Prognostic significance of BRCA1 and BRCA2 methylation status in circulating cell-free DNA of Pancreatic Cancer patients.

Introduction: Pancreatic cancer is the most fatal cancer type in the world. Its high mortality is mostly correlated to the absence of symptoms and the difficulty in early diagnosis, which in the majority of the cases occurs when the disease has already spread metastasis. Nowadays, tests that could predict early diagnosis are not available yet and the number of prognostic tests is limited. Hence, there is an urgent need for biomarkers capable of detecting early development or the rapid progression of the disease. Patients and Methods: DNA methylation represents the most frequent epigenetic event among tumor suppressor genes that are involved in various carcinogenic pathways. In the recent study we have tried to evaluate, for the first time, the prognostic value of BRCA1 and BRCA2 methylation in the cell-free DNA of pancreatic cancer patients. Using methylation-specific real-time PCR we examined the methylation status of BRCA1 and BRCA2 in 55 patients with operable and 50 patients with metastatic pancreatic cancer. In the operable disease setting, BRCA1 was found to be methylated in 33/55 (63.5%) patients examined while BRCA2 was also highly methylated in 31/55 (56.3%). In the metastatic disease, BRCA1 was found to be methylated in 26/50 (52%) while BRCA2 was found methylated in 23/50 (46%). Results: All control samples were negative for BRCA1 orBRCA2 promoter methylation. Patients with operable pancreatic cancer and a methylated BRCA1 and BRCA2 promoter status had a statistically significant poorer outcome as compared with patients with a non-methylated one (p=0.012 and p=0.001, respectively). Conclusion: In this study plasma methylation of BRCA1 and BRCA2 represents a frequent event in both the operable as well as in the metastatic setting. BRCA1 and BRCA2 methylation was significant and correlated with decreased survival in patients with operable pancreatic cancer. A larger cohort of patients is required to further explore the potential of these findings as well as to investigate whether BRCA1/2 methylation in plasma could serve as a potential prognostic biomarker in pancreatic cancer.

Implementing pharmacogenetic testing in fluoropyrimidine-treated cancer patients: DPYD genotyping to guide chemotherapy dosing in Greece.

Introduction: Dihydropyrimidine dehydrogenase (DPD), encoded by DPYD gene, is the rate-limiting enzyme responsible for fluoropyrimidine (FP) catabolism. DPYD gene variants seriously affect DPD activity and are well validated predictors of FP-associated toxicity. DPYD variants rs3918290, rs55886062, rs67376798, and rs75017182 are currently included in FP genetic-based dosing guidelines and are recommended for genotyping by the European Medicines Agency (EMA) before treatment initiation. In Greece, however, no data exist on DPYD genotyping. The aim of the present study was to analyze prevalence of DPYD rs3918290, rs55886062, rs67376798, rs75017182, and, additionally, rs1801160 variants, and assess their association with FP-induced toxicity in Greek cancer patients. Methods: Study group consisted of 313 FP-treated cancer patients. DPYD genotyping was conducted on QuantStudio ™ 12K Flex Real-Time PCR System (ThermoFisher Scientific) using the TaqMan® assays C__30633851_20 (rs3918290), C__11985548_10 (rs55886062), C__27530948_10 (rs67376798), C_104846637_10 (rs75017182) and C__11372171_10 (rs1801160). Results: Any grade toxicity (1-4) was recorded in 208 patients (66.5%). Out of them, 25 patients (12%) experienced grade 3-4 toxicity. DPYD EMA recommended variants were detected in 9 patients (2.9%), all experiencing toxicity (p = 0.031, 100% specificity). This frequency was found increased in grade 3-4 toxicity cases (12%, p = 0.004, 97.9% specificity). DPYD deficiency increased the odds of grade 3-4 toxicity (OR: 6.493, p = 0.014) and of grade 1-4 gastrointestinal (OR: 13.990, p = 0.014), neurological (OR: 4.134, p = 0.040) and nutrition/metabolism (OR: 4.821, p = 0.035) toxicities. FP dose intensity was significantly reduced in DPYD deficient patients (ß = -0.060, p <0.001). DPYD rs1801160 variant was not associated with FP-induced toxicity or dose intensity. Triple interaction of DPYD*TYMS*MTHFR was associated with grade 3-4 toxicity (OR: 3.725, p = 0.007). Conclusion: Our findings confirm the clinical validity of DPYD reduced function alleles as risk factors for development of FP-associated toxicity in the Greek population. Pre-treatment DPYD genotyping should be implemented in clinical practice and guide FP dosing. DPYD*gene interactions merit further investigation as to their potential to increase the prognostic value of DPYD genotyping and improve safety of FP-based chemotherapy.

