The spread of mammographic screening programmes around the world, including in developing countries, has substantially contributed to the diagnosis of small non-palpable lesions, which has increased the detection rate of DCIS (ductal carcinoma in situ). DCIS is heterogeneous in several ways, such as its clinical presentation, morphology and genomic profile. Excellent outcomes have been reported; however, many questions remain unanswered. For example, which patients groups are overtreated and could instead benefit from minimal intervention and which patient groups require a more traditional multidisciplinary approach. The development of a comprehensive integrated analysis that includes the radiological, morphological and genetic aspects of DCIS is necessary to answer these questions. This review focuses on discussing the significant findings about the morphological and molecular features of DCIS and its progression that have helped to uncover the biological and genetic heterogeneity of this disease. The knowledge gained in recent years might allow the development of tailored clinical management for women with DCIS in the future.
Breast cancer; cancer progression; Ductal carcinoma in situ (DCIS); epithelial cells; invasive breast carcinoma (IBC); microenvironment; BM, basal membrane; CAFs, carcinoma-associated fibroblasts; CC, comedocarcinoma; DCIS, ductal carcinoma in situ; DCIS-IBC, ductal carcinoma in situ with co-existing invasive breast carcinoma; ER, estrogen receptor; HG, high grade; IBC, invasive breast carcinoma; LCM: laser capture microdissection; LG, low grade; MEC, myoepithelial cell; miRNA, microRNAs; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; PR, progesterone receptor
Li-Fraumeni (LFS) and Li-Fraumeni-like (LFL) syndromes are associated to germline TP53 mutations, and are characterized by the development of central nervous system tumors, sarcomas, adrenocortical carcinomas, and other early-onset tumors. Due to the high frequency of breast cancer in LFS/LFL families, these syndromes clinically overlap with hereditary breast cancer (HBC). Germline point mutations in BRCA1, BRCA2, and TP53 genes are associated with high risk of breast cancer. Large rearrangements involving these genes are also implicated in the HBC phenotype.
We have screened DNA copy number changes by MLPA on BRCA1, BRCA2, and TP53 genes in 23 breast cancer patients with a clinical diagnosis consistent with LFS/LFL; most of these families also met the clinical criteria for other HBC syndromes.
We found no DNA copy number alterations in the BRCA2 and TP53 genes, but we detected in one patient a 36.4 Kb BRCA1 microdeletion, confirmed and further mapped by array-CGH, encompassing exons 9–19. Breakpoints sequencing analysis suggests that this rearrangement was mediated by flanking Alu sequences.
This is the first description of a germline intragenic BRCA1 deletion in a breast cancer patient with a family history consistent with both LFL and HBC syndromes. Our results show that large rearrangements in these known cancer predisposition genes occur, but are not a frequent cause of cancer susceptibility.
Breast cancer; Copy number variation; MLPA; BRCA1 microdeletion; Li-Fraumeni syndrome
Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2′-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5′ region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, ≥3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients.
Sequencing technologies and new bioinformatics tools have led to the complete sequencing of various genomes. However, information regarding the human transcriptome and its annotation is yet to be completed. The Human Cancer Genome Project, using ORESTES (open reading frame EST sequences) methodology, contributed to this objective by generating data from about 1.2 million expressed sequence tags. Approximately 30% of these sequences did not align to ESTs in the public databases and were considered no-match ORESTES. On the basis that a set of these ESTs could represent new transcripts, we constructed a cDNA microarray. This platform was used to hybridize against 12 different normal or tumor tissues. We identified 3421 transcribed regions not associated with annotated transcripts, representing 83.3% of the platform. The total number of differentially expressed sequences was 1007. Also, 28% of analyzed sequences could represent noncoding RNAs. Our data reinforces the knowledge of the human genome being pervasively transcribed, and point out molecular marker candidates for different cancers. To reinforce our data, we confirmed, by real-time PCR, the differential expression of three out of eight potentially tumor markers in prostate tissues. Lists of 1007 differentially expressed sequences, and the 291 potentially noncoding tumor markers were provided.
The basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' Broom Disease (WBD) in cacao (Theobroma cacao). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. M. perniciosa, together with the related species M. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9× coverage) of M. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen.
Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that M. perniciosa has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that M. perniciosa have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in M. perniciosa genome survey.
