Correction to: A systematic comparison of copy number alterations in four types of female cancer.

After publication of the original article [1] the authors found that the article contained an incorrect version of Fig. 4. This does not affect the results and conclusions of the article.

A systematic comparison of copy number alterations in four types of female cancer.

BACKGROUND: Detection and localization of genomic alterations and breakpoints are crucial in cancer research. The purpose of this study was to investigate, in a methodological and biological perspective, different female, hormone-dependent cancers to identify common and diverse DNA aberrations, genes, and pathways. METHODS: In this work, we analyzed tissue samples from patients with breast (n = 112), ovarian (n = 74), endometrial (n = 84), or cervical (n = 76) cancer. To identify genomic aberrations, the Circular Binary Segmentation (CBS) and Piecewise Constant Fitting (PCF) algorithms were used and segmentation thresholds optimized. The Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm was applied to the segmented data to identify significantly altered regions and the associated genes were analyzed by Ingenuity Pathway Analysis (IPA) to detect over-represented pathways and functions within the identified gene sets. RESULTS AND DISCUSSION: Analyses of high-resolution copy number alterations in four different female cancer types are presented. For appropriately adjusted segmentation parameters the two segmentation algorithms CBS and PCF performed similarly. We identified one region at 8q24.3 with focal aberrations that was altered at significant frequency across all four cancer types. Considering both, broad regions and focal peaks, three additional regions with gains at significant frequency were revealed at 1p21.1, 8p22, and 13q21.33, respectively. Several of these events involve known cancer-related genes, like PPP2R2A, PSCA, PTP4A3, and PTK2. In the female reproductive system (ovarian, endometrial, and cervix [OEC]), we discovered three common events: copy number gains at 5p15.33 and 15q11.2, further a copy number loss at 8p21.2. Interestingly, as many as 75% of the aberrations (75% amplifications and 86% deletions) identified by GISTIC were specific for just one cancer type and represented distinct molecular pathways. CONCLUSIONS: Our results disclose that some prominent copy number changes are shared in the four examined female, hormone-dependent cancer whereas others are definitive to specific cancer types.

Integrated analysis of high-resolution DNA methylation profiles, gene expression, germline genotypes and clinical end points in breast cancer patients.

Breast cancer is a heterogeneous disease for which alterations in DNA methylation patterns have been shown to be of biological and clinical importance. Here we report on the integrated analysis of molecular alterations including the methylation status of 27 gene promoters analyzed by highly quantitative pyrosequencing, and the association to gene expression, germline genotype and clinical parameters including survival. Breast cancer specific deregulation of DNA methylation (both hyper- and hypomethylation) was found in twenty genes including ACVR1, OGG1, IL8 and TFF1. The methylation level in the promoter regions was significantly negatively correlated to gene expression for twelve genes (such as MST1R, ST6GAL1 and TFF1) indicating that a gain of aberrant methylation (hypermethylation) inhibits gene expression. Multiple associations between molecular and clinical parameters were identified, and multivariate statistical analysis demonstrated that methylation was more strongly associated to clinical parameters than gene expression for the investigated genes. The methylation level of BCAP31 and OGG1 showed significant association to survival, and these associations were validated in a larger patient cohort (The Cancer Genome Atlas). Our study provides evidence for the promise of DNA methylation alterations for clinical applications.

Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy.

Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation (n = 20) and after receiving 10-24 Gray (Gy) (n = 19). DNA methylation analysis was performed by using Illumina Infinium 27K arrays. Fourteen genes were selected for technical validation by pyrosequencing. Eighty-two differentially methylated genes were identified in irradiated (n = 11) versus nonirradiated (n = 19) samples (false discovery rate, FDR = 1.1%). Methylation levels in pathways belonging to the immune system were most altered after RT. Based on methylation levels before irradiation, a panel of five genes (H2AFY, CTSA, LTC4S, IL5RA and RB1) were significantly associated with clinical response (p = 0.041). Furthermore, the degree of methylation changes for 2,516 probes correlated with the given radiation dose. Within the 2,516 probes, an enrichment for pathways involved in cellular immune response, proliferation and apoptosis was identified (FDR < 5%). Here, we observed clear differences in methylation levels induced by radiation, some associated with response to treatment. Our study adds knowledge on the molecular mechanisms behind radiation response.

Genome-wide association study in breast cancer survivors reveals SNPs associated with gene expression of genes belonging to MHC class I and II.