Detection of ESR1 Mutations in Primary Tumors and Plasma Cell-Free DNA in High-Grade Serous Ovarian Carcinoma Patients.

ESR1 mutations have been recently associated with resistance to endocrine therapy in metastatic breast cancer and their detection has led to the development and current evaluation of novel, highly promising therapeutic strategies. In ovarian cancer there have been just a few reports on the presence of ESR1 mutations. The aim of our study was to evaluate the frequency and the clinical relevance of ESR1 mutations in high-grade serous ovarian cancer (HGSOC). Drop-off droplet digital PCR (ddPCR) was first used to screen for ESR1 mutations in 60 primary tumors (FFPEs) from HGSOC patients and in 80 plasma cell-free DNA (cfDNA) samples from advanced and metastatic ovarian cancer patients. We further used our recently developed ESR1-NAPA assay to identify individual ESR1 mutations in drop-off ddPCR-positive samples. We report for the first time the presence of ESR1 mutations in 15% of FFPEs and in 13.8% of plasma cfDNA samples from advanced and metastatic ovarian cancer patients. To define the clinical significance of this finding, our results should be further validated in a large and well-defined cohort of ovarian cancer patients.

Hypoxia and anaerobic metabolism relate with immunologically cold breast cancer and poor prognosis.

PURPOSE: Hypoxia-Inducible Factor HIF1α and lactate dehydrogenase LDHA drive anaerobic tumor metabolism and define clinical aggressiveness. We investigated their expression in breast cancer and their role in immune response and prognosis of breast cancer. METHODS: Tissue material from 175 breast cancer patients treated in a prospective study were analyzed with immunohistochemistry for HIF1α and LDH5 expression, in parallel with the tumor-infiltrating lymphocyte TIL-density and tertiary lymphoid structure TLS-density. RESULTS: High LDH5 expression was noted in 48/175 tumors, and this was related to HIF1α overexpression (p < 0.0001), triple-negative TNBC histology (p = 0.01), poor disease-specific survival (p < 0.007), metastasis (p < 0.01), and locoregional recurrence (p = 0.03). High HIF1α expression, noted in 39/175 cases, was linked with low steroid receptor expression (p < 0.05), her2 overexpression (p = 0.01), poor survival (p < 0.04), and high metastasis rates (p < 0.004). High TIL-density in the invading tumor front (TILinv) was linked with low LDH5 and HIF expression (p < 0.0001) and better prognosis (p < 0.02). High TIL-density in inner tumor areas (TILinn) was significantly linked with TNBC. Multivariate analysis showed that PgR-status (p = 0.003, HR 2.99, 95% CI 1.4-6.0), TILinv (p = 0.02, HR 2.31, 95% CI 1.1-4.8), LDH5 (p = 0.01, HR 2.43, 95% CI 1.2-5.0), N-stage (p = 0.04, HR 2.42, 95% CI 1.0-5.8), T-stage (p = 0.04, HR 2.31, 95% CI 1.0-5.1), and her2 status (p = 0.05, HR 2.01, 95% CI 1.0-4.2) were independent variables defining death events. CONCLUSION: Overexpression of LDH5, an event directly related to HIF1α overexpression, characterizes a third of breast tumors, which is more frequent in TNBC. Both HIF1α and LDH5 define cold breast cancer microenvironment and poor prognosis. A rational is provided to study further whether metabolic manipulations targeting HIF and LDH5 may enhance the antitumor immune response in breast cancer.

MTHFR c.665C>T guided fluoropyrimidine therapy in cancer: gender-dependent effect on dose requirements.

