Association between SNP rs527616 in lncRNA AQP4-AS1 and susceptibility to breast cancer in a southern Brazilian population.

Breast cancer (BC) is the leading cause of death by this disease in women worldwide. Among the factors involved in tumorigenesis, long non-coding RNAs (lncRNAs) and their differential expression have been associated. Differences in gene expression may be triggered by variations in DNA sequence, including single nucleotide polymorphisms (SNPs). In the present study, we analyzed the rs527616 (C>G), located in the lncRNA AQP4-AS1, using PCR-SSP in 306 BC patients and 312 controls, from a Brazilian population. In the BC group, the frequency found for CG heterozygotes was above the expected and the overdominant model is the best one to explain our results (OR: 1.70, IC 95%: 1.23-2.34, P<0.001). Furthermore, the SNP were associated with age at BC diagnosis and the risk genotype more frequent in the older age group. According to TCGA data, AQP4-AS1 is down-regulated in BC tissue, and the overexpression is associated with better prognoses, including Luminal A, HER2-, stage 1 of disease and smaller tumor. In conclusion, the CG genotype is associated with increased susceptibility in the southern Brazilian population. This SNP is mapped in the lncRNA AQP4-AS1, showing differential expression in BC samples. Based on these results, we emphasize the potential of the role of AQP4-AS1 in cancer.

Copy Number Analysis of the DLX4 and ERBB2 Genes in South African Breast Cancer Patients.

Breast cancer is one of the main causes of cancer death among South African women. Although several risk factors can be attributed to the observed high mortality rate, the biology of the tumors is not extensively investigated. Copy number gain of the DLX4 homeobox gene has been observed in breast cancer in association with poor prognosis and specific racial groups. Therefore, we aimed to assess the copy number and prognostic role of DLX4 in breast cancer from South African patients. Due to the co-location of ERBB2 and DLX4 in the 17q21 region, its copy number was also evaluated. Our results in the analysis of 66 cases demonstrated copy number gains of DLX4 and ERBB2 in 24.1 and 29.7% of the cases, respectively. Linear regression analysis showed no dependency between the copy number alterations in these genes. Although not significant, patients with DLX4 and ERBB2 gains presented a higher frequency of advanced-grade tumors. In addition, copy number alterations of these genes were not significantly differently observed in the 3 main racial groups of the Western Cape population: Colored, White, and Black. These findings indicate that gains of DLX4 and ERBB2 occur in South African breast cancer patients irrespectively of their race and factors known to influence prognosis.

Amplification and deletion of the RAPH1 gene in breast cancer patients.

Lamellipodin protein (Lpd), encoded by the RAPH1 gene, modulates the assembly of actin cytoskeleton through its binding to the Ena/VASPs proteins, and acts in cellular motility and lamelipodial protrusion. The region where RAPH1 gene is located (2q33) is deleted in various types of cancer and the gene expression changes in tumors when compared to normal tissues. Amplifications and deletions of the RAPH1 gene were investigated in breast carcinoma samples, in order to determine the possible relationship of the gene with breast cancer tumorigenesis and lymph node metastasis. RAPH1 gene alterations were determined by relative quantification, standard curve method using Real-time PCR technique in samples of tumor and peripheral blood from 52 patients. Regression and correlation analyses were conducted using gene alterations and clinicopathological data. All samples analyzed were altered, with 63.5 % deletion cases and 36.5 % amplification cases. The logistic regression and correlation analysis with clinicopathological data did not show significant results. The results suggest that although the RAPH1 gene was deleted or amplified in all samples, the Lpd does not seem to play a major role in tumorigenesis of mammary carcinomas and probably other proteins, also involved in the process of cellular motility and metastasis, are acting more effectively for or against the migration of breast tumor cells.

MiR-150-5p Overexpression in Triple-Negative Breast Cancer Contributes to the In Vitro Aggressiveness of This Breast Cancer Subtype.