INTRODUCTION: We investigated the effect of genetic variation on gene expression in blood from a cohort of BC survivors. Further, we investigated the associations that were specific for BC survivors by performing identical analyses for a group of healthy women and comparing the results. METHODS: eQTL analysis was performed for 288 BC survivors (full data set). Further, using a subset of the data, eQTL analyses were performed on 288 BC survivors and on 81 healthy women separately and results were compared. RESULTS: A large number of associations were observed for the BC survivors, and the expression of human leukocyte antigen genes was found associated with SNPs in 100 genes. The comparison analyses with healthy women revealed associations occurring specifically in BC survivors, and the genes showed enrichment for immune system processes. CONCLUSIONS: The results suggest that the immune system has a different constitution in BC survivors compared to healthy women.

Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors.

BACKGROUND: Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. METHODS: Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. RESULTS: Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. CONCLUSIONS: In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above.

Detection of frequent ABCB1 polymorphisms by high-resolution melting curve analysis and their effect on breast carcinoma prognosis.

BACKGROUND: The ABCB1 gene encodes P-glycoprotein implicated in the development of cellular drug resistance. The aim of this study was to develop high-resolution melting (HRM) analysis for determination of ABCB1 polymorphisms and evaluate their associations with clinical data of breast carcinoma patients. METHODS: HRM analysis was designed to assess five single nucleotide polymorphisms (SNPs) in ABCB1 (rs2214102, rs1128503, rs2032582, rs2032583 and rs1045642) in genomic DNA from 103 breast carcinoma patients. Results were confirmed by direct DNA sequencing. RESULTS: HRM analysis revealed distinct patterns of melting curves for the respective genotypes of all followed SNPs. Sensitivity of HRM analysis compared with direct DNA sequencing was superior (97.1% vs. 93.9%). The overall accuracy of HRM was 97.6%. The coefficients of variation in replicate experiments encompassed the range 0.002%-0.038%. On the basis of the examined SNPs, one strong haplotype block containing rs2032582 and rs1128503 SNPs was identified. Significant associations of rs2032582 SNP with tumor size, negative HER-2/neu status, and family history of breast carcinoma were found. Patients carrying the ancestral homozygous genotype (GG) in rs2214102 had significantly worse progression-free survival in comparison with carriers of the non-ancestral allele (A) in the adjuvant set (p=0.005). CONCLUSIONS: A rapid, accurate, low-cost and time-effective method for screening ABCB1 SNPs was developed. Significant associations of ABCB1 rs2032582 and rs2214102 SNPs with prognostic factors and survival of patients were found.

Fatigued breast cancer survivors and gene polymorphisms in the inflammatory pathway.

Chronic fatigue (CF) in breast cancer survivors (BCSs) has been associated with increased serum C-reactive protein-levels (CRP), pro-inflammatory cytokines and cytokine gene single nucleotide polymorphisms (SNPs). Still, there are few studies on these topics, and due to small study-cohorts the possibility to adjust for other conditions related to inflammatory processes, e.g. depression, has been limited. In 302 BCSs, examined approximately four years after treatment for breast cancer stage II/III, data on high sensitivity (hs)CRP, leukocytes and mRNA interleukin (IL)1ß and IL6R expression, depression and chronic fatigue were available. Three years thereafter, 236 BCSs were re-examined. The associations between fatigue and SNPs in inflammation-related genes; IL1ß (rs16944), IL6 (rs1800795), IL6receptor (rs4129267, rs4845617, rs2228145), CRP (rs2794521, rs3091244) were investigated, together with the relations between SNPs in IL6R,IL1ß and CRP genes and mRNA blood expression levels of IL6R and IL1ß and serum hsCRP-levels, respectively. All analyses were repeated after exclusion of depressed individuals and separating BCSs with persistent fatigue from never-fatigued individuals. Even after exclusion of depressed individuals neither the SNPs nor the mRNA IL1ß and IL6R expression levels were associated with chronic or persistent fatigue. In the subset of persistent fatigued and never-fatigued individuals the CRP SNP (rs3091244) was associated with hsCRP level (p=0.02). IL1ß and IL6R mRNA expression levels were not related to the IL1ß and IL6R genotypes. In a large cohort of BCSs the investigated SNPs in inflammation-related genes were not associated with fatigue, though subset analyses indicated an association between the CRP SNP (rs3091244) and serum hsCRP.