OBJECTIVES: The fluoropyrimidine derivatives 5-Fluorouracil and Capecitabine are widely used for the treatment of solid tumors. Fluoropyrimidine metabolism involves a cascade of different enzymes, including MTHFR enzyme. MTHFR c.665C>T polymorphism, leading to decreased MTHFR activity, is a potential pharmacogenomic marker for fluoropyrimidine drug response. The aim of the present study was to analyze the association of MTHFR c.665C>T polymorphism with fluoropyrimidine response in terms of therapy induced adverse events (AEs), requirement of dose reduction and delayed drug administration or therapy discontinuation. METHODS: The study group consisted of 313 fluoropyrimidine-treated cancer patients. PCR-RFLP was used to analyze MTHFR c.665C>T polymorphism. RESULTS: In female patients, MTHFR c.665 CT and TT genotypes were associated with dose reduction (p=0.029). In gender stratification, regression analysis adjusted for age of disease onset, body surface area and AE incidence, showed that MTHFR CT and TT genotypes increased both need for fluoropyrimidine dose reduction (OR 5.050, 95% CI 1.346-18.948, p=0.016) and percentage of dose reduction (ß=3.318, 95% C.I. 1.056-5.580, p=0.004) in female patients. Such differences were not present in male patients. No other associations were found. CONCLUSIONS: MTHFR c.665C>T polymorphism was associated with fluoropyrimidine dose reduction in female cancer patients. This gender*MTHFR interaction merits further investigation.

Safety and Efficacy of RAD001 (Everolimus) Administered Upon Relapse During or After Adjuvant Treatment in Post-menopausal Women With Hormone Receptor Positive, HER2/neu Negative Locally Advanced or Metastatic Breast Cancer (CRAD001JGR08 "MELPOMENI" study).

BACKGROUND/AIM: This study aimed to provide real-world safety and effectiveness data of everolimus (EVE) plus exemestane (EXE) in estrogen receptor positive/human epidermal growth factor receptor 2 negative (ER+/HER2-) advanced breast cancer (aBC). PATIENTS AND METHODS: This prospective observational study was conducted by 19 hospital-based oncologists in Greece. Eligible patients were treated with EVE+EXE in the first-line setting; EVE was initiated according to the approved label. RESULTS: Overall, 75 eligible patients (mean age: 66.9 years; visceral metastases: 49.3%; bone-only metastases: 37.3%) were included in the effectiveness analyses. Over a median (interquartile range) of 12.1 months (range=4.2-20.5 months) of EVE treatment, the median progression-free survival was 18.0 months and the overall response rate was 22.7%. Among patients that received ≥1 EVE dose (n=80), the incidence of EVE-related adverse events was 72.5% (serious: 55.0%); stomatitis (22.5%), fatigue (22.5%), pneumonitis (18.8%); and cough (18.8%) were the most common. CONCLUSION: In the routine care in Greece, EVE demonstrates clinical benefit and a predictable safety profile.

Detection of Hepatocellular Carcinoma in a High-Risk Population by a Mass Spectrometry-Based Test.

Hepatocellular carcinoma (HCC) is one of the fastest growing causes of cancer-related death. Guidelines recommend obtaining a screening ultrasound with or without alpha-fetoprotein (AFP) every 6 months in at-risk adults. AFP as a screening biomarker is plagued by low sensitivity/specificity, prompting interest in discovering alternatives. Mass spectrometry-based techniques are promising in their ability to identify potential biomarkers. This study aimed to use machine learning utilizing spectral data and AFP to create a model for early detection. Serum samples were collected from three separate cohorts, and data were compiled to make Development, Internal Validation, and Independent Validation sets. AFP levels were measured, and Deep MALDI® analysis was used to generate mass spectra. Spectral data were input into the VeriStrat® classification algorithm. Machine learning techniques then classified each sample as "Cancer" or "No Cancer". Sensitivity and specificity of the test were >80% to detect HCC. High specificity of the test was independent of cause and severity of underlying disease. When compared to AFP, there was improved cancer detection for all tumor sizes, especially small lesions. Overall, a machine learning algorithm incorporating mass spectral data and AFP values from serum samples offers a novel approach to diagnose HCC. Given the small sample size of the Independent Validation set, a further independent, prospective study is warranted.

