origin, contaminants of concern and environmental risks.

Environmental impacts of airports are similar to those of many industries, though their operations expand over a very large area. Most international impact assessment studies and environmental management programmes have been giving less focus on the impacts to soil and groundwater than desirable. This may be the result of the large attention given to air and noise pollution, relegating other environmental descriptors to a second role, even when the first are comparatively less relevant. One reason that contributes to such "biased" evaluation is the lack of systematic information about impacts to soil and groundwater from airport activities, something the present study intends to help correct. Results presented here include the review of over seven hundred documents and online databases, with the objective of obtaining the following information to support environmental studies: (i) which operations are responsible for chemical releases?; (ii) where are these releases located?; (iii) which contaminants of concern are released?; (iv) what are the associated environmental risks? Results showed that the main impacts occur as a result of fuel storage, stormwater runoff and drainage systems, fuel hydrant systems, fuel transport and refuelling, atmospheric deposition, rescue and fire fighting training areas, winter operations, electrical substations, storage of chemical products by airport owners or tenants, and maintenance of green areas. A new method for ranking environmental risks of organic substances, based on chemical properties, is proposed and applied. Results show that the contaminants with the highest risks are the perfluorochemicals, benzene, trichloroethylene and CCl(4). The obtained information provides a basis for establishing the planning and checking phases of environmental management systems, and may also help in the best design of pollution prevention measures in order to avoid or reduce significant environmental impacts from airports.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay.

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Association of ERBB2 gene status with histopathological parameters and disease-specific survival in gastric carcinoma patients.

The clinical significance of ERBB2 amplification/overexpression in gastric cancer remains unclear. In this study, we evaluated the ERBB2 status in 463 gastric carcinomas using immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH), and compared the findings with histopathological characteristics and with disease-specific survival. ERBB2 overexpression (2+ and 3+) and amplification (ratio ERBB2/CEP17 >or= 2) were found in 43 (9.3%) and 38 (8.2%) gastric carcinomas, respectively. Perfect IHC/FISH correlation was found for the 19 cases scored as 0 (all negative by FISH), and also for the 25 cases scored as 3+ (all positive by FISH). One out of six carcinomas scored as 1+ and 12 out of 18 carcinomas scored as 2+ were positive by FISH. ERBB2 amplification was associated with gastric carcinomas of intestinal type (P=0.007) and with an expansive growth pattern (P=0.021). ERBB2 amplification was detected in both histological components of two mixed carcinomas, indicating a common clonal origin. A statistically significant association was found between ERBB2 amplification and worse survival in patients with expansive gastric carcinomas (P=0.011). We conclude that ERBB2 status may have clinical significance in subsets of gastric cancer patients, and that further studies are warranted to evaluate whether patients whose gastric carcinomas present ERBB2 amplification/overexpression may benefit from therapy targeting this surface receptor.

Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma.

Ewing's sarcoma (ES) is characterized by specific chromosome translocations, the most common being t(11;22)(q24;q12). Additionally, other type of genetic abnormalities may occur and be relevant for explaining the variable tumour biology and clinical outcome. We have carried out a high-resolution array CGH and expression profiling on 25 ES tumour samples to characterize the DNA copy number aberrations (CNA) occurring in these tumours and determine their association with gene-expression profiles and clinical outcome. CNA were observed in 84% of the cases. We observed a median number of three aberrations per case. Besides numerical chromosome changes, smaller aberrations were found and defined at chromosomes 5p, 7q and 9p. All CNA were compiled to define the smallest overlapping regions of imbalance (SORI). A total of 35 SORI were delimited. Bioinformatics analyses were conducted to identify subgroups according to the pattern of genomic instability. Unsupervised and supervised clustering analysis (using SORI as variables) segregated the tumours in two distinct groups: one genomically stable (< or =3 CNA) and other genomically unstable (>3 CNA). The genomic unstable group showed a statistically significant shorter overall survival and was more refractory to chemotherapy. Expression profile analysis revealed significant differences between both groups. Genes related with chromosome segregation, DNA repair pathways and cell-cycle control were upregulated in the genomically unstable group. This report elucidates, for the first time, data about genomic instability in ES, based on CNA and expression profiling, and shows that a genomically unstable group of Ewing's tumours is correlated with a significant poor prognosis.