NOTCH2 in breast cancer: association of SNP rs11249433 with gene expression in ER-positive breast tumors without TP53 mutations.

BACKGROUND: A recent genome-wide association study (GWAS) has identified a single nucleotide polymorphism (SNP) rs11249433 in the 1p11.2 region as a novel genetic risk factor for breast cancer, and this association was stronger in patients with estrogen receptor (ER)+ versus ER- cancer. RESULTS: We found association between SNP rs11249433 and expression of the NOTCH2 gene located in the 1p11.2 region. Examined in 180 breast tumors, the expression of NOTCH2 was found to be lowest in tumors with TP53 mutations and highest in TP53 wild-type/ER+ tumors (p = 0.0059). In the latter group, the NOTCH2 expression was particularly increased in carriers of the risk genotypes (AG/GG) of rs11249433 when compared to the non-risk AA genotype (p = 0.0062). Similar association between NOTCH2 expression and rs11249433 was observed in 60 samples of purified monocytes from healthy controls (p = 0.015), but not in total blood samples from 302 breast cancer patients and 76 normal breast tissue samples. We also identified the first possible dominant-negative form of NOTCH2, a truncated version of NOTCH2 consisting of only the extracellular domain. CONCLUSION: This is the first study to show that the expression of NOTCH2 differs in subgroups of breast tumors and by genotypes of the breast cancer-associated SNP rs11249433. The NOTCH pathway has key functions in stem cell differentiation of ER+ luminal cells in the breast. Therefore, increased expression of NOTCH2 in carriers of rs11249433 may promote development of ER+ luminal tumors. Further studies are needed to investigate possible mechanisms of regulation of NOTCH2 expression by rs11249433 and the role of NOTCH2 splicing forms in breast cancer development.

Linkage disequilibrium between the CYP2C19*17 allele and wildtype CYP2C8 and CYP2C9 alleles: identification of CYP2C haplotypes in healthy Nordic populations.

PURPOSE: To determine the distribution of clinically important CYP2C genotypes and allele frequencies in healthy Nordic populations with special focus on linkage disequilibrium. METHODS: A total of 896 healthy subjects from three Nordic populations (Danish, Faroese, and Norwegian) were genotyped for five frequent and clinically important CYP2C allelic variants: the defective CYP2C8*3, CYP2C9*2, CYP2C9*3, and CYP2C19*2 alleles, and the CYP2C19*17 allele that causes rapid drug metabolism. Linkage disequilibrium was evaluated and CYP2C haplotypes were inferred in the entire population. RESULTS: Ten CYP2C haplotypes were inferred, the most frequent of which (49%) was the CYP2C wildtype haplotype carrying CYP2C8*1, CYP2C9*1, and CYP2C19*1. The second most frequent haplotype (19%) is composed of CYP2C19*17, CYP2C8*1, and CYP2C9*1. This predicted haplotype accounts for 99.7% of the CYP2C19*17 alleles found in the 896 subjects. CONCLUSION: CYP2C19*17 is a frequent genetic variant in Nordic populations that exists in strong linkage disequilibrium with wildtype CYP2C8*1 and CYP2C9*1 alleles, which effectively makes it a determinant for a haplotype exhibiting an efficient CYP2C substrate metabolism.

Germline glutathione S-transferase variants in breast cancer: relation to diagnosis and cutaneous long-term adverse effects after two fractionation patterns of radiotherapy.

PURPOSE: To explore whether certain glutathione S-transferase (GST) polymorphisms are associated with an increased risk of breast cancer or the level of radiation-induced adverse effects after two fractionation patterns of adjuvant radiotherapy. METHODS AND MATERIALS: The prevalence of germline polymorphic variants in GSTM1, GSTP1, and GSTT1 was determined in 272 breast cancer patients and compared with that in a control group of 270 women from the general population with no known history of breast cancer. The genetic variants were determined using multiplex polymerase chain reaction followed by restriction enzyme fragment analysis. In 253 of the patients surveyed for radiotherapy-induced side effects after a median observation time of 13.7 years (range, 7-22.8 years), the genotypes were related to the long-term effects observed after two fractionation patterns (treatment A, 4.3 Gy in 10 fractions for 156 patients; and treatment B, 2.5 Gy in 20 fractions for 97; both administered within a 5-week period). RESULTS: None of the GST polymorphisms conferred an increased risk of breast cancer, either alone or in combination. Compared with treatment B, treatment A was followed by an increased level of moderate to severe radiation-induced side effects for all the endpoints studied (i.e., degree of telangiectasia, subcutaneous fibrosis and atrophy, lung fibrosis, costal fractures, and pleural thickening; p <0.001 for all endpoints). A significant association was found between the level of pleural thickening and the GSTP1 Ile105Val variant. CONCLUSION: The results of this study have illustrated the impact of hypofractionation on the level of adverse effects and indicated that the specific alleles of GSTP1, M1, and T1 studied here may be significant in determining the level of adverse effects after radiotherapy.

