Genomic-transcriptomic evolution in lung cancer and metastasis.

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy1. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study2,3. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis.

Neoantigen-directed immune escape in lung cancer evolution.

The interplay between an evolving cancer and a dynamic immune microenvironment remains unclear. Here we analyse 258 regions from 88 early-stage, untreated non-small-cell lung cancers using RNA sequencing and histopathology-assessed tumour-infiltrating lymphocyte estimates. Immune infiltration varied both between and within tumours, with different mechanisms of neoantigen presentation dysfunction enriched in distinct immune microenvironments. Sparsely infiltrated tumours exhibited a waning of neoantigen editing during tumour evolution, indicative of historical immune editing, or copy-number loss of previously clonal neoantigens. Immune-infiltrated tumour regions exhibited ongoing immunoediting, with either loss of heterozygosity in human leukocyte antigens or depletion of expressed neoantigens. We identified promoter hypermethylation of genes that contain neoantigenic mutations as an epigenetic mechanism of immunoediting. Our results suggest that the immune microenvironment exerts a strong selection pressure in early-stage, untreated non-small-cell lung cancers that produces multiple routes to immune evasion, which are clinically relevant and forecast poor disease-free survival.

Quantification of tumour evolution and heterogeneity via Bayesian epiallele detection.

BACKGROUND: Epigenetic heterogeneity within a tumour can play an important role in tumour evolution and the emergence of resistance to treatment. It is increasingly recognised that the study of DNA methylation (DNAm) patterns along the genome - so-called 'epialleles' - offers greater insight into epigenetic dynamics than conventional analyses which examine DNAm marks individually. RESULTS: We have developed a Bayesian model to infer which epialleles are present in multiple regions of the same tumour. We apply our method to reduced representation bisulfite sequencing (RRBS) data from multiple regions of one lung cancer tumour and a matched normal sample. The model borrows information from all tumour regions to leverage greater statistical power. The total number of epialleles, the epiallele DNAm patterns, and a noise hyperparameter are all automatically inferred from the data. Uncertainty as to which epiallele an observed sequencing read originated from is explicitly incorporated by marginalising over the appropriate posterior densities. The degree to which tumour samples are contaminated with normal tissue can be estimated and corrected for. By tracing the distribution of epialleles throughout the tumour we can infer the phylogenetic history of the tumour, identify epialleles that differ between normal and cancer tissue, and define a measure of global epigenetic disorder. CONCLUSIONS: Detection and comparison of epialleles within multiple tumour regions enables phylogenetic analyses, identification of differentially expressed epialleles, and provides a measure of epigenetic heterogeneity. R code is available at github.com/james-e-barrett.

Tumor response and patient outcome after preoperative radiotherapy in locally advanced non-inflammatory breast cancer patients.

PURPOSE: The purpose of this analysis was to assess the tumor response and long-term outcome in patients treated with preoperative radiotherapy (PRT) without systemic therapy. METHODS: Between 1997 and 2000, 134 patients with non-inflammatory locally advanced breast cancer (LABC) were treated with PRT. The tumor dose was 45 Gy in 15 fractions to the breast and to regional lymph nodes over 6 weeks. Radical mastectomy was performed 6 weeks after PRT to all patients and adjuvant systemic therapy was administered as per protocol. The measures of disease outcome were overall survival (OS) and disease-free survival (DFS) which estimated using the Kaplan-Meier method. RESULTS: Median follow-up was 74 months (range 4-216). Objective clinical tumor response after PRT was observed in 77.6% of the patients. Clinical complete tumor response (cCR) was achieved in 21.6% of the patients. Pathological CR in the breast was achieved in 15% of the patients. The 5- and 10-year OS were 55.1 and 37.8%, respectively. The 5- and 10-year DFS were 39.2 and 27%, respectively. Patients who achieved cCR had significantly longer OS in comparison with patients achieving clinical partial response (cPR) and clinical stable disease (cSD). Similarly, DFS of patients in the cCR group was longer compared with patients with cPR and cSD, yet without statistical significance. CONCLUSIONS: Our results showed that local control in LABC patients achieved by primary PRT, followed by radical mastectomy was comparable with the results reported in the literature. Complete pathologic response to PRT identified a subgroup of patients with a trend toward better DFS and OS.