MSH6 germline mutations in early-onset colorectal cancer patients without family history of the disease.

Germline MLH1 and MSH2 mutations are scarce in young colorectal cancer patients with negative family history of the disease. To evaluate the contribution of germline MSH6 mutations to early-onset colorectal cancer, we have analysed peripheral blood of 38 patients diagnosed with this disease before 45 years of age and who presented no family history of hereditary nonpolyposis colorectal cancer-related cancers. Blood samples from 108 healthy volunteers were analysed for those genetic alterations suspected to affect the function of MSH6. Of the seven (18.4%) MSH6 alterations found, we have identified three novel germline mutations, one 8 bp deletion leading to a truncated protein and two missense mutations resulting in the substitution of amino acids belonging to different polarity groups. High-frequency microsatellite instability was found in the patient with the MSH6 deletion, but not in the other 27 carcinomas analysed. No MLH1 promoter methylation was detected in tumour tissue. Our findings suggest that germline MSH6 mutations contribute to a subset of early-onset colorectal cancer. Further studies are warranted to understand the genetic and environmental factors responsible for the variable penetration of MSH6 germline mutations, as well as to identify other causes of early-onset colorectal cancer.

SEPT2 is a new fusion partner of MLL in acute myeloid leukemia with t(2;11)(q37;q23).

We have identified a new mixed lineage leukemia (MLL) gene fusion partner in a patient with treatment-related acute myeloid leukemia (AML) presenting a t(2;11)(q37;q23) as the only cytogenetic abnormality. Fluorescence in situ hybridization demonstrated a rearrangement of the MLL gene and molecular genetic analyses identified a septin family gene, SEPT2, located on chromosome 2q37, as the fusion partner of MLL. RNA and DNA analyses showed the existence of an in-frame fusion of MLL exon 7 with SEPT2 exon 3, with the genomic breakpoints located in intron 7 and 2 of MLL and SEPT2, respectively. Search for DNA sequence motifs revealed the existence of two sequences with 94.4% homology with the topoisomerase II consensus cleavage site in MLL intron 7 and SEPT2 intron 2. SEPT2 is the fifth septin family gene fused with MLL, making this gene family the most frequently involved in MLL-related AML (about 10% of all known fusion partners). The protein encoded by SEPT2 is highly homologous to septins 1, 4 and 5 and is involved in the coordination of several key steps of mitosis. Further studies are warranted to understand why the septin protein family is particularly involved in the pathogenesis of MLL-associated leukemia.

Assessment of clonal relationships in ipsilateral and bilateral multiple breast carcinomas by comparative genomic hybridisation and hierarchical clustering analysis.

The issue of whether multiple, ipsilateral or bilateral, breast carcinomas represent multiple primary tumours or dissemination of a single carcinomatous process has been difficult to resolve, especially for individual patients. We have addressed the problem by comparative genomic hybridisation analysis of 26 tumours from 12 breast cancer patients with multiple ipsilateral and/or bilateral carcinoma lesions. Genomic imbalances were detected in 25 of the 26 (96%) tumours. Using the genomic imbalances detected in these 26 lesions as well as those previously found by us in an independent series of 35 unifocal breast carcinomas, we compared a probabilistic model for likelihood of independence with unsupervised hierarchical clustering methodologies to determine the clonal relatedness of multiple tumours in breast cancer patients. We conclude that CGH analysis of multiple breast carcinomas followed by unsupervised hierarchical clustering of the genomic imbalances is more reliable than previous criteria to determine the tumours' clonal relationship in individual patients, that most ipsilateral breast carcinomas arise through intramammary spreading of a single breast cancer, and that most patients with bilateral breast carcinomas have two different diseases.

Aberrant cellular retinol binding protein 1 (CRBP1) gene expression and promoter methylation in prostate cancer.