This genome survey gives an overview of the M. perniciosa genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the M. perniciosa/cacao pathosystem.
Massively Parallel Signature Sequencing (MPSS) is a powerful technique for genome-wide gene expression analysis, which, similar to SAGE, relies on the production of short tags proximal to the 3′end of transcripts. A single MPSS experiment can generate over 107 tags, providing a 10-fold coverage of the transcripts expressed in a human cell. A significant fraction of MPSS tags cannot be assigned to known transcripts (orphan tags) and are likely to be derived from transcripts expressed at very low levels (∼1 copy per cell). In order to explore the potential of MPSS for the characterization of the human transcriptome, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3′ cDNA fragments. GLGI-MPSS was applied to 83 orphan tags and 41 cDNA fragments were obtained. The analysis of these 41 fragments allowed the identification of novel transcripts, alternative tags generated from polymorphic and alternatively spliced transcripts, as well as the detection of artefactual MPSS tags. A systematic large-scale analysis of the genome by MPSS, in combination with the use of GLGI-MPSS protocol, will certainly provide a complementary approach to generate the complete catalog of human transcripts.
A new method, which allows for the identification and prioritization of predicted cancer genes for future analysis, is presented. This method generates a gene-specific score called the “S-Score” by incorporating data from different types of analysis including mutation screening, methylation status, copy-number variation and expression profiling. The method was applied to the data from The Cancer Genome Atlas and allowed the identification of known and potentially new oncogenes and tumor suppressors associated with different clinical features including shortest term of survival in ovarian cancer patients and hormonal subtypes in breast cancer patients. Furthermore, for the first time a genome-wide search for genes that behave as oncogenes and tumor suppressors in different tumor types was performed. We envisage that the S-score can be used as a standard method for the identification and prioritization of cancer genes for follow-up studies.
Genetic counselling and testing for Lynch syndrome have recently been introduced in several South American countries, though yet not available in the public health care system.
We compiled data from publications and hereditary cancer registries to characterize the Lynch syndrome mutation spectrum in South America. In total, data from 267 families that fulfilled the Amsterdam criteria and/or the Bethesda guidelines from Argentina, Brazil, Chile, Colombia and Uruguay were included.
Disease-predisposing mutations were identified in 37% of the families and affected MLH1 in 60% and MSH2 in 40%. Half of the mutations have not previously been reported and potential founder effects were identified in Brazil and in Colombia.
The South American Lynch syndrome mutation spectrum includes multiple new mutations, identifies potential founder effects and is useful for future development of genetic testing in this continent.
Lynch syndrome; MLH1; MSH2; South America; Mutation
Alzheimer’s disease (AD) is the most common cause of dementia in the human population, characterized by a spectrum of neuropathological abnormalities that results in memory impairment and loss of other cognitive processes as well as the presence of non-cognitive symptoms. Transcriptomic analyses provide an important approach to elucidating the pathogenesis of complex diseases like AD, helping to figure out both pre-clinical markers to identify susceptible patients and the early pathogenic mechanisms to serve as therapeutic targets. This study provides the gene expression profile of postmortem brain tissue from subjects with clinic-pathological AD (Braak IV, V, or V and CERAD B or C; and CDR ≥1), preclinical AD (Braak IV, V, or VI and CERAD B or C; and CDR = 0), and healthy older individuals (Braak ≤ II and CERAD 0 or A; and CDR = 0) in order to establish genes related to both AD neuropathology and clinical emergence of dementia. Based on differential gene expression, hierarchical clustering and network analysis, genes involved in energy metabolism, oxidative stress, DNA damage/repair, senescence, and transcriptional regulation were implicated with the neuropathology of AD; a transcriptional profile related to clinical manifestation of AD could not be detected with reliability using differential gene expression analysis, although genes involved in synaptic plasticity, and cell cycle seems to have a role revealed by gene classifier. In conclusion, the present data suggest gene expression profile changes secondary to the development of AD-related pathology and some genes that appear to be related to the clinical manifestation of dementia in subjects with significant AD pathology, making necessary further investigations to better understand these transcriptional findings on the pathogenesis and clinical emergence of AD.
