Methylation signature of lymph node metastases in breast cancer patients.

BACKGROUND: Invasion and metastasis are two important hallmarks of malignant tumors caused by complex genetic and epigenetic alterations. The present study investigated the contribution of aberrant methylation profiles of cancer related genes, APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14 (ARF), P16 (CDKN2A), P21 (CDKN1A), PTEN, and TIMP3, in the matched axillary lymph node metastasis in comparison to the primary tumor tissue and the adjacent normal tissue from the same breast cancer patients to identify the potential of candidate genes methylation as metastatic markers. METHODS: The quantitative methylation analysis was performed using the SEQUENOM's EpiTYPER? assay which relies on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The quantitative DNA methylation analysis of the candidate genes showed higher methylation proportion in the primary tumor tissue than that of the matched normal tissue and the differences were significant for the APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 promoter regions (P<0.05). Among those candidate methylated genes, APC, BMP6, BRCA1 and P16 displayed higher methylation proportion in the matched lymph node metastasis than that found in the normal tissue (P<0.05). The pathway analysis revealed that BMP6, BRCA1 and P16 have a role in prevention of neoplasm metastasis. CONCLUSIONS: The results of the present study showed methylation heterogeneity between primary tumors and metastatic lesion. The contribution of aberrant methylation alterations of BMP6, BRCA1 and P16 genes in lymph node metastasis might provide a further clue to establish useful biomarkers for screening metastasis.

Interpreting Breast International Group (BIG) 1-98: a randomized, double-blind, phase III trial comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with hormone receptor-positive, early breast cancer.

The Breast International Group (BIG) 1-98 study is a four-arm trial comparing 5 years of monotherapy with tamoxifen or with letrozole or with sequences of 2 years of one followed by 3 years of the other for postmenopausal women with endocrine-responsive early invasive breast cancer. From 1998 to 2003, BIG -98 enrolled 8,010 women. The enhanced design f the trial enabled two complementary analyses of efficacy and safety. Collection of tumor specimens further enabled treatment comparisons based on tumor biology. Reports of BIG 1-98 should be interpreted in relation to each individual patient as she weighs the costs and benefits of available treatments. Clinicaltrials.gov ID: NCT00004205.

Concurrent or Sequential Adjuvant Letrozole and Radiotherapy after Conservative Surgery for Early-stage Breast Cancer: Long-term Results of the Cohort Phase 2 Randomized Trial

Purpose/Objective(s): Letrozole radiosensitizes breast cancer cells in vitro. In clinical settings, no data exist for the combination of letrozole and radiotherapy. We assessed concurrent and sequential radiotherapy and letrozole in the adjuvant setting.Materials/Methods: The present study is registered with ClinicalTrials.gov, number NCT00208273. This Phase 2 randomized trial was undertaken in two centers in France and one in Switzerland between January 12, 2005, and February 21, 2007. One hundred fifty postmenopausal women with early-stage breast cancer were randomly assigned after conserving surgery to either concurrent radiotherapy and letrozole (n = 75) or sequential radiotherapy and letrozole (n = 75). Randomization was open label with a minimization technique, stratified by investigational centers, chemotherapy (yes vs. no), radiation boost (yes vs. no), and value of radiation-induced lymphocyte apoptosis (#16% vs. .16%). The whole breast was irradiated to a total dose of 50 Gy in 25 fractions over 5 weeks. In the case of supraclavicular and internal mammary node irradiation, the dose was 44 - 50 Gy. Letrozole was administered orally once daily at a dose of 2 - 5 mg for 5 years (beginning 3 weeks pre-radiotherapy in the concomitant group, and 3 weeks postradiotherapy in the sequential group). The primary endpoint was the occurrence of acute (during and within 6 weeks of radiotherapy) and late (within 2 years) radiation-induced Grade 2 or worse toxic effects of the skin and lung (functional pulmonary test and lung CT-scan). Analyses were by intention-to-treat. The long-term follow-up after 2 years was only performed in Montpellier (n = 121) and evaluated skin toxicity (clinical examination every 6 months), lung fibrosis (one CT-scan yearly), cosmetic outcome.Results: All patients were analyzed apart from 1 in the concurrent group who withdrew consent before any treatment.Within the first 2 years (n = 149), no lung toxicity was identified by CT scan and no modification from baseline was noted by the lung diffusion capacity test. Two patients in each group had Grade 2 or worse late effects (both radiation-induced subcutaneous fibrosis [RISF]). After 2 years (n = 121), and with a median follow-up of 50 months (38-62), 2 patients (1 in each arm) presented a Grade 3 RISF. No lung toxicity was identified by CT scan. Cosmetic results (photographies) and quality of life was good to excellent. All patients who had Grade 3 subcutaneous fibrosis had an RILA value of 16% or less, irrespective of the sequence with letrozole.Conclusions:With long-term follow-up, letrozole can be safely delivered shortly after surgery and concomitantly with radiotherapy.