Methylation Status of Corticotropin-Releasing Factor (CRF) Receptor Genes in Colorectal Cancer.

The corticotropin-releasing factor (CRF) system has been strongly associated with gastrointestinal pathophysiology, including colorectal cancer (CRC). We previously showed that altered expression of CRF receptors (CRFRs) in the colon critically affects CRC progression and aggressiveness through regulation of colonic inflammation. Here, we aimed to assess the potential of CRFR methylation levels as putative biomarkers in CRC. In silico methylation analysis of CRF receptor 1 (CRFR1) and CRF receptor 2 (CRFR2) was performed using methylome data derived by CRC and Crohn's disease (CD) tissues and CRC-derived circulating cell-free DNAs (ccfDNAs). In total, 32 and 33 differentially methylated sites of CpGs (DMCs) emerged in CRFR1 and CRFR2, respectively, between healthy and diseased tissues. The methylation patterns were verified in patient-derived ccfDNA samples by qMSP and associated with clinicopathological characteristics. An automated machine learning (AutoML) technology was applied to ccfDNA samples for classification analysis. In silico analysis revealed increased methylation of both CRFRs in CRC tissue and ccfDNA-derived datasets. CRFR1 hypermethylation was also noticed in gene body DMCs of CD patients. CRFR1 hypermethylation was further validated in CRC adjuvant-derived ccfDNA samples, whereas CRFR1 hypomethylation, observed in metastasis-derived ccfDNAs, was correlated to disease aggressiveness and adverse prognostic characteristics. AutoML analysis based on CRFRs methylation status revealed a three-feature high-performing biosignature for CRC diagnosis with an estimated AUC of 0.929. Monitoring of CRFRs methylation-based signature in CRC tissues and ccfDNAs may be of high diagnostic and prognostic significance in CRC.

Gender-dependent association of TYMS-TSER polymorphism with 5-fluorouracil or capecitabine-based chemotherapy toxicity.

Aim:TYMS gene encodes for TS enzyme involved in 5-fluorouracil (5-FU) and capecitabine (CAP) metabolism. This study assessed the association of TYMS-TSER and 3RG>C polymorphisms with 5-FU/CAP adverse event (AE) incidence. Materials & methods:TYMS-TSER and 3RG>C polymorphisms were analyzed by use of PCR/PCR-RFLP in 313 5-FU/CAP-treated cancer patients. Results: Female TYMS-TSER 2R carriers were at increased risk for 5-FU/CAP AEs (odds ratio: 2.195; p = 0.032). 2R/2R genotype was the only factor that increased risk for delayed drug administration or therapy discontinuation (odds ratio: 5.049; p = 0.016). No other associations were found. Conclusion:TYMS-TSER 3R/2R polymorphism was associated with incidence of AEs in female cancer patients. This gender-driven association potentially implicates the ER that, in female patients, potentially regulates TS expression.

Prognostic Role of RASSF1A, SOX17 and Wif-1 Promoter Methylation Status in Cell-Free DNA of Advanced Gastric Cancer Patients.

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A, SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A, SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer.

Prognostic Significance of SLFN11 Methylation in Plasma Cell-Free DNA in Advanced High-Grade Serous Ovarian Cancer.

BACKGROUND: Epigenetic alterations in ctDNA are highly promising as a source of novel potential liquid biopsy biomarkers and comprise a very promising liquid biopsy approach in ovarian cancer, for early diagnosis, prognosis and response to treatment. METHODS: In the present study, we examined the methylation status of six gene promoters (BRCA1, CST6, MGMT, RASSF10, SLFN11 and USP44) in high-grade serous ovarian cancer (HGSOC). We evaluated the prognostic significance of DNA methylation of these six gene promoters in primary tumors (FFPEs) and plasma cfDNA samples from patients with early, advanced and metastatic HGSOC. RESULTS: We report for the first time that the DNA methylation of SLFN11 in plasma cfDNA was significantly correlated with worse PFS (p = 0.045) in advanced stage HGSOC. CONCLUSIONS: Our results strongly indicate that SLFN11 epigenetic inactivation could be a predictor of resistance to platinum drugs in ovarian cancer. Our results should be further validated in studies based on a larger cohort of patients, in order to further explore whether the DNA methylation of SLFN11 promoter could serve as a potential prognostic DNA methylation biomarker and a predictor of resistance to platinum-based chemotherapy in ovarian cancer.