Genetic factors predisposing individuals to cancer remain elusive in the majority of patients with a familial or clinical history suggestive of hereditary breast cancer. Germline DNA copy number variation (CNV) has recently been implicated in predisposition to cancers such as neuroblastomas as well as prostate and colorectal cancer. We evaluated the role of germline CNVs in breast cancer susceptibility, in particular those with low population frequencies (rare CNVs), which are more likely to cause disease."
Using whole-genome comparative genomic hybridization on microarrays, we screened a cohort of women fulfilling criteria for hereditary breast cancer who did not carry BRCA1/BRCA2 mutations.
The median numbers of total and rare CNVs per genome were not different between controls and patients. A total of 26 rare germline CNVs were identified in 68 cancer patients, however, a proportion that was significantly different (P = 0.0311) from the control group (23 rare CNVs in 100 individuals). Several of the genes affected by CNV in patients and controls had already been implicated in cancer.
This study is the first to explore the contribution of germline CNVs to BRCA1/2-negative familial and early-onset breast cancer. The data suggest that rare CNVs may contribute to cancer predisposition in this small cohort of patients, and this trend needs to be confirmed in larger population samples.
MUTYH-associated polyposis (MAP) is a recessive, hereditary, colorectal cancer-predisposing syndrome caused by biallelic mutations in the MUTYH gene. Most MUTYH pathogenic variants are missense mutations, and until recently no gross genomic deletions had been described.
We have identified a large deletion in the MUTYH gene: a > 4.2 kb deletion encompassing exons 4-16. This is the second description of this rearrangement, which has been recently described as the first large deletion in this gene. The clinically suspected MAP patient was homozygous for this mutation and presented with no amplification products for 14 exons of MUTYH on initial screening. Deletion breakpoints were refined to base pair level through array comparative genomic hybridization (aCGH) analysis followed by sequencing. The identified breakpoints were located within intron 3 and 146 bp downstream of the 3' end of the gene, with the presence of an AluJr element adjacent to the distal breakpoint. The presence of a 2 bp insertion at the junction suggests the involvement of the non-homologous end joining (NHEJ) repair mechanism, possibly facilitated by rearrangement-promoting elements. Examination of the MUTYH locus revealed a high Alu density that may make this region prone to rearrangements.
Large deletions are a possible mechanism for loss of function of the MUTYH gene, and investigation of such mutations may be important in identifying causative mutations in MAP patients.
Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression.
The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system.
In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.
Ductal carcinoma in situ (DCIS) of the breast includes a heterogeneous group of preinvasive tumors with uncertain evolution. Definition of the molecular factors necessary for progression to invasive disease is crucial to determining which lesions are likely to become invasive. To obtain insight into the molecular basis of DCIS, we compared the gene expression pattern of cells from the following samples: non-neoplastic, pure DCIS, in situ component of lesions with co-existing invasive ductal carcinoma, and invasive ductal carcinoma.
Forty-one samples were evaluated: four non-neoplastic, five pure DCIS, 22 in situ component of lesions with co-existing invasive ductal carcinoma, and 10 invasive ductal carcinoma. Pure cell populations were isolated using laser microdissection. Total RNA was purified, DNase treated, and amplified using the T7-based method. Microarray analysis was conducted using a customized cDNA platform. The concept of molecular divergence was applied to classify the sample groups using analysis of variance followed by Tukey's test.
Among the tumor sample groups, cells from pure DCIS exhibited the most divergent molecular profile, consequently identifying cells from in situ component of lesions with co-existing invasive ductal carcinoma as very similar to cells from invasive lesions. Additionally, we identified 147 genes that were differentially expressed between pure DCIS and in situ component of lesions with co-existing invasive ductal carcinoma, which can discriminate samples representative of in situ component of lesions with co-existing invasive ductal carcinoma from 60% of pure DCIS samples. A gene subset was evaluated using quantitative RT-PCR, which confirmed differential expression for 62.5% and 60.0% of them using initial and partial independent sample groups, respectively. Among these genes, LOX and SULF-1 exhibited features that identify them as potential participants in the malignant process of DCIS.
We identified new genes that are potentially involved in the malignant transformation of DCIS, and our findings strongly suggest that cells from the in situ component of lesions with co-existing invasive ductal carcinoma exhibit molecular alterations that enable them to invade the surrounding tissue before morphological changes in the lesion become apparent.
This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.