Microvascular fragment spheroids: Three-dimensional vascularization units for tissue engineering and regeneration.

Adipose tissue-derived microvascular fragments (MVF) serve as vascularization units in tissue engineering and regenerative medicine. Because a three-dimensional cellular arrangement has been shown to improve cell function, we herein generated for the first time MVF spheroids to investigate whether this further increases their vascularization potential. These spheroids exhibited a morphology, size, and viability comparable to that of previously introduced stromal vascular fraction (SVF) spheroids. However, MVF spheroids contained a significantly higher number of CD31-positive endothelial cells and α-smooth muscle actin (SMA)-positive perivascular cells, resulting in an enhanced angiogenic sprouting activity. Accordingly, they also exhibited an improved in vivo vascularization and engraftment after transplantation into mouse dorsal skinfold chambers. These findings indicate that MVF spheroids are superior to SVF spheroids and, thus, may be highly suitable to improve the vascularization of tissue defects and implanted tissue constructs.

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Full in-frame exon 3 skipping of BRCA2 confers high risk of breast and/or ovarian cancer.

Germline pathogenic variants in the BRCA2 gene are associated with a cumulative high risk of breast/ovarian cancer. Several BRCA2 variants result in complete loss of the exon-3 at the transcript level. The pathogenicity of these variants and the functional impact of loss of exon 3 have yet to be established. As a collaboration of the COVAR clinical trial group (France), and the ENIGMA consortium for investigating breast cancer gene variants, this study evaluated 8 BRCA2 variants resulting in complete deletion of exon 3. Clinical information for 39 families was gathered from Portugal, France, Denmark and Sweden. Multifactorial likelihood analyses were conducted using information from 293 patients, for 7 out of the 8 variants (including 6 intronic). For all variants combined the likelihood ratio in favor of causality was 4.39*1025. These results provide convincing evidence for the pathogenicity of all examined variants that lead to a total exon 3 skipping, and suggest that other variants that result in complete loss of exon 3 at the molecular level could be associated with a high risk of cancer comparable to that associated with classical pathogenic variants in BRCA1 or BRCA2 gene. In addition, our functional study shows, for the first time, that deletion of exon 3 impairs the ability of cells to survive upon Mitomycin-C treatment, supporting lack of function for the altered BRCA2 protein in these cells. Finally, this study demonstrates that any variant leading to expression of only BRCA2 delta-exon 3 will be associated with an increased risk of breast and ovarian cancer.

BRCA Share: A Collection of Clinical BRCA Gene Variants.

As next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.

Mutation analysis of PALB2 gene in French breast cancer families.

Several population-based and family-based studies have demonstrated that germline mutations of the PALB2 gene (Partner and Localizer of BRCA2) are associated with an increased risk of breast cancer. Distinct mutation frequencies and spectrums have been described depending on the population studied. Here we describe the first complete PALB2 coding sequence screening in the French population. We screened the complete coding sequence and intron-exon boundaries of PALB2, using the EMMA technique, to assess the contribution of pathogenic mutations in a set of 835 familial breast cancer cases and 662 unrelated controls from the French national study GENESIS and the Paul Strauss Cancer Centre, all previously tested negative for BRCA1 and BRCA2 pathogenic mutations. Our analysis revealed the presence of four novel deleterious mutations: c.1186insT, c.1857delT and c.2850delC in three cases, c.3418dupT in one control. In addition, we identified two in-frame insertion/deletion, 19 missense substitutions (two of them predicted as pathogenic), 9 synonymous variants, 28 variants located in introns and 2 in UTRs, as well as frequent variants. Truncating PALB2 mutations were found in 0.36% of familial breast cancer cases, a frequency lower than the one detected in comparable studies in other populations (0.73-3.40%). This suggests a small but significant contribution of PALB2 mutations to the breast cancer susceptibility in the French population.

An entire exon 3 germ-line rearrangement in the BRCA2 gene: pathogenic relevance of exon 3 deletion in breast cancer predisposition.