A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer.

To better understand the relationship between tumor-host interactions and the efficacy of chemotherapy, we have developed an analytical approach to quantify several biological processes observed in gene expression data sets. We tested the approach on tumor biopsies from individuals with estrogen receptor-negative breast cancer treated with chemotherapy. We report that increased stromal gene expression predicts resistance to preoperative chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC) in subjects in the EORTC 10994/BIG 00-01 trial. The predictive value of the stromal signature was successfully validated in two independent cohorts of subjects who received chemotherapy but not in an untreated control group, indicating that the signature is predictive rather than prognostic. The genes in the signature are expressed in reactive stroma, according to reanalysis of data from microdissected breast tumor samples. These findings identify a previously undescribed resistance mechanism to FEC treatment and suggest that antistromal agents may offer new ways to overcome resistance to chemotherapy.

Sindrome metabolica e rischio di tumore mammario. [Metabolic syndrome and the risk of breast cancer].

Metabolic syndrome has been associated with an increased risk of various cancers. A multicenter study conducted in Italy and Switzerland on 3,869 cases of breast cancer in post-menopause reported a relative risk of 1.75 in women with the metabolic syndrome, confirming the results of other smaller epidemiological studies.

Premenopausal serum androgens and breast cancer risk: a nested case-control study.

ABSTRACT: INTRODUCTION: Prospective epidemiologic studies have consistently shown that levels of circulating androgens in postmenopausal women are positively associated with breast cancer risk. However, data in premenopausal women are limited. METHODS: A case-control study nested within the New York University Women's Health Study was conducted. A total of 356 cases (276 invasive and 80 in situ) and 683 individually-matched controls were included. Matching variables included age and date, phase, and day of menstrual cycle at blood donation. Testosterone, androstenedione, dehydroandrosterone sulfate (DHEAS) and sex hormone-binding globulin (SHBG) were measured using direct immunoassays. Free testosterone was calculated. RESULTS: Premenopausal serum testosterone and free testosterone concentrations were positively associated with breast cancer risk. In models adjusted for known risk factors of breast cancer, the odds ratios for increasing quintiles of testosterone were 1.0 (reference), 1.5 (95% confidence interval (CI), 0.9 to 2.3), 1.2 (95% CI, 0.7 to 1.9), 1.4 (95% CI, 0.9 to 2.3) and 1.8 (95% CI, 1.1 to 2.9; Ptrend = 0.04), and for free testosterone were 1.0 (reference), 1.2 (95% CI, 0.7 to 1.8), 1.5 (95% CI, 0.9 to 2.3), 1.5 (95% CI, 0.9 to 2.3), and 1.8 (95% CI, 1.1 to 2.8, Ptrend = 0.01). A marginally significant positive association was observed with androstenedione (P = 0.07), but no association with DHEAS or SHBG. Results were consistent in analyses stratified by tumor type (invasive, in situ), estrogen receptor status, age at blood donation, and menopausal status at diagnosis. Intra-class correlation coefficients for samples collected from 0.8 to 5.3 years apart (median 2 years) in 138 cases and 268 controls were greater than 0.7 for all biomarkers except for androstenedione (0.57 in controls). CONCLUSIONS: Premenopausal concentrations of testosterone and free testosterone are associated with breast cancer risk. Testosterone and free testosterone measurements are also highly reliable (that is, a single measurement is reflective of a woman's average level over time). Results from other prospective studies are consistent with our results. The impact of including testosterone or free testosterone in breast cancer risk prediction models for women between the ages of 40 and 50 years should be assessed. Improving risk prediction models for this age group could help decision making regarding both screening and chemoprevention of breast cancer.

