Joaquina Martínez-Galán

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

IntroductionRadiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects.MethodsIntrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments.ResultsRadiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects.ConclusionAfter radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

BackgroundTumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients.MethodsPatients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique).ResultsOur results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively.ConclusionSilencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.

Pancreatic ductal adenocarcinoma: metastatic disease

Abstract The treatment of choice of metastatic PADC is systemic chemotherapy. In the last decade, there have been significant advances in this area. New combination poli-chemotherapy schemes have shown a significant increase in overall survival and progression-free survival without impairing quality of life. In addition, the value of second-line chemotherapy treatment has consolidated and a new concept called “therapeutic sequencing” has also emerged. The aim of this article is to review the different therapeutic options in metastatic PDAC based on patient’s characteristics.

P1-05-06: Hypermethilation 14-3-3- Sigma Promoter as a Biomarker to Screening for Metastasis and Potencial Prognostic Factor in Breast Cancer Patients.

Background: 14-3-3sigma is an epithelial marker whose expression is induced by DNA damage through a p53-dependent pathway. 14-3-3sigma functions sequesters cyclin B1-CDC2 complexes outside the nucleus and thereby contributes to a G2 arrest. Epigenetic silencing by CpG methylation, p53 inactivation, and proteasome-dependent proteolysis leads to loss of 14-3-3sigma and is often observed in precancerous lesions and likely to be causally linked to the onset of cancer Objetive: To correlation methylation levels of promoter 14-3-3 sigma with association prognostic factors in breast cancer and their correlation with phenotype luminal. Material and Methods: This is a prospective study we quantified methylation levels of promoter 14-3-3 sigma gene in 107 women with breast cancer and 108 control subjects by Real Time QMS-PCR SYBR green (methylation-specific PCR) and analyzed association with prognostics factor in breast cancer. Results: Median age was 58 years (32-88); 69% were postmenopausal women. Nodal involvement N0; 63%,N1;30%,N2;7%), tumor size (T1;58%,T2;35%,T3;4%,T4;4%) and grade G1; 20%,G2;37%,G3;30%). Significant differences between breast cancer patients (pts) and healthy controls in relative serum levels of methylated gene promoters 14-3-3s (p=0.0047) were detected. Presence of methylated 14-3-3-s in serum of breast cancer patients was associated with T3-4 stage (OMS) (p 0.05). Conclusions: Hypermethylation of the 14-3-3 sigma promoter is an early and frequent event in breast neoplastic transformation, leading to the suggestion that silencing of 14-3-3 sigma may be an important event in tumor progression and particularly in breast carcinogenesis. This study identifies the presence of variations in global levels of methylation promoters genes in patients breast cancer with different phenotype classes and shows that these differences have clinical significance. Perhaps in the detection of CpG methylation of 14-3-3sigma may be used for diagnostic and prognostic purposes. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-05-06.

Proteomic biomarkers in body fluids associated with pancreatic cancer

Pancreatic cancer (PC) is a highly malignant disease that represents the fourth leading cancer-related death worldwide. There has been very little improvement in survival rates over recent years, and surgical resection remains the only reliable curative approach. Factors that contribute to this dismal prognosis for PC include its rapid progression and invasion, the absence of specific symptoms, and the little impact of available chemotherapy. Importantly, the management of this malignancy is also limited by the lack of highly specific and sensitive biomarkers for its diagnosis and follow-up, and their identification is therefore considered a promising strategy to improve outcomes in these patients. Numerous translational studies have explored the usefulness of body fluids as a non-invasive source of PC-specific biomarkers, and innovations in proteomic methods and technologies have provided a myriad of protein biomarkers for different cancers. The adoption of a proteomic approach has improved understanding of the biology of PC and contributed to the potential identification of protein biomarkers for this disease. This review considers the most recent research efforts to develop novel proteomic biomarkers in body fluids for PC.