Pharmacogenetics in cancer therapy - 8 years of experience at the Institute for Oncology and Radiology of Serbia.

PURPOSE: Pharmacogenetics is a study of possible mechanism by which an individual's response to drugs is genetically determined by variations in their DNA sequence. The aim of pharmacogenetics is to identify the optimal drug and dose for each individual based on their genetic constitution, i.e. to individualize drug treatment. This leads to achieving the maximal therapeutic response for each patient, while reducing adverse side effects of therapy and the cost of treatment. A centralized pharmacogenetics service was formed at the Institute for Oncology and Radiology of Serbia (IORS) with the aim to provide a personalized approach to cancer treatment of Serbian patients. METHODS: Analyses of KRAS mutations in metastatic colorectal cancer, EGFR mutations in advanced non-small cell lung cancer, CYP2D6 polymorphism in breast cancer, DPD polymorphism in colorectal cancer and MTHFR polymorphism in osteosarcoma have been performed by real time polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Mutation testing analyses were successful for 1694 KRAS samples and 1821 EGFR samples, while polymorphism testing was successful for 9 CYP2D6 samples, 65 DPD samples and 35 MTHFR samples. CONCLUSIONS: Pharmacogenetic methods presented in this paper provide cancer patients in Serbia the best possible choice of treatment at the moment.

MicroRNA expression signatures for the prediction of BRCA1/2 mutation-associated hereditary breast cancer in paraffin-embedded formalin-fixed breast tumors.

Screening for germline mutations in breast cancer-associated genes BRCA1 and BRCA2 is indicated for patients with breast cancer from high-risk breast cancer families and influences both treatment options and clinical management. However, only 25% of selected patients test positive for BRCA1/2 mutation, indicating that additional diagnostic biomarkers are necessary. We analyzed 124 formalin-fixed paraffin-embedded (FFPE) tumor samples from patients with hereditary (104) and sporadic (20) invasive breast cancer, divided into two series (A and B). Microarray expression profiling of 829 human miRNAs was performed on 76 samples (Series A), and bioinformatics tool Prophet was used to develop and test a microarray classifier. Samples were stratified into a training set (n = 38) for microarray classifier generation and a test set (n = 38) for signature validation. A 35-miRNA microarray classifier was generated for the prediction of BRCA1/2 mutation status with a reported 95% (95% CI = 0.88-1.0) and 92% (95% CI: 0.84-1.0) accuracy in the training and the test set, respectively. Differential expression of 12 miRNAs between BRCA1/2 mutation carriers versus noncarriers was validated by qPCR in an independent tumor series B (n = 48). Logistic regression model based on the expression of six miRNAs (miR-142-3p, miR-505*, miR-1248, miR-181a-2*, miR-25* and miR-340*) discriminated between tumors from BRCA1/2 mutation carriers and noncarriers with 92% (95% CI: 0.84-0.99) accuracy. In conclusion, we identified miRNA expression signatures predictive of BRCA1/2 mutation status in routinely available FFPE breast tumor samples, which may be useful to complement current patient selection criteria for gene testing by identifying individuals with high likelihood of being BRCA1/2 mutation carriers.

Short telomeres are frequent in hereditary breast tumors and are associated with high tumor grade.

Telomere shortening is a common event involved in malignant transformation. Critically short telomeres may trigger chromosomal aberrations and produce genomic instability leading to cancer development. Therefore, telomere shortening is a frequent molecular alteration in early stages of many epithelial tumors and in breast cancer correlates with stage and prognosis. A better understanding of the involvement of short telomeres in tumors may have a significant impact on patient management and the design of more specific treatments. To understand the role of telomere length (TL) in breast cancer etiology we measured the length of individual telomere signals in single cells by using quantitative telomere in situ hybridization in paraffin-embedded tissue from hereditary and sporadic breast cancers. A total of 104 tumor tissue samples from 75 familial breast tumors (BRCA1, n = 14; BRCA2, n = 13; non-BRCA1/2, n = 48) and 29 sporadic tumors were analyzed. Assessment of telomere signal intensity allowed estimation of the mean TL and related variables, such as percentage of critically short telomeres and percentage of cells with short telomeres. These data were correlated with the immunohistochemical expression of molecular breast cancer markers. Hereditary BRCA1, BRCA2, and non-BRCA1/2 tumors were characterized by shorter TL comparing to sporadic tumors. Considering all tumors, tumor grade was a strong risk factor determining the proportion of short telomeres or short telomere cells. Moreover, some histopathological features appeared to be differentially associated to hereditary or sporadic subgroups. Short telomeres correlated with ER-negative tumors in sporadic cases but not in familial cases, whereas a high level of apoptosis was associated with shorter telomeres in hereditary BRCA1 and BRCA2 tumors. In addition, TL helped to define a subset of non-BRCA1/2 tumors with short telomeres associated with increased expression of antiapoptotic proteins. These findings highlight the potential interest of TL measurements as markers of aggressiveness in breast cancer.