Can resistance to trastuzumab be reversed by endocrine therapy? Combination of trastuzumab and letrozole after resistance to sequential trastuzumab and aromatase inhibitor monotherapies in patients with ER-positive, HER-2 positive, advanced breast cancer: a proof-of-concept trial (SAKK 23/03)

Introduction: Trastuzumab (T) is a cornerstone in the treatment of patients with HER2-overexpressing advanced breast cancer and development of resistance to T is a major therapeutic problem. HER-2 is part of a highly interactive signaling network that may impair efficacy of endocrine therapy. A sequential treatment design was chosen in this trial to ensure complete resistance to single agent therapy before receiving both a non-steroidal aromatase inhibitor (AI) and T. Any kind of clinical activity with combined treatment of AI and T after progression of single agent treatments could indicate restoration of sensitivity as a consequence of cross-talking and networking between both pathways. Methods: Key eligibility criteria included postmenopausal patients (pts.) with advanced, measurable, HER-2 positive (assessed by FISH, ratio (≥2)), HR positive disease and progression on prior treatment with a non-steroidal AI, e.g. letrozole or anastrozole, either in an adjuvant or advanced setting. Pts. received standard dose T monotherapy either weekly or three-weekly in step 1 and upon disease progression, continued T in combination with letrozole in step 2. The primary endpoint was clinical benefit response (CBR: CR, PR or SD for at least 24 weeks (+/- 1 week) according to RECIST) in step 2. Results: Thirteen pts. were enrolled in five centers in Switzerland. In step 1, six pts. (46%) achieved CBR. Median time to progression (TTP) was 161 days (Range: 50 - 627). Based on data collected until the end of May 2010, CBR was observed in seven out of the eleven evaluable pts. (64%) in step 2, including one pt. with partial response. Four of the seven pts. within step 2 that achieved CBR also had CBR in step 1. Seven out of eleven pts. have documented tumor progression during step 2 treatment. Median TTP for all eleven pts. was 184 days (range 61 - 471). Mean time on study treatment (TTP in step 1 plus TTP in step 2) for pts. reaching step 2 was 380 days (range 174 - 864). Adverse events were generally mild. Conclusion: Results of this proof-of-principle trial suggest that complete resistance to both AI and T can be overcome in a proportion of pts. by combined treatment of AI and T, as all pts. served as their own control. Our results appear promising for a new treatment strategy which offers a chemotherapy-free and well-tolerated option for at least a subset of the pts. with HR positive, HER-2 positive breast cancer. Further trials will need to corroborate this finding.

Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.

Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2's tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2's function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair.

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.

Breast cancer incidence and overdiagnosis in Catalonia (Spain)

Introduction: Early detection of breast cancer (BC) with mammography may cause overdiagnosis and overtreatment, detecting tumors which would remain undiagnosed during a lifetime. The aims of this study were: first, to model invasive BC incidence trends in Catalonia (Spain) taking into account reproductive and screening data; and second, to quantify the extent of BC overdiagnosis. Methods: We modeled the incidence of invasive BC using a Poisson regression model. Explanatory variables were: age at diagnosis and cohort characteristics (completed fertility rate, percentage of women that use mammography at age 50, and year of birth). This model also was used to estimate the background incidence in the absence of screening. We used a probabilistic model to estimate the expected BC incidence if women in the population used mammography as reported in health surveys. The difference between the observed and expected cumulative incidences provided an estimate of overdiagnosis. Results: Incidence of invasive BC increased, especially in cohorts born from 1940 to 1955. The biggest increase was observed in these cohorts between the ages of 50 to 65 years, where the final BC incidence rates more than doubled the initial ones. Dissemination of mammography was significantly associated with BC incidence and overdiagnosis. Our estimates of overdiagnosis ranged from 0.4% to 46.6%, for women born around 1935 and 1950, respectively. Conclusions: Our results support the existence of overdiagnosis in Catalonia attributed to mammography usage, and the limited malignant potential of some tumors may play an important role. Women should be better informed about this risk. Research should be oriented towards personalized screening and risk assessment tools.