The development of bronchoscopy in China: a national cross-sectional study.

Objective: To investigate the development of bronchoscopy in China and compare it with its application in the early 21st century. Methods: The data collection was based on questionnaires. Three hundred and nineteen hospitals, which distributed across 30 provinces and 130 cities, were included in the study. Data about the application of bronchoscopy in Shanghai and Hunan province in the early 21st century are also involved for comparison. Results: The median period of performing diagnostic and therapeutic bronchoscopy was 19.7±11.0 and 7.4±7.0 years, respectively. On average, about 155.2 cases and 28.4 cases received diagnostic and therapeutic bronchoscopy in each hospital per month. The average area and number of the examination room was 122.7m2 and 2.2m2, respectively. More examination items were performed in specialty hospitals than those in general hospitals (P<0.05) and specialty hospitals owned more rooms exclusively for bronchoscopy (P<0.05), while no difference of the number of allocated doctors was found (P>0.05). On the other side, the whole amount of diagnosis and therapeutic items in teaching hospitals was slightly higher than that in non-teaching hospitals (P<0.01). Comparison of diagnosis and therapeutic endoscopy in Shanghai and Hunan province shows that the number of flexible bronchoscopy increased by 5.8 times in Shanghai from 2002 to 2017, while that increased by 3.4 times in Hunan province from 2005 to 2017. Furthermore, the average number of allocated doctors increased by 0.85 times in Shanghai, which was more rapidly compared with that of Hunan province (0.66 times) (P<0.05). Besides, the development rate of the diagnosis and therapeutic projects in Shanghai was significantly higher than that in Hunan province (P<0.05). Conclusion: All different classes of hospitals in China are capable of carrying out conventional bronchoscopy diagnosis and therapeutic projects, and newly developed bronchoscopy technology has gradually spread in high-level hospitals since 21st century. The higher class the hospital was, the earlier bronchoscopy was performed. Respiratory endoscopy in China has developed rapidly since the early 21st century and the construction of respiratory endoscopy center and the personnel training are on the right track, but it is also faced with inadequate equipment, unbalanced regional development and insufficient personnel allocation.

Circulating cell-free DNA in breast cancer: size profiling, levels, and methylation patterns lead to prognostic and predictive classifiers.

Blood circulating cell-free DNA (ccfDNA) is a suggested biosource of valuable clinical information for cancer, meeting the need for a minimally-invasive advancement in the route of precision medicine. In this paper, we evaluated the prognostic and predictive potential of ccfDNA parameters in early and advanced breast cancer. Groups consisted of 150 and 16 breast cancer patients under adjuvant and neoadjuvant therapy respectively, 34 patients with metastatic disease and 35 healthy volunteers. Direct quantification of ccfDNA in plasma revealed elevated concentrations correlated to the incidence of death, shorter PFS, and non-response to pharmacotherapy in the metastatic but not in the other groups. The methylation status of a panel of cancer-related genes chosen based on previous expression and epigenetic data (KLK10, SOX17, WNT5A, MSH2, GATA3) was assessed by quantitative methylation-specific PCR. All but the GATA3 gene was more frequently methylated in all the patient groups than in healthy individuals (all p < 0.05). The methylation of WNT5A was statistically significantly correlated to greater tumor size and poor prognosis characteristics and in advanced stage disease with shorter OS. In the metastatic group, also SOX17 methylation was significantly correlated to the incidence of death, shorter PFS, and OS. KLK10 methylation was significantly correlated to unfavorable clinicopathological characteristics and relapse, whereas in the adjuvant group to shorter DFI. Methylation of at least 3 or 4 genes was significantly correlated to shorter OS and no pharmacotherapy response, respectively. Classification analysis by a fully automated, machine learning software produced a single-parametric linear model using ccfDNA plasma concentration values, with great discriminating power to predict response to chemotherapy (AUC 0.803, 95% CI [0.606, 1.000]) in the metastatic group. Two more multi-parametric signatures were produced for the metastatic group, predicting survival and disease outcome. Finally, a multiple logistic regression model was constructed, discriminating between patient groups and healthy individuals. Overall, ccfDNA emerged as a highly potent predictive classifier in metastatic breast cancer. Upon prospective clinical evaluation, all the signatures produced could aid accurate prognosis.