MiR-150-5p is frequently deregulated in cancer, with expression and mode of action varying according to the tumor type. Here, we investigated the expression levels and role of miR-150-5p in the aggressive breast cancer subtype triple-negative breast cancer (TNBC). MiR-150-5p expression levels were analyzed in tissue samples from 113 patients with invasive breast cancer (56 TNBC and 57 non-TNBC) and 41 adjacent non-tumor tissues (ANT). Overexpression of miR-150-5p was observed in tumor tissues compared with ANT tissues and in TNBC compared with non-TNBC tissues. MiR-150-5p expression levels were significantly associated with high tumor grades and the Caucasian ethnicity. Interestingly, high miR-150-5p levels were associated with prolonged overall survival. Manipulation of miR-150-5p expression in TNBC cells modulated cell proliferation, clonogenicity, migration, and drug resistance. Manipulation of miR-150-5p expression also resulted in altered expression of its mRNA targets, including epithelial-to-mesenchymal transition markers, MYB, and members of the SRC pathway. These findings suggest that miR-150-5p is overexpressed in TNBC and contributes to the aggressiveness of TNBC cells in vitro.

Simultaneous CXCL12 and ESR1 CpG island hypermethylation correlates with poor prognosis in sporadic breast cancer.

BACKGROUND: CXCL12 is a chemokine that is constitutively expressed in many organs and tissues. CXCL12 promoter hypermethylation has been detected in primary breast tumours and contributes to their metastatic potential. It has been shown that the oestrogen receptor alpha (ESR1) gene can also be silenced by DNA methylation. In this study, we used methylation-specific PCR (MSP) to analyse the methylation status in two regions of the CXCL12 promoter and ESR1 in tumour cell lines and in primary breast tumour samples, and correlated our results with clinicopathological data. METHODS: First, we analysed CXCL12 expression in breast tumour cell lines by RT-PCR. We also used 5-aza-2'-deoxycytidine (5-aza-CdR) treatment and DNA bisulphite sequencing to study the promoter methylation for a specific region of CXCL12 in breast tumour cell lines. We evaluated CXCL12 and ESR1 methylation in primary tumour samples by methylation-specific PCR (MSP). Finally, promoter hypermethylation of these genes was analysed using Fisher's exact test and correlated with clinicopathological data using the Chi square test, Kaplan-Meier survival analysis and Cox regression analysis. RESULTS: CXCL12 promoter hypermethylation in the first region (island 2) and second region (island 4) was correlated with lack of expression of the gene in tumour cell lines. In the primary tumours, island 2 was hypermethylated in 14.5% of the samples and island 4 was hypermethylated in 54% of the samples. The ESR1 promoter was hypermethylated in 41% of breast tumour samples. In addition, the levels of ER alpha protein expression diminished with increased frequency of ESR1 methylation (p < 0.0001). This study also demonstrated that CXCL12 island 4 and ESR1 methylation occur simultaneously at a high frequency (p = 0.0220). CONCLUSIONS: This is the first study showing a simultaneous involvement of epigenetic regulation for both CXCL12 and ESR1 genes in Brazilian women. The methylation status of both genes was significantly correlated with histologically advanced disease, the presence of metastases and death. Therefore, the methylation pattern of these genes could be used as a molecular marker for the prediction of breast cancer outcome.

Frequency of the TP53 R337H variant in sporadic breast cancer and its impact on genomic instability.

The R337H is a TP53 germline pathogenic variant that has been associated with several types of cancers, including breast cancer. Our main objective was to determine the frequency of the R337H variant in sporadic breast cancer patients from Paraná state, South Brazil, its association with prognosis and its impact in genomic instability. The genotyping of 805 breast cancer tissues revealed a genotypic and allelic frequency of the R337H variant of 2.36% and 1.18%, respectively. In these R337H+ cases a lower mean age at diagnosis was observed when compared to the R337H-cases. Array-CGH analysis showed that R337H+ patients presented a higher number of copy number alterations (CNAs), compared to the R337H-. These CNAs affected genes and miRNAs that regulate critical cancer signaling pathways; a number of these genes were associated with survival after querying the KMplot database. Furthermore, homozygous (R337H+/R337H+) fibroblasts presented increased levels of copy number variants when compared to heterozygous or R337H- cells. In conclusion, the R337H variant may contribute to 2.36% of the breast cancer cases without family cancer history in Paraná. Among other mechanisms, R337H increases the level of genomic instability, as evidenced by a higher number of CNAs in the R337H+ cases compared to the R337H-.