Fatigue During and After Breast Cancer Therapy-A Prospective Study.

CONTEXT: Chronic fatigue (CF) in breast cancer (BC) survivors is multifactorial and may be caused by immune activation triggered by BC or its treatment. In the Neoadjuvant Avastin in Breast Cancer study, BC patients received neoadjuvant chemotherapy (FEC100→taxane) ± bevacizumab, a monoclonal antibody with fatigue as a potential side effect. OBJECTIVES: To examine fatigue levels and prevalence of CF before and during chemotherapy and at follow-up, and their associations with C-reactive protein (CRP) and clinical variables. METHODS: Eighty-four HER2-negative patients with cT2-4N0-3M0 BC responded to questionnaires and had CRP measured before treatment (T0), after FEC100 (T1), after taxanes before surgery (T2), and at two-year follow-up (T3). RESULTS: The prevalence of CF increased from 8% at T0 to 36% at T3, P < 0.0001. Fatigue levels peaked during chemotherapy from 12.0 at T0 to 20.0 at T2, and declined to 16.7 at T3, P < 0.001. Women with CF at T3 had higher fatigue levels at T0, T2, and T3 than those without CF (P ≤ 0.01). Psychological distress (P = 0.03) and pain (P = 0.04) at T3 were associated with CF at T3. Only psychological distress remained a significant predictor in multivariate analysis. CRP increased from T0 to T1 (P < 0.01) and declined to baseline values at T3, but changes were not associated with bevacizumab treatment. No association was found between bevacizumab or CRP, and fatigue levels or CF. CONCLUSION: Neither bevacizumab treatment nor low-grade systemic inflammation as measured by CRP was associated with the increased fatigue levels and raised prevalence of CF, observed during and after BC therapy. Increased fatigue levels at baseline and psychological distress at T3 were associated with CF at T3.

Analysis of SNP-expression association matrices.

High throughput expression profiling and genotyping technologies provide the means to study the genetic determinants of population variation in gene expression variation. In this paper we present a general statistical framework for the simultaneous analysis of gene expression data and SNP genotype data measured for the same cohort. The framework consists of methods to associate transcripts with SNPs affecting their expression, algorithms to detect subsets of transcripts that share significantly many associations with a subset of SNPs, and methods to visualize the identified relations. We apply our framework to SNP-expression data collected from 50 breast cancer patients. Our results demonstrate an overabundance of transcript-SNP associations in this data, and pinpoint SNPs that are potential master regulators of transcription. We also identify several statistically significant transcript-subsets with common putative regulators that fall into well-defined functional categories.

ATM variants and cancer risk in breast cancer patients from Southern Finland.

BACKGROUND: Individuals heterozygous for germline ATM mutations have been reported to have an increased risk for breast cancer but the role for ATM genetic variants for breast cancer risk has remained unclear. Recently, a common ATM variant, ATMivs38 -8T>C in cis with the ATMex39 5557G>A (D1853N) variant, was suggested to associate with bilateral breast cancer among familial breast cancer patients from Northern Finland. We have here evaluated the 5557G>A and ivs38-8T>C variants in an extensive case-control association analysis. We also aimed to investigate whether there are other ATM mutations or variants contributing to breast cancer risk in our population. METHODS: Two common ATM variants, 5557G>A and ivs38-8T>C, previously suggested to associate with bilateral breast cancer, were genotyped in an extensive set of 786 familial and 884 unselected breast cancer cases as well as 708 healthy controls. We also screened the entire coding region and exon-intron boundaries of the ATM gene in 47 familial breast cancer patients and constructed haplotypes of the patients. The identified variants were also evaluated for increased breast cancer risk among additional breast cancer cases and controls. RESULTS: Neither of the two common variants, 5557G>A and ivs38-8T>C, nor any haplotype containing them, was significantly associated with breast cancer risk, bilateral breast cancer or multiple primary cancers in any of the patient groups or subgoups. Three rare missense alterations and one intronic change were each found in only one patient of over 250 familial patients studied and not among controls. The fourth missense alteration studied further was found with closely similar frequencies in over 600 familial cases and controls. CONCLUSION: Altogether, our results suggest very minor effect, if any, of ATM genetic variants on familial breast cancer in Southern Finland. Our results do not support association of the 5557G>A or ivs38-8T>C variant with increased breast cancer risk or with bilateral breast cancer.