Circulating cell-free DNA release in vitro: kinetics, size profiling, and cancer-related gene methylation.

Circulating cell-free DNA (ccfDNA) is a biological entity of great interest due to its potential as liquid biopsy biomaterial carrying clinically valuable information. To better understand its nature, we studied ccfDNA in vitro in two human cancer cell lines MCF-7 and HeLa. Normalized indexes of ccfDNA per cell population decreased over time of culture but were significantly elevated after exposure to IC50 doses of the demethylating/apoptotic agent 5-azacytidine (5-AZA-CR). Fragment-size profiling was indicative of active release, whereas exposure to 5-AZA-CR induced the release of additional shorter fragments, indicative of apoptosis. Finally, the methylation profile of a panel of cancer-specific genes as assessed by quantitative methylation analysis in ccfDNA was identical to the corresponding genomic DNA and followed accurately changes caused by 5-AZA-CR. Overall, our in vitro findings support that ccfDNA can be a reliable biosource of clinically relevant information that can be further studied in these cell culture models.

Tumor molecular profiling of NSCLC patients using next generation sequencing.

Non­small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with a broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using next generation sequencing (NGS) technology, has become a key tool for facilitating treatment decisions and the clinical management of NSCLC patients. The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq™ technology, was evaluated for the analysis of tumor DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissues. Furthermore, the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. The mutation spectrum of the tumors was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. The panel used for tumor DNA analysis in this study exhibited high rates (100%) of sensitivity, specificity and reproducibility at a mutation allelic frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer-driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 23% presented a mutation in a gene associated with approved or emerging targeted therapy. More specifically, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK, ROS1) and 9.4% (47/502) had an alteration in a gene related to emerging targeted therapies (ERBB2, BRAF, MET and RET). Furthermore, 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or indicative for access to a clinical trial. Thus, the targeted NGS panel used in this study is a reliable approach for tumor molecular profiling and can be applied in personalized treatment decision making for NSCLC patients.

Sequential vs concurrent epirubicin and docetaxel as adjuvant chemotherapy for high-risk, node-negative, early breast cancer: an interim analysis of a randomised phase III study from the Hellenic Oncology Research Group.

BACKGROUND: Sequential anthracyclines and taxanes are standard adjuvant chemotherapy for patients with high-risk axillary node-positive breast cancer. We compared a sequential to a concurrent regimen in high-risk node-negative early breast cancer. METHODS: Patients were eligible if they had tumours >2 cm or T1c with two of the following characteristics: no oestrogen receptor (ER) and progesterone receptor (PR) expression, histological grade III, Ki67 >40% and vascular, lymphovascular or perineural invasion. They were randomised to receive four cycles of epirubicin 90 mg m-2 followed by four cycles of docetaxel 75 mg m-2 (sequential regimen) or six cycles of epirubicin 75 mg m-2 plus docetaxel 75 mg m-2 (concurrent regimen). All chemotherapy cycles were administered every 21 days with G-CSF prophylaxis only for the concurrent arm. The primary endpoint was disease-free survival (DFS). RESULTS: Between 2001 and 2013, 658 women received the sequential (n=329) or the concurrent (n=329) regimen. The median age was 53 years, 43.9% of the patients were premenopausal and of the tumours 44.2% were ⩽2 cm, 52.7% histological grade 3 and 35.3% hormone receptor-negative. After a median follow-up of 70.5 months, there were 29 (8.8%) vs 42 (12.8%) disease relapses (P=0.102) and 11 (3.3%) vs 19 (5.8%) deaths (P=0.135), in the sequential and concurrent arm, respectively. The 5-year DFS rates were 92.6% vs 88.2% for sequential and concurrent arm, respectively (hazard ratio (HR): 1.591; 95% confidence interval (CI): 0.990-2.556; P=0.055). Toxicity included grade 2-4 neutropenia in 54% vs 41% (P=0.001), febrile neutropenia 2.7% vs 6.1% (P=0.06), nausea/vomiting 18.5% vs 12.4% (P=0.03) of patients in the sequential and concurrent arm. There were no toxic deaths. CONCLUSIONS: Sequential compared with the concurrent administration of anthracyclines and taxanes is associated with a non-significant but possibly clinically meaningful improvement in DFS. In the era of molecular selection of patients for adjuvant chemotherapy, this study offers valuable information for the optimal administration of anthracyclines and taxanes in patients with node-negative disease.