From First Line to Sequential Treatment in the Management of Metastatic Pancreatic Cancer

The current management of patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) is based on systemic chemotherapy. The results of the MPACT and PRODIGE clinical trials have demonstrated that the combination of nab-paclitaxel and gemcitabine (GEM) as well as FOLFIRINOX regimen result in improvement in overall survival when compared to GEM alone. Treatment guidelines now recommend either one of these two regimens as first line treatment for fit patients with mPDAC. Because no head-to-head comparison between the two regimens exists, the selection of one versus the other is based on clinical criteria. The design and eligibility criteria of these two clinical trials are dissimilar, making the results of the MPACT trial more applicable to the general population of patients with mPDAC. In addition, the combination of nab-paclitaxel and GEM is better tolerated and easier to administer in clinical practice than FOLFIRINOX. Furthermore, when the regimens are studied in comparable patient populations the efficacy results are very similar. Nanoliposomal irinotecan plus 5FU has recently demonstrated a significant increase in efficacy rates after a GEM-based treatment. Importantly, treatment of mPDAC should now be considered as a continuum care for patients who are fit, with second and even third line treatments. Different sequential treatment algorithms are proposed based on available data. In retrospective studies, patients who were managed with GEM-based regimens followed by fluoropyrimidine-based regimens appear to have the most favorable outcome.

170PMODIFICATION OF METHYLATION LEVEL DNA PROMOTER IN SERUM OF A PANEL OF GENE BEFORE AND AFTER TREATMENT AND IMPACT IN PROGNOSTIC FACTOR IN PATIENTS WITH BREAST CANCER

ABSTRACT Aim: Alterations of genetic and epigenetic feature can provide important insghts into the natural history of breast cancer. Although DNA methylation analysis is a rapidly developing field, a reproducible epigenetic blood-based assay for diagnosis and follow up of breast cancer has yet to be successfully developed into a routine clinical test. The aim of ths study was to review DNA methylation of promoter methylation of 14.3.3 sigma and ESR1 in circulating DNA of breast cancer patients and to highlight the value of those novel biomarkers in prediction of therapeutic outcom Methods: Plasma was sampled prospectively from 110 patients diagnosed of breast cancer. A PCR quantitative technique was used to analyzed the utility of circulating DNA with CpG island hypermethylation of ESR1, 14.3.3 sigma, APC and Rar Beta genes promoter regions as breast cancer biomarkers Results: The cutt-off points for the genes methylated promoters were established from the ROC curves, selecting values that gave the maximal likelihood ratio. Mean serum values of methylated genes before treatment was for ESR1:0.009 microg/ml, 14.3.3 sigma: 0.047 microg/ml, RarBeta: 0.0001 microg/ml and APC: 0.012 microg/ml. Mean values of methylated genes after treatment was for ESR: 0.003 microg/ml, 14.3.3 sigma: 0.038 microg/ml, RarBeta: 0 microg/ml and APC:0.001microg/ml. In the light of the discriminatory power of the ESR1 and 14.3.3 sigma and the finding that serum levels of methylated gene promoter changed after breast cancer treatment, post treatment modifications in these two promoters were analyzed. We observed lower methylated ESR1, 14.3.3 sigma and APC values after surgery respect pretreatment levels, but without an overall statistically significant difference. With a median follow up of 8 years we found that patients with a significant decrease of sera methylated levels of these genes after surgery had better interval free progression and overall survival respect patients without this observation. Conclusions: These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation os specific gene promoters in DNA extracted from serum. Althought numerous issues remain to be resolved the quantitative measurement of circulating methylated DNA is a promising tool for cancer prognostic assessment. Disclosure: All authors have declared no conflicts of interest.

Significance and clinical outcome of lymph node level assessment in biliary tract cancers.

274 Background: Biliary tract cancers (BTCs) are relatively rare neoplasms encompass both cholangiocarcinoma (CC).The role of routine lymphadenectomy at the time of surgical resection remains poorly defined.We sought to identify factors associated with outcome following and examine the impact of lymph node (LN) assessment on survival. Methods: 43 pts who underwent curative intent surgery between 2000-10 were identified from a database.We calculated prognostic factors and impact lymph node assessment for survival. Results: A total 43 pts were identified with no metastatic BTCs.The median age was 65 years (29–82 years); PS0:33/43 (76%); PS1:8/43 (19%) and PS2:2/43 (5%) pts.A histological diagnosis of adenocarcinoma was confirmed in 100%.Surgical resection was performed in all patients.After resection 42% (18/43) had positive nodes.Adjuvant chemotherapy had 31/43(72%),preferred with gemcitabine and a median number of cycles 6. Grade 3 or 4 toxicities rarely occurred.During median follow-up of 6.6 years tumor...