AIMS: Retinoids are involved in cell growth, differentiation, and carcinogenesis. Their effects depend on cytosolic transport and binding to nuclear receptors. CRBP1 encodes a protein involved in this process. Because altered CRBP1 expression and promoter hypermethylation occur in several tumours, these changes were investigated in prostate tumorigenesis. METHODS: The CRBP1 promoter was assessed by methylation specific polymerase chain reaction on tissue samples from 36 radical prostatectomy specimens (paired normal tissue, adenocarcinoma, and high grade prostatic intraepithelial neoplasia (HGPIN)), 32 benign prostatic hyperplasias (BPHs), and 13 normal prostate tissue samples from cystoprostatectomies. Methylation of DNA extracted from microdissected tissue was examined blindly. CRBP1 expression was assessed by immunohistochemistry on formalin fixed, paraffin wax embedded tissue. RESULTS: Loss of CRBP1 expression was seen in 15 of 36 adenocarcinomas and 18 of 36 HGPINs. Fifteen adenocarcinomas and nine HGPINs showed overexpression, whereas the remainder showed normal expression. BPH displayed normal expression. No significant associations were found between CRBP1 expression and Gleason score or stage. CRBP1 promoter hypermethylation was found in 17 of 36 adenocarcinomas, three of 35 HGPINs, one of 36 normal prostate tissues from the same patients, none of 32 BPHs, and none of 13 normal prostate tissues from cystoprostatectomies. Loss of expression and hypermethylation of CRBP1 were not significantly associated. CONCLUSIONS: Altered CRBP1 expression and hypermethylation are common in prostate carcinoma, although CRBP1 hypermethylation is not an early event in tumorigenesis. Moreover, both adenocarcinoma and HGPIN show frequent CRBP1 overexpression. The molecular mechanisms underlying altered CRBP1 expression in prostate cancer deserve further study.

CRD-BP: a c-Myc mRNA stabilizing protein with an oncofetal pattern of expression.

The Coding Region Determinant-Binding Protein (CRD-BP) is an RRM and KH-domain-containing protein that recognizes specifically at least three RNAs. It binds to one of the two c-myc mRNA instability elements, to the 5'Un Translated Region (UTR) of the leader 3 IGF-II mRNA and to the oncofetal H19 RNA. CRD-BP has been assigned a role in stabilizing c-myc mRNA by preventing its endonucleolytic cleavage and in repressing the translation of the leader 3 IGF-II mRNA, the major embryonic species of this message. CRD-BP is normally expressed only in fetal tissues. However, its expression is detected in primary tumors and transformed cell lines of different origins. The vast majority of colon (80%) and breast (60%) tumors and sarcomas (73%) express CRD-BP whereas in other tumor types, for example prostate carcinomas, its expression is rare. CRD-BP expression has also been detected in benign tumors such as breast fibroadenomas, meningiomas and other benign mesenchymal tumors, implying a role for this gene in abnormal cell proliferation. In breast carcinomas, CRD-BP expression and or gene copy number gains in the region encompassing the c-myc locus were detected in approximately 75% of tumors, implying that the deregulated expression of c-myc may be more widespread than previously believed. Infiltrated lymph nodes, corresponding to CRD-BP-positive primary tumors, were also found positive indicating that monitoring for CRD-BP could prove useful for the detection and monitoring of disseminated disease.

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding, CGH, M-FISH, and chromosome- and locus-specific FISH.

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.

Combined classical and molecular cytogenetic analysis of cancer.

While chromosome-banding analysis has set the standard for karyotyping from 1970 onwards, fluorescent in situ hybridisation (FISH) has more recently been used to complement the study of chromosomal rearrangements. Especially useful has been the appearance of FISH methodologies with screening abilities, namely comparative genome hybridisation (CGH), multicolour-FISH (m-FISH), and cross-species colour banding (RxFISH). These FISH-based screening techniques are reviewed here together with methodologies using chromosome- or locus-specific probes. Emphasis is put on the strengths and limitations of these FISH techniques to complement standard chromosome banding analysis. Judicious choice from the molecular cytogenetic techniques now available has significantly improved our ability to characterise the genomic rearrangements of cancer cells.

Complete cytogenetic characterization of the human breast cancer cell line MA11 combining G-banding, comparative genomic hybridization, multicolor fluorescence in situ hybridization, RxFISH, and chromosome-specific painting.