BACKGROUND: Germ-line mutations in the BRCA1 and BRCA2 genes are major contributors to hereditary breast/ovarian cancer. Large rearrangements are less frequent in the BRCA2 gene than in BRCA1. We report, here, the first total deletion of exon 3 in the BRCA2 gene that was detected during screening of 2058 index cases from breast/ovarian cancer families for BRCA2 large rearrangements. Deletion of exon 3, which is in phase, does not alter the reading frame. Low levels of alternative transcripts lacking exon 3 (Δ3 delta3 transcript) have been reported in normal tissues, which raises the question whether deletion of exon 3 is pathogenic. METHODS: Large BRCA2 rearrangements were analysed by QMPSF (Quantitative Multiplex PCR of Short Fluorescent Fragments) or MLPA (Multiplex Ligation-Dependent Probe Amplification). The exon 3 deletion was characterized with a "zoom-in" dedicated CGH array to the BRCA2 gene and sequencing. To determine the effect of exon 3 deletion and assess its pathogenic effect, three methods of transcript quantification were used: fragment analysis of FAM-labelled PCR products, specific allelic expression using an intron 2 polymorphism and competitive quantitative RT-PCR. RESULTS: Large rearrangements of BRCA2 were detected in six index cases out of 2058 tested (3% of all deleterious BRCA2 mutations). This study reports the first large rearrangement of the BRCA2 gene that includes all of exon 3 and leads to an in frame deletion of exon 3 at the transcriptional level. Thirty five variants in exon 3 and junction regions of BRCA2 are also reported, that contribute to the interpretation of the pathogenicity of the deletion. The quantitative approaches showed that there are three classes of delta3 BRCA2 transcripts (low, moderate and exclusive). Exclusive expression of the delta3 transcript by the mutant allele and segregation data provide evidence for a causal effect of the exon 3 deletion. CONCLUSION: This paper highlights that large rearrangements and total deletion of exon 3 in the BRCA2 gene could contribute to hereditary breast and/or ovarian cancer. In addition, our findings suggest that, to interpret the pathogenic effect of any variants of exon 3, both accurate transcript quantification and co-segregation analysis are required.

Evidence for SMAD3 as a modifier of breast cancer risk in BRCA2 mutation carriers.

INTRODUCTION: Current attempts to identify genetic modifiers of BRCA1 and BRCA2 associated risk have focused on a candidate gene approach, based on knowledge of gene functions, or the development of large genome-wide association studies. In this study, we evaluated 24 SNPs tagged to 14 candidate genes derived through a novel approach that analysed gene expression differences to prioritise candidate modifier genes for association studies. METHODS: We successfully genotyped 24 SNPs in a cohort of up to 4,724 BRCA1 and 2,693 BRCA2 female mutation carriers from 15 study groups and assessed whether these variants were associated with risk of breast cancer in BRCA1 and BRCA2 mutation carriers. RESULTS: SNPs in five of the 14 candidate genes showed evidence of association with breast cancer risk for BRCA1 or BRCA2 carriers (P < 0.05). Notably, the minor alleles of two SNPs (rs7166081 and rs3825977) in high linkage disequilibrium (r² = 0.77), located at the SMAD3 locus (15q22), were each associated with increased breast cancer risk for BRCA2 mutation carriers (relative risk = 1.25, 95% confidence interval = 1.07 to 1.45, P(trend) = 0.004; and relative risk = 1.20, 95% confidence interval = 1.03 to 1.40, P(trend) = 0.018). CONCLUSIONS: This study provides evidence that the SMAD3 gene, which encodes a key regulatory protein in the transforming growth factor beta signalling pathway and is known to interact directly with BRCA2, may contribute to increased risk of breast cancer in BRCA2 mutation carriers. This finding suggests that genes with expression associated with BRCA1 and BRCA2 mutation status are enriched for the presence of common genetic modifiers of breast cancer risk in these populations.

No association of TGFB1 L10P genotypes and breast cancer risk in BRCA1 and BRCA2 mutation carriers: a multi-center cohort study.

BACKGROUND: The transforming growth factor beta-1 gene (TGFB1) is a plausible candidate for breast cancer susceptibility. The L10P variant of TGFB1 is associated with higher circulating levels and secretion of TGF-beta, and recent large-scale studies suggest strongly that this variant is associated with breast cancer risk in the general population. METHODS: To evaluate whether TGFB1 L10P also modifies the risk of breast cancer in BRCA1 or BRCA2 mutation carriers, we undertook a multi-center study of 3,442 BRCA1 and 2,095 BRCA2 mutation carriers. RESULTS: We found no evidence of association between TGFB1 L10P and breast cancer risk in either BRCA1 or BRCA2 mutation carriers. The per-allele HR for the L10P variant was 1.01 (95%CI: 0.92-1.11) in BRCA1 carriers and 0.92 (95%CI: 0.81-1.04) in BRCA2 mutation carriers. CONCLUSIONS: These results do not support the hypothesis that TGFB1 L10P genotypes modify the risk of breast cancer in BRCA1 or BRCA2 mutation carriers.