Identification of miRNAs Enriched in Extracellular Vesicles Derived from Serum Samples of Breast Cancer Patients.

MicroRNAs derived from extracellular vesicles (EV-miRNAs) are circulating miRNAs considered as potential new diagnostic markers for cancer that can be easily detected in liquid biopsies. In this study, we performed RNA sequencing analysis as a screening strategy to identify EV-miRNAs derived from serum of clinically well-annotated breast cancer (BC) patients from the south of Brazil. EVs from three groups of samples (healthy controls (CT), luminal A (LA), and triple-negative (TNBC)) were isolated from serum using a precipitation method and analyzed by RNA-seq (screening phase). Subsequently, four EV-miRNAs (miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p) were selected to be quantified by quantitative real-time PCR (RT-qPCR) in individual samples (test phase). A panel composed of miR-142-5p, miR-320a, and miR-4433b-5p distinguished BC patients from CT with an area under the curve (AUC) of 0.8387 (93.33% sensitivity, 68.75% specificity). The combination of miR-142-5p and miR-320a distinguished LA patients from CT with an AUC of 0.9410 (100% sensitivity, 93.80% specificity). Interestingly, decreased expression of miR-142-5p and miR-150-5p were significantly associated with more advanced tumor grades (grade III), while the decreased expression of miR-142-5p and miR-320a was associated with a larger tumor size. These results provide insights into the potential application of EVs-miRNAs from serum as novel specific markers for early diagnosis of BC.

New recurrent deletions in the PPARgamma and TP53 genes are associated with childhood myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a rare hematological malignancy in children. It was performed FISH analysis in 19 pediatric MDS patients to investigate deletions involving the PPARgamma and TP53 genes. Significant losses in the PPARgamma gene and deletions in the tumor suppressor gene TP53 were observed in 17 and 18 cases, respectively. Using quantitative RT-PCR, it was detected PPARgamma transcript downexpression in a subset of these cases. G-banding analysis revealed 17p deletions in a small number of these cases. One MDS therapy-related patient had neither a loss of PPARgamma nor TP53. These data suggest that the PPARgamma and TP53 genes may be candidates for molecular markers in pediatric MDS, and that these potentially recurrent deletions could contribute to the identification of therapeutic approaches in primary pediatric MDS.

ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma.

BACKGROUND: ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer. METHODS: First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test. RESULTS: The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was statistically significant (p = 0.0002). CONCLUSION: ADAM33 gene silencing may be related to the discohesive histological appearance of ILCs. We suggest that ADAM33 promoter methylation may be a useful molecular marker for differentiating ILC and IDC.

Integrated copy number and miRNA expression analysis in triple negative breast cancer of Latin American patients.

Triple negative breast cancer (TNBC), a clinically aggressive breast cancer subtype, affects 15-35% of women from Latin America. Using an approach of direct integration of copy number and global miRNA profiling data, performed simultaneously in the same tumor specimens, we identified a panel of 17 miRNAs specifically associated with TNBC of ancestrally characterized patients from Latin America, Brazil. This panel was differentially expressed between the TNBC and non-TNBC subtypes studied (p ≤ 0.05, FDR ≤ 0.25), with their expression levels concordant with the patterns of copy number alterations (CNAs), present mostly frequent at 8q21.3-q24.3, 3q24-29, 6p25.3-p12.2, 1q21.1-q44, 5q11.1-q22.1, 11p13-p11.2, 13q12.11-q14.3, 17q24.2-q25.3 and Xp22.33-p11.21. The combined 17 miRNAs presented a high power (AUC = 0.953 (0.78-0.99);95% CI) in discriminating between the TNBC and non-TNBC subtypes of the patients studied. In addition, the expression of 14 and 15 of the 17miRNAs was significantly associated with tumor subtype when adjusted for tumor stage and grade, respectively. In conclusion, the panel of miRNAs identified demonstrated the impact of CNAs in miRNA expression levels and identified miRNA target genes potentially affected by both CNAs and miRNA deregulation. These targets, involved in critical signaling pathways and biological functions associated specifically with the TNBC transcriptome of Latina patients, can provide biological insights into the observed differences in the TNBC clinical outcome among racial/ethnic groups, taking into consideration their genetic ancestry.