Gene promoter methylation and protein expression of BRMS1 in uterine cervix in relation to high-risk human papilloma virus infection and cancer.

Cervical cancer is strongly related to certain high-risk types of human papilloma virus infection. Breast cancer metastasis suppressor 1 (BRMS1) is a tumor suppressor gene, its expression being regulated by DNA promoter methylation in several types of cancers. This study aims to evaluate the methylation status of BRMS1 promoter in relation to high-risk types of human papilloma virus infection and the development of pre-cancerous lesions and describe the pattern of BRMS1 protein expression in normal, high-risk types of human papilloma virus-infected pre-cancerous and malignant cervical epithelium. We compared the methylation status of BRMS1 in cervical smears of 64 women with no infection by high-risk types of human papilloma virus to 70 women with proven high-risk types of human papilloma virus infection, using real-time methylation-specific polymerase chain reaction. The expression of BRMS1 protein was described by immunohistochemistry in biopsies from cervical cancer, pre-cancerous lesions, and normal cervices. Methylation of BRMS1 promoter was detected in 37.5% of women with no high-risk types of human papilloma virus infection and was less frequent in smears with high-risk types of human papilloma virus (11.4%) and in women with pathological histology (cervical intraepithelial neoplasia) (11.9%). Methylation was detected also in HeLa cervical cancer cells. Immunohistochemistry revealed nuclear BRMS1 protein staining in normal high-risk types of human papilloma virus-free cervix, in cervical intraepithelial neoplasias, and in malignant tissues, where staining was occasionally also cytoplasmic. In cancer, expression was stronger in the more differentiated cancer blasts. In conclusion, BRMS1 promoter methylation and aberrant protein expression seem to be related to high-risk types of human papilloma virus-induced carcinogenesis in uterine cervix and is worthy of further investigation.

Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with methylated RASSF1A. The observed significant correlations between APC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.

SOX17 promoter methylation in plasma circulating tumor DNA of patients with non-small cell lung cancer.

BACKGROUND: SOX17 belongs to the high-mobility group-box transcription factor superfamily and down-regulates the Wnt pathway. The aim of our study was to evaluate the prognostic significance of SOX17 promoter methylation in circulating tumor DNA (ctDNA) in plasma of non-small cell lung cancer (NSCLC) patients. METHODS: We examined the methylation status of SOX17 promoter in 57 operable NSCLC primary tumors and paired adjacent non-cancerous tissues and in ctDNA isolated from 48 corresponding plasma samples as well as in plasma from 74 patients with advanced NSCLC and 49 healthy individuals. SOX17 promoter methylation was examined by Methylation Specific PCR (MSP). RESULTS: In operable NSCLC, SOX17 promoter was fully methylated in primary tumors (57/57, 100%), and in corresponding ctDNA (27/48, 56.2%) while it was detected in only 1/49 (2.0%) healthy individuals. In advanced NSCLC, SOX17 promoter was methylated in ctDNA in 27/74 (36.4%) patients and OS was significantly different in favor of patients with non-methylated SOX17 promoter (p=0.012). Multivariate analysis revealed that SOX17 promoter methylation in ctDNA was an independent prognostic factor associated with OS in patients with advanced but not operable NSCLC. CONCLUSIONS: Our results show that SOX17 promoter is highly methylated in primary tumors and in corresponding plasma samples both in operable and advanced NSCLC. In the advanced setting, SOX17 promoter methylation in plasma ctDNA has a statistical significant influence on NSCLC patient's survival time. Detection of SOX17 promoter methylation in plasma provides prognostic information and merits to be further evaluated as a circulating tumor biomarker in patients with operable and advanced NSCLC.