Breast cancer risk in BRCA1 and BRCA2 mutation carriers and polyglutamine repeat length in the AIB1 gene.

Marked variation in phenotypic expression among BRCA1 and BRCA2 mutation carriers may be partly explained by modifier genes that influence mutation penetrance. Variation in CAG/CAA repeat lengths coding for stretches of glutamines in the C-terminus of the AIB1 protein (amplified in breast cancer 1, a steroid receptor coactivator) has been proposed to modify the breast cancer risk in women carrying germline BRCA1 mutations. We genotyped the AIB1 repeat length polymorphism from the genomic DNA of a group of 851 BRCA1 and 324 BRCA2 female germline mutation carriers to estimate an association with breast cancer risk modification. Hazard ratios (HR) were calculated using a Cox proportional hazards model. For BRCA1 and BRCA2 mutation carriers, analyzed separately and together, we found that women who carried alleles with 28 or more polyglutamine repeats had no increased risk of breast cancer compared to those who carried alleles with fewer repeats (HR for BRCA1/2 carriers = 0.88, 95% CI [confidence interval] = 0.75-1.04). Analyzing average repeat lengths as a continuous variable showed no excess risk of breast cancer (BC) in BRCA1 or BRCA2 mutation carriers (HR for average repeat length in BRCA1/2 carriers = 1.01, 95% CI = 0.92-1.11). These results strongly suggest that contrary to previous studies, there is no significant effect of AIB1 genetic variation on BC risk in BRCA1 mutation carriers and provide an indication that there is also no strong risk modification in BRCA2 carriers.

Common BRCA2 variants and modification of breast and ovarian cancer risk in BRCA1 mutation carriers.

The HH genotype of the nonconservative amino acid substitution polymorphism N372H in the BRCA2 gene was reported to be associated with a 1.3- to 1.5-fold increase in risk of both breast and ovarian cancer. As these studies concerned sporadic cancer cases, we investigated whether N372H and another common variant located in the 5'-untranslated region (203G > A) of the BRCA2 gene modify breast or ovarian cancer risk in BRCA1 mutation carriers. The study includes 778 women carrying a BRCA1 germ-line mutation belonging to 403 families. The two BRCA2 variants were analyzed by the TaqMan allelic discrimination technique. Genotypes were analyzed by disease-free survival analysis using a Cox proportional hazards model. We found no evidence of a significant modification of breast cancer penetrance in BRCA1 mutation carriers by either polymorphism. In respect of ovarian cancer risk, we also saw no effect with the N372H variant but we did observe a borderline association with the 5'-untranslated region 203A allele (hazard ratio, 1.43; CI, 1.01-2.00). In contrast to the result of Healey et al. on newborn females and adult female controls, we found no departure from Hardy-Weinberg equilibrium in the distribution of N372H alleles for our female BRCA1 carriers. We conclude that if these single-nucleotide polymorphisms do modify the risk of cancer in BRCA1 mutation carriers, their effects are not significantly larger than that of N372H previously observed in the general population.

BRCA1 testing in breast and/or ovarian cancer families from northeastern France identifies two common mutations with a founder effect.

OBJECTIVE: The purpose of this study was to determine whether two mutations detected frequently in a population of breast and/or ovarian cancer families originating from the northeastern part of France could be due to a founder effect. METHODS: 83 index cases of families ascertained to have a familial breast and/or ovarian cancer history, were screened for mutations in all coding exons of the BRCA1 gene, using combined DGGE and direct sequencing. For haplotype analysis, six polymorphic markers were used for allelotyping of mutation carriers and non carriers from nine families with 3600del11 mutation and four families with G1710X mutation. RESULTS: Of 83 index cases, 27 (32%) had 14 different BRCA1 mutations, one of which (G1710X), had not been reported in other populations. Two mutations were particularly common: 3600del11 in exon 11 accounted for 37% and the nonsense mutation G1710X in exon 18 for 15% of all mutations. We identified a common haplotype for each mutation suggesting a common founder for each recurrent mutation. No specific phenotype could be assigned to any of the common mutations. CONCLUSIONS: These data demonstrate geographical clustering and suggest a founder effect for particular BRCA1 mutations, which identification will facilitate carrier detection in French families with breast cancer and breast and/or ovarian cancer.