Long Non-Coding RNA TUG1 Expression Is Associated with Different Subtypes in Human Breast Cancer.

Taurine upregulated 1 gene (TUG1) is a long non-coding RNA associated with several types of cancer. Recently, differential expression of TUG1 was found in cancerous breast tissues and associated with breast cancer malignancy features. Although this is evidence of a potential role in breast cancer, TUG1 expression could not be associated with different subtypes, possibly due to the small number of samples analyzed. Breast cancer is a heterogeneous disease and, based on molecular signatures, may be classified into different subtypes with prognostic implications. In the present study, we include analysis of TUG1 expression in 796 invasive breast carcinoma and 105 normal samples of RNA sequencing (RNA-seq) datasets from The Cancer Genome Atlas (TCGA) and describe that TUG1 expression is increased in HER2-enriched and basal-like subtypes compared to luminal A. Additionally, TUG1 expression is associated with survival in HER2-enriched patients. These results reinforce the importance of TUG1 in breast cancer and outline its potential impact on specific subtypes.

Copy number and expression analysis of FOSL1, GSTP1, NTSR1, FADD and CCND1 genes in primary breast tumors with axillary lymph node metastasis.

In breast cancer, lymph node (LN) metastasis is one of the strongest prognostic factors at diagnosis. Therefore the identification of molecular markers with metastatic potential that promote the development of LN metastasis is of critical clinical relevance. In this study, we evaluated the copy number status of the FOSL1, GSTP1, NTSR1, FADD and CCND1 genes by TaqMan assays in 137 breast cancer patients, 84 with LN metastasis (LN+) and 53 with no LN metastasis (LN-). The copy number data for four of these genes (FOSL1, GSTP1, FADD and CCND1) were integrated with their mRNA expression levels in 31 patients. In both groups of patients, gains were the most frequent copy number alteration (CNA) observed, involving mainly the CCND1, NTSR1 and FADD genes; mRNA overexpression was more commonly observed for the CCND1 and FADD genes. For the FADD gene in the LN+ group, gene expression was shown to be dependent on CNAs; for the other genes no association was found. In conclusion, increase copy number and mRNA overexpression of FOSL1, GSTP1, FADD, NTSR1 and CCND1 genes are frequently observed in primary breast tumors, and except for the FADD gene, they occur independently and irrespectively of the patients' LN axillary metastatic status.

Worse prognosis in breast cancer patients can be predicted by immunohistochemical analysis of positive MMP-2 and negative estrogen and progesterone receptors.

INTRODUCTION:: Breast cancer is the most cause of death, and approximately 90% of these deaths are due to metastases. Matrix metalloproteinase-2 (MMP-2) gelatinase activity is able to degrade a major constituent of the tumor microenvironment, type IV collagen. Two well-established proteins used as markers in clinical practice for breast cancer are the receptors for estrogen (ER) and progesterone (PR). Although the presence of these receptors has been associated with a better prognosis, loss of these proteins can occur during tumor progression, with subsequent resistance to hormone therapy. OBJECTIVE:: To study the correlation among MMP-2, ER, and PR, as well as the establishment of the metastatic process in primary breast tumors. METHOD:: Breast cancer samples (n=44) were analyzed by immunohistochemistry for MMP-2, ER, and PR. RESULTS:: We observed that 90% of patients who had metastases and died showed positive staining for MMP-2 (p=0.0082 for both). Using Kaplan-Meier analysis, we found that negative ER patients who were also positive for MMP-2 had even worse disease-free survival (DFS) and overall survival (OS) (p= 0.012 and p=0.005, respectively). Similar results were found in PR-negative patients for DFS (a trend p=0.077) and OS (p=0.038). CONCLUSION:: Regardless of our small sample size (n=44), the data obtained strongly suggest that MMP-2 in combination with already well-established markers could help to predict the emergence of metastases and death in patients with breast cancer.