EGFR mutation testing from pleural effusions of non-small cell lung cancer patients at the institute for oncology and radiology of Serbia.

BACKGROUND: The use tumor-derived cell-free DNA extracted from body fluids is being evaluated for genetic testing in lung cancer. The aim of this study was to explore the feasibility and utility of implementation of EGFR molecular testing from pleural effusions in non-small cell lung cancer in the clinical diagnostics workflow. PATIENTS AND METHODS: This study included patients diagnosed with primary lung adenocarcinoma in the period July 2016 to June 2023. EGFR mutation testing was performed by qPCR (Cobas®) and dPCR. Testing was performed from 211 plasma samples when tissue was unavailable at diagnosis, and from 301 plasma samples and 18 pleural effusions at progression on first/second generation of EGFR TKIs. Descriptive methods of statistical analysis were used to summarize the sample data. Fisher's exact test, McNemar's test, Cohen's kappa tests were used for statistical analyses. Two-sided p-values <0.05 were considered statistically significant. RESULTS: A significantly higher detection rate of the T790M mutation in pleural effusion was obtained compared to blood (50% and 20%, p=0.047). When comparing the detection success rate of the resistant T790M mutation in pleural effusion and blood, a statistically significant difference was obtained in favor of pleural effusion (50% vs. 21.87%, p=0.01). CONCLUSIONS: Superior performance of pleural effusions compared to blood plasma was shown both in the analysis of success rate and in the detection of the resistant T790M mutation, at progression on EGFR TKIs. Pleural effusion should be considered in this setting whenever available, especially in countries with limited health resources.

Nischarin expression may have differing roles in male and female melanoma patients.

Due to the development of resistance to previously effective therapies, there is a constant need for novel treatment modalities for metastatic melanoma. Nischarin (NISCH) is a druggable scaffolding protein reported as a tumor suppressor and a positive prognostic marker in breast and ovarian cancers through regulation of cancer cell survival, motility and invasion. The aim of this study was to examine the expression and potential role of nischarin in melanoma. We found that nischarin expression was decreased in melanoma tissues compared to the uninvolved skin, and this was attributed to the presence of microdeletions and hyper-methylation of the NISCH promoter in the tumor tissue. In addition to the previously reported cytoplasmic and membranous localization, we observed nischarin in the nuclei in melanoma patients' tissues. NISCH expression in primary melanoma had favorable prognostic value for female patients, but, unexpectedly, high NISCH expression predicted worse prognosis for males. Gene set enrichment analysis suggested significant sex-related disparities in predicted association of NISCH with several signaling pathways, as well as with different tumor immune infiltrate composition in male and female patients. Taken together, our results imply that nischarin may have a role in melanoma progression, but that fine-tuning of the pathways it regulates is sex-dependent. KEY MESSAGES: Nischarin is a tumor suppressor whose role has not been investigated in melanoma. Nischarin expression was downregulated in melanoma tissue compared to the normal skin. Nischarin had the opposite prognostic value in male and female melanoma patients. Nischarin association with signaling pathways differed in females and males. Our findings challenge the current view of nischarin as a universal tumor suppressor.

Prediction of Cervical Lymph Node Metastasis in Clinically Node-Negative T1 and T2 Papillary Thyroid Carcinoma Using Supervised Machine Learning Approach.