Identification of genes associated with tumorigenesis and metastatic potential of hypopharyngeal cancer by microarray analysis.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world. There is a need, for both clinical and scientific reasons, to find markers to identify patients with aggressive disease as early as possible, and to understand the events leading to malignant transformation and susceptibility to metastasis. We report the first large-scale gene expression analysis of a unique HNSCC location, the hypopharynx. Four normal and 34 tumour samples were analysed with 12 600 gene microarrays. Clusters of differentially expressed genes were identified in the chromosomal regions 3q27.3, 17q21.2-q21.31, 7q11.22-q22.1 and 11q13.1-q13.3, which, interestingly, have already been identified by comparative genomic hybridization (CGH) as major regions of gene amplification. We showed that six overexpressed genes (EIF4G1, DVL3, EPHB4, MCM7, BRMS1 and SART1) located in these regions are indeed amplified. We report 119 genes that are highly differentially expressed between 'early' tumours and normal samples. Of these, we validated by quantitative PCR six novel poorly characterized genes. These genes are potential new markers of HNSCC. Comparing patients with relatively nonaggressive and aggressive tumours (without or with clinical evidence of metastasis 3 years after surgery), we identified 164 differentially expressed genes potentially involved in the acquisition of metastatic potential. This study contributes to the understanding of HNSCC, staging patients into prognostic groups and identifying high-risk patients who may benefit from more aggressive treatment.

BRCA1 wild-type allele modifies risk of ovarian cancer in carriers of BRCA1 germ-line mutations.

Strong inter- and intrafamilial variation of penetrance of breast and ovarian cancer is observed in BRCA1 mutation carriers. The wild-type copy of the BRCA1 gene is a plausible candidate as a cancer risk modifier given that the residual function corresponding to the intact BRCA1 allele may influence the process of tumor formation in BRCA1 carriers. Indeed, growing evidence is now becoming available on impaired reparation of double-strand DNA breaks in cells heterozygous for BRCA1 mutations, implying an enhanced mutability of BRCA1(+/-) cells. To determine whether certain variant forms of the wild-type BRCA1 allele are implicated in variation of the BRCA1-related cancer risk, their effect was studied in a panel of 591 women with BRCA1 germ-line mutations. We found that BRCA1 carriers with the wild-type BRCA1 copy bearing a common Gly1038 variant were at increased risk of ovarian cancer (hazards ratio, 1.50; 95% confidence interval, 1.03-2.19). The results of our study imply that a quite significant proportion of the interindividual variability in ovarian cancer penetrance in BRCA1 carriers may be explained by a common BRCA1 Gly1038 wild-type allele, given its high frequency (0.27).

Amplicon mapping and transcriptional analysis pinpoint cyclin L as a candidate oncogene in head and neck cancer.

DNA gains targeting the 3q chromosome are common in head and neck squamous cell carcinomas, as well as in lung, ovarian, and cervical cancer. Several candidate oncogenes located on 3q were proposed, i.e., PIK3CA, p63, and eIF-5A2. However, none of these genes was found included in a narrow high-level amplification. Recently, microarray-based comparative genomic hybridization (array CGH) was developed for high-resolution screening of deletions and amplifications in tumor genomes. In this study, by microarray-based comparative genomic hybridization, we found a narrow 3q25.3 high-level amplification in a head and neck cancer cell line. We precisely delimited the 3-Mb length-amplified segment by semiquantitative PCR and measured the transcriptional level of every gene (RefSeq full-length mRNA) located inside this segment by cDNA microarray and quantitative reverse transcription-PCR. Four genes were overexpressed in three head and neck cancer cell lines with increased DNA copy number, compared with a control tongue cell line. We extended the transcriptional analysis of these four genes to 20 head and neck squamous cell carcinomas. Only one gene, cyclin L (ania-6a), is commonly overexpressed in primary tumors compared with corresponding normal tissues. This cyclin was previously pinpointed as a candidate for a role in promoting cell cycle entry. Thus, we propose cyclin L as a candidate oncogene in head and neck cancer.