The MA11 cell line was established from malignant cells isolated from the bone marrow of a breast cancer patient. It metastasizes selectively to the brain in athymic mice. Since the genomic rearrangements of only a few breast cancer cell lines have been characterized completely, we analyzed MA11 cytogenetically. Because the G-banding analysis revealed a very complex karyotype with several markers and chromosomes with additional material of unknown origin, we used multicolor fluorescence in situ hybridization (M-FISH), cross-species color banding (RxFISH), comparative genomic hybridization (CGH), and chromosome-specific probes to better characterize the chromosome abnormalities. The use of these FISH-based screening techniques allowed us to detect previously unsuspected chromosomal changes and determine the identity of chromosomal markers. Multicolor FISH was especially useful to identify the rearranged chromosomes, whereas RxFISH, G-banding, and CGH were instrumental in determining breakpoint positions, although some uncertainties were removed only after hybridization with chromosome-specific probes. The combined analysis revealed a near-triploid karyotype with no less than 20 chromosomes demonstrating structural rearrangements. The resulting imbalances included several of those known to be common in primary breast carcinomas (gain of 1q, 8q, and 20q and loss of 8p, 11q, and 13q), indicating that the MA11 cell line may serve as a good model to study breast carcinogenesis. The full cytogenetic characterization we present may guide future searches for the mechanism of organ-selective metastasis in this model system and, possibly, also in vivo.

Evaluation of breast cancer polyclonality by combined chromosome banding and comparative genomic hybridization analysis.

Cytogenetically unrelated clones have been detected by chromosome banding analysis in many breast carcinomas. Because these karyotypic studies were performed on short-term cultured samples, it may be argued that in vitro selection occurred or that small clones may have arisen during culturing. To address this issue, we analyzed 37 breast carcinomas by G-banding and comparative genomic hybridization (CGH), a fluorescent in situ hybridization--based screening technique that does not require culturing or tumor metaphases. All but two of the 37 karyotypically abnormal cases presented copy number changes by CGH. The picture of genomic alterations revealed by the two techniques overlapped only partly. Sometimes the CGH analysis revealed genomic imbalances that belonged to cell populations not picked up by the cytogenetic analysis and in other cases, especially when the karyotypes had many markers and chromosomes with additional material of unknown origin, CGH gave a more reliable overall picture of the copy number gains and losses. However, besides sometimes revealing cell populations with balanced chromosome aberrations or unbalanced changes that nevertheless remained undetected by CGH, G-banding analysis was essential to understand how the genomic imbalances arose in the many cases in which both techniques detected the same clonal abnormalities. Furthermore, because CGH pictures only imbalances present in a significant proportion of the test sample, the very detection by this technique of imbalances belonging to apparently small, cytogenetically unrelated clones of cells proves that these clones must have been present in vivo. This constitutes compelling evidence that the cytogenetic polyclonality observed after short-term culturing of breast carcinomas is not an artifact.

Detailed genome-wide screening for inter- and intrachromosomal abnormalities by sequential G-banding and RxFISH color banding of the same metaphase cells.

While the now-classic chromosome banding methods, such as G-banding, remain the techniques of choice for the initial screening for karyotypic abnormalities, sometimes chromosomal rearrangements involve segments too small or too similarly banded to be detected or described adequately by these techniques. The necessity to use a genome-wide, fluorescence in situ hybridization (FISH)-based screening technique as a complement to G-banding is especially obvious in cases where the information obtained by the latter analysis does not provide an adequate guide to the choice of probes for chromosome-specific FISH. Furthermore, the same metaphase cells should ideally be used for both G-banding and FISH analysis to overcome the scarcity of metaphases observed in many cases and to ensure the correct interpretation of chromosomal aberrations in cytogenetically unstable neoplasms with massive cell-to-cell karyotypic variability. We describe a protocol which enables cross-species color banding (RxFISH), a new FISH-based screening technique that simultaneously imparts specific color banding patterns on all chromosomes, of preparations that have been G-banded and mounted for up to several years, as well as a procedure allowing chromosome-specific painting of the same metaphase cells to resolve whatever doubts persist after the preceding G-banding and RxFISH analyses. This approach makes possible a detailed, genome-wide screening for inter- and intrachromosomal abnormalities including archival cases whose karyotypic rearrangements had been incompletely identified by G-banding.