Competing risks to breast cancer mortality in Catalonia

Background: Breast cancer mortality has experienced important changes over the last century. Breast cancer occurs in the presence of other competing risks which can influence breast cancer incidence and mortality trends. The aim of the present work is: 1) to assess the impact of breast cancer deaths among mortality from all causes in Catalonia (Spain), by age and birth cohort and 2) to estimate the risk of death from other causes than breast cancer, one of the inputs needed to model breast cancer mortality reduction due to screening or therapeutic interventions. Methods: The multi-decrement life table methodology was used. First, all-cause mortality probabilities were obtained by age and cohort. Then mortality probability for breast cancer was subtracted from the all-cause mortality probabilities to obtain cohort life tables for causes other than breast cancer. These life tables, on one hand, provide an estimate of the risk of dying from competing risks, and on the other hand, permit to assess the impact of breast cancer deaths on all-cause mortality using the ratio of the probability of death for causes other than breast cancer by the all-cause probability of death. Results: There was an increasing impact of breast cancer on mortality in the first part of the 20th century, with a peak for cohorts born in 1945–54 in the 40–49 age groups (for which approximately 24% of mortality was due to breast cancer). Even though for cohorts born after 1955 there was only information for women under 50, it is also important to note that the impact of breast cancer on all-cause mortality decreased for those cohorts. Conclusion: We have quantified the effect of removing breast cancer mortality in different age groups and birth cohorts. Our results are consistent with US findings. We also have obtained an estimate of the risk of dying from competing-causes mortality, which will be used in the assessment of the effect of mammography screening on breast cancer mortality in Catalonia.

Estimation of age- and stage-specific Catalan breast cancer survival functions using US and Catalan survival data

Background: During the last part of the 1990s the chance of surviving breast cancer increased. Changes in survival functions reflect a mixture of effects. Both, the introduction of adjuvant treatments and early screening with mammography played a role in the decline in mortality. Evaluating the contribution of these interventions using mathematical models requires survival functions before and after their introduction. Furthermore, required survival functions may be different by age groups and are related to disease stage at diagnosis. Sometimes detailed information is not available, as was the case for the region of Catalonia (Spain). Then one may derive the functions using information from other geographical areas. This work presents the methodology used to estimate age- and stage-specific Catalan breast cancer survival functions from scarce Catalan survival data by adapting the age- and stage-specific US functions. Methods: Cubic splines were used to smooth data and obtain continuous hazard rate functions. After, we fitted a Poisson model to derive hazard ratios. The model included time as a covariate. Then the hazard ratios were applied to US survival functions detailed by age and stage to obtain Catalan estimations. Results: We started estimating the hazard ratios for Catalonia versus the USA before and after the introduction of screening. The hazard ratios were then multiplied by the age- and stage-specific breast cancer hazard rates from the USA to obtain the Catalan hazard rates. We also compared breast cancer survival in Catalonia and the USA in two time periods, before cancer control interventions (USA 1975–79, Catalonia 1980–89) and after (USA and Catalonia 1990–2001). Survival in Catalonia in the 1980–89 period was worse than in the USA during 1975–79, but the differences disappeared in 1990–2001. Conclusion: Our results suggest that access to better treatments and quality of care contributed to large improvements in survival in Catalonia. On the other hand, we obtained detailed breast cancer survival functions that will be used for modeling the effect of screening and adjuvant treatments in Catalonia.