Papillary thyroid carcinoma (PTC) is generally considered an indolent cancer. However, patients with cervical lymph node metastasis (LNM) have a higher risk of local recurrence. This study evaluated and compared four machine learning (ML)-based classifiers to predict the presence of cervical LNM in clinically node-negative (cN0) T1 and T2 PTC patients. The algorithm was developed using clinicopathological data from 288 patients who underwent total thyroidectomy and prophylactic central neck dissection, with sentinel lymph node biopsy performed to identify lateral LNM. The final ML classifier was selected based on the highest specificity and the lowest degree of overfitting while maintaining a sensitivity of 95%. Among the models evaluated, the k-Nearest Neighbor (k-NN) classifier was found to be the best fit, with an area under the receiver operating characteristic curve of 0.72, and sensitivity, specificity, positive and negative predictive values, F1 and F2 scores of 98%, 27%, 56%, 93%, 72%, and 85%, respectively. A web application based on a sensitivity-optimized kNN classifier was also created to predict the potential of cervical LNM, allowing users to explore and potentially build upon the model. These findings suggest that ML can improve the prediction of LNM in cN0 T1 and T2 PTC patients, thereby aiding in individual treatment planning.

Integration of BRCA1-mediated miRNA and mRNA profiles reveals microRNA regulation of TRAF2 and NFκB pathway.

Microarray-based techniques are being useful to obtain miRNA and gene expression signatures associated with different tumors. BRCA1 deregulation is a frequent event in the pathogenesis of breast as well as other cancers. In addition to DNA repair functions of BRCA1, it is involved in a wide range of cellular processes such as cell cycle, chromatin remodeling or transcription. However, the molecular events underlying BRCA1-associated tumorigenesis are still largely unknown. In order to deepen our understanding of BRCA1-associated tumorigenesis, we integrated data from mRNA and miRNA microarray experiments on HCC1937 breast cancer cell line, and the isogenic HCC1937 stably expressing BRCA1, to obtain significant miRNA-mRNA relationships associated with the presence of BRCA1 gene. By using bioinformatic integration of gene and miRNA expression data, we found significant miRNA-gene relationships underlying the array signatures. We additionally evaluated the role of these statistically significant pairs at the biological pathways level and identified MAPK and NF-κB pathways associated with these expression changes. Furthermore, we experimentally validated miRNAs induced by BRCA1 that commonly regulate TRAF2, a key regulator of NF-κB and MAPK pathways. We demonstrate that miR-146a, miR-99b and miR-205, induced in HCC1937 BRCA1-expressing cells, bind and regulate TRAF2 gene. In addition, re-expression of miR-146a, miR-99b or miR-205 in HCC1937 BRCA1-null cells was sufficient to modulate NF-κB activity. Our results demonstrate that integration of mRNA and miRNA associated with BRCA1 expression was useful to discover new miRNA-gene interactions as molecular events underlying BRCA1-mediated tumorigenesis.

Comparison and imputation-aided integration of five commercial platforms for targeted DNA methylome analysis.

Targeted bisulfite sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. In this study, we benchmarked five commercially available TBS platforms-three hybridization capture-based (Agilent, Roche and Illumina) and two reduced-representation-based (Diagenode and NuGen)-across 11 samples. Two samples were also compared with whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. Although all platforms produced robust and reproducible data, major differences in the number and identity of the CpG sites covered make it difficult to compare datasets generated on different platforms. To overcome this limitation, we applied imputation and show that it improves interoperability from an average of 10.35% (0.8 million) to 97% (7.6 million) common CpG sites. Our study provides guidance on which TBS platform to use for different methylome features and offers an imputation-based harmonization solution that allows comparative, integrative analysis.

EGFR mutation testing from liquid biopsy of non-small cell lung cancer at the Institute for Oncology and Radiology of Serbia.