Differentially expressed miRNAs in triple negative breast cancer between African-American and non-Hispanic white women.

Triple Negative Breast Cancer (TNBC), a clinically aggressive subtype of breast cancer, disproportionately affects African American (AA) women when compared to non-Hispanic Whites (NHW). MiRNAs(miRNAs) play a critical role in these tumors, through the regulation of cancer driver genes. In this study, our goal was to characterize and compare the patterns of miRNA expression in TNBC of AA (n = 27) and NHW women (n = 30). A total of 256 miRNAs were differentially expressed between these groups, and distinct from the ones observed in their respective non-TNBC subtypes. Fifty-five of these miRNAs were mapped in cytobands carrying copy number alterations (CNAs); 26 of them presented expression levels concordant with the observed CNAs. Receiving operating characteristic (ROC) analysis showed a good power (AUC ≥ 0.80; 95% CI) for over 65% of the individual miRNAs and a high combined power with superior sensitivity and specificity (AUC = 0.88 (0.78-0.99); 95% CI) of the 26 miRNA panel in discriminating TNBC between these populations. Subsequent miRNA target analysis revealed their involvement in the interconnected PI3K/AKT, MAPK and insulin signaling pathways. Additionally, three miRNAs of this panel were associated with early age at diagnosis. Altogether, these findings indicated that there are different patterns of miRNA expression between TNBC of AA and NHW women and that their mapping in genomic regions with high levels of CNAs is not merely physical, but biologically relevant to the TNBC phenotype. Once validated in distinct cohorts of AA women, this panel can potentially represent their intrinsic TNBC genome signature.

Comparative Proteomics of Tumor and Paired Normal Breast Tissue Highlights Potential Biomarkers in Breast Cancer.

BACKGROUND/AIM: Breast cancer is the most common type of cancer among women worldwide, and about 57,000 new cases are expected for the Brazilian population in 2015. Elucidation of protein expression and modification is essential for the biological understanding, early diagnosis and therapeutics of breast cancer. The main objectives of the study are comparison between the proteome of tumor and paired non-tumor breast cancer tissues, describing all identified proteins, highlighting the ones most differentially expressed and comparing the data with existing literature. MATERIALS AND METHODS: The five paired samples from patients with invasive ductal carcinoma were analyzed by 2-DE and MS. RESULTS: We collected 161 identified spots corresponding to 110 distinct proteins. Forty-three differentially-expressed spots were common to at least two samples, and the ten proteins with the highest-fold changes were CASPE, ENOG, TPM1, CAPG, VIME, TPM3, TRFE, PDIA6, WDR61 and PDIA3. Metabolic enzymes and proteins with binding functions were the most representative functional classes of proteins with increased and decreased expression in tumor tissue respectively. CONCLUSION: Taking the fold change as a parameter, we point to future targets to be studied by functional methods in a search for biomarkers for initiation and progress of breast cancer.

Prevalence of an inherited cancer predisposition syndrome associated with the germ line TP53 R337H mutation in Paraguay.