Methylation ESR1 as a potential factor of resistence to HER2/neu therapy in patients with breast cancer.

24 Background: Estrogen receptor (ER)-positive breast cancers are considered prognostically more favorable than ER-negative tumors and are often responsive to anti-estrogen therapy. Whereas tumors overexpressing HER2 are endocrine resistant and they require the blockage of the HER2 pathway in addition to estrogen deprivation. To evaluate epigenetics differences in tumor-related genes to ESR1 and HER2/neu status in primary breast cancer is the objective of this study. METHODS We quantified methylation levels of promoter of ERS1 which are to confer growth adventage to cells in 107 women with breast cancer. Real Time QMS-PCR SYBR green (methylation-specific PCR) was used to analyze the hypermethylation. Tumours were classified as phenotype basal, luminal A, Luminal B, and phenotype HER2+. RESULTS Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p=0.0179). Of the available cases, 60 pts (56%) were Luminal A, 10 pts (9.3%) Luminal B, 13 pts (12%) Basal like, and 9 pts (8.4%) HER2+. We observed that methylated ERS1 was preferably associated with phenotype Basal Like and worse interval progression free and survival global though p > 0.05 and the amplification HER2+ was correlation with significant more frequent methylation gene (p<0.05). Thet hypermethylation of normal ERS1 and 14-3-3σ combined differentiated between breast cancer patients and healthy controls (p = 0.0001) with a sensitivity of 81% (95% CI: 72-88%) and specificity of 88% (95% CI: 78-94%). CONCLUSIONS This study identifies the presence of variations in global levels of methylation promoters genes in healthy controls and breast cancer with different phenotype classes and shows that these differences have clinical significance. These showed that frequent methylation had a strong association with molecular phenotype of breast cancer and perhaps in the future can explain therapy resistance related to RE and HER2/neu status and this may be of significance in the assessment of targeted therapy resistance related to ER and HER2/neu status in breast cancer patients.

Abstract A67: Methylation of ESR1 and 14-3-3 sigma promoter in serum as possible biomarkers for screening and diagnosis for breast cancer

Background: To investigate the association between gene hypermethylation and the major clinico-pathological features of breast cancer, including diagnosis and treatment response. Material and Methods: 107 women with breast cancer and 108 control subjects were recruited. Real Time QMS-PCR SYBR green (methylation-specific PCR) was used to analyze the utility of circulating DNA with CpG island hypermethylation of APC, RAR-β, E-Cadherin, ESR1 and 14-3-3 sigma; gene promoter regions as breast cancer biomarkers. Sera were collected in 107 operable breast cancer patients (pts) previously surgery and in 60 of those pts after treatment. Respect controls, 34 had benign breast disease and 74 with no evidence of breast disease. Results: We found significant differences between breast cancer patients and healthy controls in relative serum levels of methylated gene promoters ESR1 (p = 0.0112) and 14-3-3sigma (p=0.0047). We observed that hypermethylation of these combined two genes differentiated between breast cancer patients and healthy controls (p <0.0001) with a sensitivity of 81% (95% CI: 72–88%) and specificity of 88% (95% CI: 78–94 %). Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p = 0.0179); and presence of methylated 14-3-3-sigma was associated with T3-4 stage (OMS) (p< 0.05) and nodal positive status (p< 0.05). We observed lower methylated ERS1 or 14-3-3-sigma values after surgery, respect pretreatment levels, but without an overall statistically significant difference. With a median follow up of 6 years, we found that patients with a significant decrease of sera methylated levels of both genes after surgery had better time to progression an overall survival respect patients without this observation. Conclusions: this panel of genes detected in ductal lavage and blood specimens could be useful as markers for early detection of breast carcinoma. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA is a promising tool for cancer risk assessment. Note: This abstract was not presented at the conference. Citation Format: Joaquina Martinez-Galan, Blanca Torres-Torres, Juan Ramon Delgado. Methylation of ESR1 and 14-3-3 sigma promoter in serum as possible biomarkers for screening and diagnosis for breast cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A67.