Genome-wide DNA methylation analyses in lung adenocarcinomas: Association with EGFR, KRAS and TP53 mutation status, gene expression and prognosis.

BACKGROUND: DNA methylation alterations are early events in tumorigenesis and important in the regulation of gene expression in cancer cells. Lung cancer patients have in general a poor prognosis, and a deeper insight into the epigenetic landscape in lung adenocarcinoma tumors and its prognostic implications is needed. RESULTS: We determined whole-genome DNA methylation profiles of 164 fresh frozen lung adenocarcinoma samples and 19 samples of matched normal lung tissue using the Illumina Infinium 450K array. A large number of differentially methylated CpGs in lung adenocarcinoma tissue were identified, and specific methylation profiles were observed in tumors with mutations in the EGFR-, KRAS- or TP53 genes and according to the patients' smoking status. The methylation levels were correlated with gene expression and both positive and negative correlations were seen. Methylation profiles of the tumor samples identified subtypes of tumors with distinct prognosis, including one subtype enriched for TP53 mutant tumors. A prognostic index based on the methylation levels of 33 CpGs was established, and was significantly associated with prognosis in the univariate analysis using an independent cohort of lung adenocarcinoma patients from The Cancer Genome Atlas project. CpGs in the HOX B and HOX C gene clusters were represented in the prognostic signature. CONCLUSIONS: Methylation differences mirror biologically important features in the etiology of lung adenocarcinomas and influence prognosis.

Interaction between p53 mutation and a somatic HDMX biomarker better defines metastatic potential in breast cancer.

TP53 gene mutation is associated with poor prognosis in breast cancer, but additional biomarkers that can further refine the impact of the p53 pathway are needed to achieve clinical utility. In this study, we evaluated a role for the HDMX-S/FL ratio as one such biomarker, based on its association with other suppressor mutations that confer worse prognosis in sarcomas, another type of cancer that is surveilled by p53. We found that HDMX-S/FL ratio interacted with p53 mutational status to significantly improve prognostic capability in patients with breast cancer. This biomarker pair offered prognostic utility that was comparable with a microarray-based prognostic assay. Unexpectedly, the utility tracked independently of DNA-damaging treatments and instead with different tumor metastasis potential. Finally, we obtained evidence that this biomarker pair might identify patients who could benefit from anti-HDM2 strategies to impede metastatic progression. Taken together, our work offers a p53 pathway marker, which both refines our understanding of the impact of p53 activity on prognosis and harbors potential utility as a clinical tool.

DNA methylation status of key cell-cycle regulators such as CDKNA2/p16 and CCNA1 correlates with treatment response to doxorubicin and 5-fluorouracil in locally advanced breast tumors.

PURPOSE: To explore alterations in gene promoter methylation as a potential cause of acquired drug resistance to doxorubicin or combined treatment with 5-fluorouracil and mitomycin C in human breast cancers. EXPERIMENTAL DESIGN: Paired tumor samples from locally advanced breast cancer patients treated with doxorubicin and 5-fluorouracil-mitomycin C were used in the genome-wide DNA methylation analysis as discovery cohort. An enlarged cohort from the same two prospective studies as those in the discovery cohort was used as a validation set in pyrosequencing analysis. RESULTS: A total of 469 genes were differentially methylated after treatment with doxorubicin and revealed a significant association with canonical pathways enriched for immune cell response and cell-cycle regulating genes including CDKN2A, CCND2, CCNA1, which were also associated to treatment response. Treatment with FUMI resulted in 343 differentially methylated genes representing canonical pathways such as retinoate biosynthesis, gαi signaling, and LXR/RXR activation. Despite the clearly different genes and pathways involved in the metabolism and therapeutic effect of both drugs, 46 genes were differentially methylated before and after treatment with both doxorubicin and FUMI. DNA methylation profiles in genes such as BRCA1, FOXC1, and IGFBP3, and most notably repetitive elements like ALU and LINE1, were associated with TP53 mutations status. CONCLUSION: We identified and validated key cell-cycle regulators differentially methylated before and after neoadjuvant chemotherapy such as CDKN2A and CCNA1 and reported that methylation patterns of these genes may be potential predictive markers to anthracycline/mitomycine sensitivity.

Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis.

BACKGROUND: Ductal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma. DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development. RESULTS: We generate genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer, and validate methylation changes between normal and DCIS in an independent dataset of 15 normal and 40 DCIS samples. We also validate a prognostic signature on 583 breast cancer samples from The Cancer Genome Atlas. Our analysis reveals that DNA methylation profiles of DCIS are radically altered compared to normal breast tissue, involving more than 5,000 genes. Changes between DCIS and invasive breast carcinoma involve around 1,000 genes. In tumors, DNA methylation is associated with gene expression of almost 3,000 genes, including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs is associated with survival of breast cancer patients with invasive tumors, as well as with survival of patients with DCIS and mixed lesions of DCIS and invasive breast carcinoma. CONCLUSIONS: This work demonstrates that changes in the epigenome occur early in the neoplastic progression, provides evidence for the possible utilization of DNA methylation-based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis.

SNP in TXNRD2 associated with radiation-induced fibrosis: a study of genetic variation in reactive oxygen species metabolism and signaling.

PURPOSE: The aim of the study was to identify noninvasive markers of treatment-induced side effects. Reactive oxygen species (ROS) are generated after irradiation, and genetic variation in genes related to ROS metabolism might influence the level of radiation-induced adverse effects (AEs). METHODS AND MATERIALS: 92 breast cancer (BC) survivors previously treated with hypofractionated radiation therapy were assessed for the AEs subcutaneous atrophy and fibrosis, costal fractures, lung fibrosis, pleural thickening, and telangiectasias (median follow-up time 17.1 years). Single-nucleotide polymorphisms (SNPs) in 203 genes were analyzed for association to AE grade. SNPs associated with subcutaneous fibrosis were validated in an independent BC survivor material (n=283). The influence of the studied genetic variation on messenger ribonucleic acid (mRNA) expression level of 18 genes previously associated with fibrosis was assessed in fibroblast cell lines from BC patients. RESULTS: Subcutaneous fibrosis and atrophy had the highest correlation (r=0.76) of all assessed AEs. The nonsynonymous SNP rs1139793 in TXNRD2 was associated with grade of subcutaneous fibrosis, the reference T-allele being more prevalent in the group experiencing severe levels of fibrosis. This was confirmed in another sample cohort of 283 BC survivors, and rs1139793 was found significantly associated with mRNA expression level of TXNRD2 in blood. Genetic variation in 24 ROS-related genes, including EGFR, CENPE, APEX1, and GSTP1, was associated with mRNA expression of 14 genes previously linked to fibrosis (P≤.005). CONCLUSION: Development of subcutaneous fibrosis can be associated with genetic variation in the mitochondrial enzyme TXNRD2, critically involved in removal of ROS, and maintenance of the intracellular redox balance.

Gefitinib in Combination with Weekly Docetaxel in Patients with Metastatic Breast Cancer Caused Unexpected Toxicity: Results from a Randomized Phase II Clinical Trial.

In patients with metastatic breast cancer, taxane treatment demonstrates activity but is not curative. Targeted treatment modalities are therefore necessary in order to improve outcomes in this group. A randomized placebo-controlled phase II trial was initiated to evaluate effect and toxicity of gefitinib (250 mg QD) and docetaxel 35 mg/m(2) (six of seven weeks) (NCT 00319618). The inclusion of 66 patients was planned. The study was closed due to treatment-related toxicity. Of the 18 included patients, seven (of which three received gefitinib) were withdrawn from the study due to toxicity. Of the nine patients receiving gefitinib and chemotherapy, one achieved a partial response and four stable disease. In the chemotherapy of nine patients, four had a partial response and four stable disease. The breast cancer patients in this study were genotyped using a panel of 14 single-nucleotide polymorphisms (SNPs), previously found associated with docetaxel clearance in a cohort of lung cancer patients. We were unable to identify genes related to toxicity in this study. Nevertheless, toxicity was aggravated by the addition of the tyrosine kinase inhibitor. In conclusion, despite adequately tolerated as monotherapy, combination regimens should be carefully considered for overlapping adverse events in order to avoid increased treatment-related toxicity.