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer, and the germline TP53 R337H mutation is the most common mutation reported to date. However, this mutation is associated with a lower cumulative lifetime cancer risk than other mutations in the p53 DNA-binding domain. A detailed statistical analysis of 171,500 DNA tests in Brazilian neonates found that 0.27% of the general population is positive for this mutation, and some of the estimated 200,000 Brazilian R337H carriers in southern and southeastern Brazil have already developed cancer. The present study was designed to estimate R337H prevalence in neighboring Paraguay. To address this question, 10,000 dried blood samples stored in Guthrie cards since 2008 were randomly selected from the Paraguayan municipalities located at the border with Brazil. These samples were tested for R337H mutation using the PCR-restriction fragment length polymorphism assay. This germline mutation was detected in five samples (5/10,000), indicating that the total number of R337H carriers in Paraguay may be as high as 3500. Previous studies have shown that other countries (i.e., Portugal, Spain, and Germany) presented one family with this mutation, leading us to conclude that, besides Brazil and Paraguay, other countries may have multiple families carrying this mutation, which is an inherited syndrome that is difficult to control.

Two different karyotypes with 1q abnormalities in a patient with Fanconi anemia.

We report a case of Fanconi anemia in which cytogenetic analysis of bone marrow (BM) samples revealed two distinct karyotypes: 46,XY,dup(1)(q21q42), in the first sample and 46,XY,del(1)(q32) in the second, aspirated 7 months later after acute myeloid leukemia (AML) developed. We discuss the cytogenetic clonal fluctuation common in Fanconi anemia, with the Fanconi's anemia (FA) reports available in the literature. Interestingly, we have identified that del(1)(q32) has not been reported before in FA.

Lack of DNA copy number alterations revealed with comparative genomic hybridization in fibroadenomas of the breast.

There are very few studies reporting DNA copy number changes in fibroadenomas of the breast. Using comparative genomic hybridization, we analyzed 20 paraffin-embedded samples of fibroadenomas of the breast from patients with no familial or previous history of breast cancer. No alterations in the DNA copy number were observed in any of the tumors analyzed, regardless of the chromosomal alterations observed using conventional cytogenetic analysis. We discuss our results and compare them to other reports on fibroadenomas.

Cytogenetic findings in phyllodes tumor and fibroadenomas of the breast.

The cytogenetic data on fibroadenomas and cystosarcoma phyllodes tumor of the breast, which are both biphasic breast tumors composed of epithelial and stromal components, are quite limited. In this study, we report on clonal chromosomal alterations in three fibroadenomas and one cystosarcoma phyllodes analyzed by GTG banding. The fibroadenomas presented mostly numerical abnormalities involving chromosomes 16, 18, and 21. One case presented a deletion on 17p. The cystosarcoma phyllodes presented numerous numerical abnormalities, mostly chromosome gains, and several marker chromosomes.

Genetic and epigenetic alterations in sentinel lymph nodes metastatic lesions compared to their corresponding primary breast tumors.

The accumulation of genetic and epigenetic changes plays a pivotal role in tumor development and progression. In this study, we investigated these changes using comparative genomic hybridization and bisulfite polymerase chain reaction analysis for CpG island hypermethylation of the following genes: TP16, THBS2, E-Cadherin (ECAD), RARbeta2, MINT1, MINT2, and MINT31 in six paired primary breast tumors and their matched sentinel lymph nodes (SLN). The most frequent chromosomal alterations observed were the following: losses of 6q13 approximately q23 and 13q13 approximately q32 and gains of 9q31 approximately qter, 11p15 approximately q21, 12q23 approximately qter, and 20q12 approximately qter. Gain of 6p21 approximately pter was observed in the SLN but in none of the primary tumors. Overall, 71% (30/42) of the methylation measurements were identical between the primary tumors and the SLN. Of the six cases, two showed no differences between the primary tumors and SLN, one tumor with 4 of 7 genes hypermethylated in the primary tumor showed loss of all four hypermethylation events in the SLN, and the remaining three tumors showed loss of one methylation event and simultaneous gain of one to two methylation changes in the SLN. This is the first study reporting genetic and epigenetic alterations in breast sentinel lymph nodes compared to their corresponding primary tumors. Characterization of such alterations may lead to identification of initial events associated with the metastatic dissemination process.