PURPOSE: Resistance to tyrosine kinase inhibitors (TKIs) in lung cancer often occurs, so mutation testing from liquid biopsy is the method of choice as a minimally invasive approach that quickly provides information for additional therapeutic options. The purpose of this study was to assess the success rate and usefulness of EGFR testing from liquid biopsy at the Institute for Oncology and Radiology of Serbia (IORS). METHODS: EGFR mutation testing was performed by real-time qPCR in 4750 tumor samples using the Cobas® EGFR Mutation Test v2. EGFR testing from 104 liquid biopsy samples was used to track the resistance on first-line EGFR-TKIs as well as for initial testing of 124 patients without tissue biopsies. RESULTS: Liquid biopsy samples were tested in cases with inadequate material for DNA isolation or without tissue biopsy at diagnosis. Nine mutated samples were detected (7.3 %) with a 99.2 % testing success rate. Testing liquid biopsy samples of patients who progressed on EGFR-TKIs showed an accordance rate of 67% with driver mutations, and 49% of mutated patients had the T790M mutation which rendered them eligible for third-generation EGFR-TKIs. An additional 5 patients tested EGFR wild type from plasma after progression were rebiopsied and 3 of them had the T790M mutation. CONCLUSIONS: EGFR mutation testing from liquid biopsy has been successfully implemented in Serbia and has proven invaluable for detecting molecular resistance mechanisms to EGFR-TKIs and as an alternative sample source for patients with scarce biopsy material or without any at all.

Molecular signature of response to preoperative radiotherapy in locally advanced breast cancer.

BACKGROUND: Radiation therapy is an indispensable part of various treatment modalities for breast cancer. Specifically, for non-inflammatory locally advanced breast cancer (LABC) patients, preoperative radiotherapy (pRT) is currently indicated as a second line therapy in the event of lack of response to neoadjuvant chemotherapy. Still approximately one third of patients fails to respond favourably to pRT. The aim of this study was to explore molecular mechanisms underlying differential response to radiotherapy (RT) to identify predictive biomarkers and potential targets for increasing radiosensitivity. METHODS: The study was based on a cohort of 134 LABC patients, treated at the Institute of Oncology and Radiology of Serbia (IORS) with pRT, without previous or concomitant systemic therapy. Baseline transcriptional profiles were established using Agilent 60 K microarray platform in a subset of 23 formalin-fixed paraffin-embedded (FFPE) LABC tumour samples of which 11 radiotherapy naïve and 3 post-radiotherapy samples passed quality control and were used for downstream analysis. Biological networks and signalling pathways underlying differential response to RT were identified using Ingenuity Pathways Analysis software. Predictive value of candidate genes in the preoperative setting was further validated by qRT-PCR in an independent subset of 60 LABC samples of which 42 had sufficient quality for data analysis, and in postoperative setting using microarray data from 344 node-negative breast cancer patients (Erasmus cohort, GSE2034 and GSE5327) treated either with surgery only (20%) or surgery with RT (80%). RESULTS: We identified 192 significantly differentially expressed genes (FDR < 0.10) between pRT-responsive and non-responsive tumours, related to regulation of cellular development, growth and proliferation, cell cycle control of chromosomal replication, glucose metabolism and NAD biosynthesis II route. APOA1, MAP3K4, and MMP14 genes were differentially expressed (FDR < 0.20) between pRT responders and non-responders in preoperative setting, while MAP3K4 was further validated as RT-specific predictive biomarker of distant metastasis free survival (HR = 2.54, [95%CI:1.42-4.55], p = 0.002) in the postoperative setting. CONCLUSIONS: This study pinpoints MAP3K4 as a putative biomarker of response to RT in both preoperative and postoperative settings and a potential target for radiosensitising combination therapy, warranting further pre-clinical studies and prospective clinical validation.

Epigenome-wide association studies for cancer biomarker discovery in circulating cell-free DNA: technical advances and challenges.

Since introducing the concept of epigenome-wide association studies (EWAS) in 2011, there has been a vast increase in the number of published EWAS studies in common diseases, including in cancer. These studies have increased our understanding of epigenetic events underlying carcinogenesis and have enabled the discovery of cancer-specific methylation biomarkers. In this mini-review, we have focused on the state of the art in EWAS applied to cell-free circulating DNA for epigenetic biomarker discovery in cancer and discussed associated technical advances and challenges, and our expectations for the future of the field.

Cell cycle, apoptosis, cellular uptake and whole-transcriptome microarray gene expression analysis of HeLa cells treated with a ruthenium(II)-arene complex with an isoquinoline-3-carboxylic acid ligand.

Ruthenium(II)-arene complexes are promising drug candidates for the therapy of solid tumors. In previous work, seven new compounds of the general formula [Ru(η6-p-cymene)(L1-7)Cl] were synthesized and characterized, of which the complex with L=isoquinoline-3-carboxylic acid (RuT7) was two times as active on HeLa cells compared to normal cell line MRC-5, as indicated by IC50 values determined after 48h of incubation (45.4±3.0 vs. 84.2±5.7µM, respectively). In the present study, cell cycle analysis of HeLa cells treated with RuT7 showed S phase arrest and an increase in sub-G1 population. The apoptotic potential of the title compound was confirmed with the Annexin V-FITC/PI assay together with a morphological evaluation of cells using fluorescent microscopy. Analysis of the intracellular accumulation of ruthenium showed 8.9ng Ru/106 cells after 6h of incubation. To gain further insight in the molecular mechanism of action of RuT7 on HeLa cells, a whole-transcriptome microarray gene expression analysis was performed. Analysis of functional categories and signaling and biochemical pathways associated with the response of HeLa cells to treatment with RuT7 showed that it leads the cells through the intrinsic (mitochondrial) apoptotic pathway, via indirect DNA damage due to the action of reactive oxygen species, and through direct DNA binding of RuT7. Statistical analysis for enrichment of gene sets associated with known drug-induced toxicities identified fewer associated toxicity profiles in RuT7-treated cells compared to cisplatin treatment. Altogether these results provide the basis for further development of RuT7 in animal and pre-clinical studies as a potential drug candidate.

miRNA expression profiling of formalin-fixed paraffin-embedded (FFPE) hereditary breast tumors.

Hereditary breast cancer constitutes only 5-10% of all breast cancer cases and is characterized by strong family history of breast and/or other associated cancer types. Only ~ 25% of hereditary breast cancer cases carry a mutation in BRCA1 or BRCA2 gene, while mutations in other rare high and moderate-risk genes and common low penetrance variants may account for additional 20% of the cases. Thus the majority of cases are still unaccounted for and designated as BRCAX tumors. MicroRNAs are small non-coding RNAs that play important roles as regulators of gene expression and are deregulated in cancer. To characterize hereditary breast tumors based on their miRNA expression profiles we performed global microarray miRNA expression profiling on a retrospective cohort of 80 FFPE breast tissues, including 66 hereditary breast tumors (13 BRCA1, 10 BRCA2 and 43 BRCAX), 10 sporadic breast carcinomas and 4 normal breast tissues, using Exiqon miRCURY LNA™ microRNA Array v.11.0. Here we describe in detail the miRNA microarray expression data and tumor samples used for the study of BRCAX tumor heterogeneity (Tanic et al., 2013) and biomarkers associated with positive BRCA1/2 mutation status (Tanic et al., 2014). Additionally, we provide the R code for data preprocessing and quality control.

Deregulated miRNAs in hereditary breast cancer revealed a role for miR-30c in regulating KRAS oncogene.

Aberrant miRNA expression has been previously established in breast cancer and has clinical relevance. However, no studies so far have defined miRNAs deregulated in hereditary breast tumors. In this study we investigated the role of miRNAs in hereditary breast tumors comparing with normal breast tissue. Global miRNA expression profiling using Exiqon microarrays was performed on 22 hereditary breast tumors and 15 non-tumoral breast tissues. We identified 19 miRNAs differentially expressed, most of them down-regulated in tumors. An important proportion of deregulated miRNAs in hereditary tumors were previously identified commonly deregulated in sporadic breast tumors. Under-expression of these miRNAs was validated by qRT-PCR in additional 18 sporadic breast tumors and their normal breast tissue counterparts. Pathway enrichment analysis revealed that deregulated miRNAs collectively targeted a number of genes belonging to signaling pathways such as MAPK, ErbB, mTOR, and those regulating cell motility or adhesion. In silico prediction detected KRAS oncogene as target of several deregulated miRNAs. In particular, we experimentally validated KRAS as a miR-30c target. Luciferase assays confirmed that miR-30c binds the 3'UTR of KRAS transcripts and expression of pre-miR-30c down-regulated KRAS mRNA and protein. Furthermore, miR-30c overexpression inhibited proliferation of breast cancer cells. Our results identify miRNAs associated to hereditary breast cancer, as well as miRNAs commonly miss-expressed in hereditary and sporadic tumors, suggesting common underlying mechanisms of tumor progression. In addition, we provide evidence that KRAS is a target of miR-30c, and that this miRNA suppresses breast cancer cell growth potentially through inhibition of KRAS signaling.