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Background C-reactive protein is an acute phase response marker and, in an epidemiological context, a predictor of cardiovascular risk. 25-Hydroxy-vitamin D is the best indicator for vitamin D status, but it can be altered by the presence of acute phase response. Our aim was to evaluate the association between serum concentrations of 25-hydroxy-vitamin D and C-reactive protein to assist the interpretation of vitamin D status in a clinical context. Methods We evaluated retrospectively 5076 patients ( n = 4087 women) assessed for 25-hydroxy-vitamin D and C-reactive protein simultaneously. Subjects were classified according to the origin as hospitalized patients ( n = 410) and outpatients ( n = 4666). Outpatients included patients from specialized ( n = 3943) and primary ( n = 723) care. Serum 25-hydroxy-vitamin D was determined by using liquid chromatography and serum C-reactive protein by using immunoturbidimetry. Results Concentrations of 25-hydroxy-vitamin D and C-reactive protein were significantly different between hospitalized subjects and outpatients but not for specialized and primary care settings. Serum concentrations of 25-hydroxy-vitamin D decreased as C-reactive protein increased. Hospitalized patients with C-reactive protein concentrations >30 mg/L showed a significant reduction of 25-hydroxy-vitamin D. In outpatients with C-reactive protein within the reference range (≤10 mg/L), C-reactive protein concentrations were not significantly different for serum 25-hydroxy-vitamin D concentrations >37.5 nmol/L. Conclusions Our data question the reliability and usefulness of assessing 25-hydroxy-vitamin D status as a biomarker of nutritional status in patients displaying acute phase response, especially at concentrations of C-reactive protein >30 mg/L. In addition, the present study shows that in subjects displaying C-reactive protein values within the reference range, serum concentrations of 25-hydroxy-vitamin D >37.5 nmol/L were not associated with lower concentrations of cardiovascular risk (as assessed by C-reactive protein concentrations).

Objectives: Epidermal growth factor receptor (EGFR) biomarker testing using blood-based liquid biopsies remains challenging due to the low concentration of circulating tumor DNA (ctDNA) in certain plasma samples. The aim of this study is to evaluate the usefulness for EGFR biomarker testing of ctDNA from pleural effusions, cerebrospinal fluids, ascites and pericardial effusions obtained during the clinical management of lung adenocarcinoma patients. Methods: For comparison purposes, 23 paired plasma and body fluid samples were collected from 17 patients with EGFR-positive lung adenocarcinoma. After circulating free DNA (cfDNA) isolation, samples were evaluated for the initial EGFR-sensitizing mutation and the p.T790M resistance mutation by array-based digital PCR (dPCR). Results: Body fluids had more cfDNA than plasma samples (1.90 vs. 0.36 ng/µL; p=0.0130), and more samples tested positive for EGFR mutations (21 vs. 16 samples), with a total of 28 vs. 22 variants detected. Furthermore, mutant allele frequencies (MAFs) observed in body fluids were significantly higher than those assessed in the paired plasma samples for EGFR-sensitizing mutations (median MAFs = 15.8 vs. 0.8%; p=0.0004) as well as for the p.T790M resistance mutation (median MAFs = 8.69 vs. 0.16%; p=0.0390). Importantly, two patients who had progressed on first-generation EGFR-tyrosine kinase inhibitors with a dubious result for p.T790M plasma (MAFs = 0.11%) had an indisputably positive result in their respective body fluid samples (MAFs = 10.25 and 9.66%). Conclusions: ctDNA derived from body fluids is an informative source for EGFR biomarker testing, with greater sensitivity than plasma samples., MB is supported by an i-PFIS predoctoral fellowship (Grant Number IFI18/00051) from ISCIII. ES is funded by the Consejería de Ciencia, Universidades e Innovación de la Comunidad de Madrid (Doctorados Industriales de la Comunidad de Madrid IND2019/BMD-17258), Spain.

Genetic screening for BRCA mutations should be offered to all women diagnosed with epithelial ovarian, fallopian tube, and/or peritoneal cancers given the implications for treatment options and cancer risk assessments. Yet, while germline breast cancer susceptibility gene 1 (BRCA1) and breast cancer susceptibility gene 2 (BRCA2) testing is commonly performed, BRCA1/2 somatic mutations testing is rather challenging since the poor quality of DNA extracted from formalin fixed paraffin embedded (FFPE) samples can significantly impair this process. Peritoneal lavage is routinely performed in surgeries of suspected ovarian malignancies. We have analyzed fresh tumor, peritoneal lavage and blood samples from ten patients and we have found an excellent agreement (88%) between fresh tumor and peritoneal lavage for BRCA mutation testing. Importantly, 112 of the 114 genomic alterations detected in fresh tumor samples were also found in peritoneal lavage fluids. Our data suggest that peritoneal washings can indeed streamline BRCA genes mutation testing procedures.

Background Non-small cell lung cancer (NSCLC) patients benefit from targeted therapies both in first- and second-line treatment. Nevertheless, molecular profiling of lung cancer tumors after first disease progression is seldom performed. The analysis of circulating tumor DNA (ctDNA) enables not only non-invasive biomarker testing but also monitoring tumor response to treatment. Digital PCR (dPCR), although a robust approach, only enables the analysis of a limited number of mutations. Next-generation sequencing (NGS), on the other hand, enables the analysis of significantly greater numbers of mutations. Methods A total of 54 circulating free DNA (cfDNA) samples from 52 NSCLC patients and two healthy donors were analyzed by NGS using the Oncomine™ Lung cfDNA Assay kit and dPCR. Results Lin’s concordance correlation coefficient and Pearson’s correlation coefficient between mutant allele frequencies (MAFs) assessed by NGS and dPCR revealed a positive and linear relationship between the two data sets (ρc = 0.986; 95% confidence interval [CI] = 0.975–0.991; r = 0.987; p Conclusions MAFs assessed by NGS were highly correlated with MAFs assessed by dPCR, demonstrating that NGS is a robust technique for ctDNA quantification using clinical samples, thereby allowing for dynamic genomic surveillance in the era of precision medicine.

Background: We examined the associations between germline variants and breast cancer mortality using a large meta-analysis of women of European ancestry. Methods: Meta-analyses included summary estimates based on Cox models of twelve datasets using ~10.4 million variants for 96,661 women with breast cancer and 7697 events (breast cancer-specific deaths). Oestrogen receptor (ER)-specific analyses were based on 64,171 ER-positive (4116) and 16,172 ER-negative (2125) patients. We evaluated the probability of a signal to be a true positive using the Bayesian false discovery probability (BFDP). Results: We did not find any variant associated with breast cancer-specific mortality at P < 5 × 10 −8 . For ER-positive disease, the most significantly associated variant was chr7:rs4717568 (BFDP = 7%, P = 1.28 × 10 −7 , hazard ratio [HR] = 0.88, 95% confidence interval [CI] = 0.84–0.92); the closest gene is AUTS2. For ER-negative disease, the most significant variant was chr7:rs67918676 (BFDP = 11%, P = 1.38 × 10 −7 , HR = 1.27, 95% CI = 1.16–1.39); located within a long intergenic non-coding RNA gene (AC004009.3), close to the HOXA gene cluster. Conclusions: We uncovered germline variants on chromosome 7 at BFDP < 15% close to genes for which there is biological evidence related to breast cancer outcome. However, the paucity of variants associated with mortality at genome-wide significance underpins the challenge in providing genetic-based individualised prognostic information for breast cancer patients.

// Mariano Provencio 1 , Maria Torrente 1 , Virginia Calvo 1 , David Perez-Callejo 1 , Lourdes Gutierrez 1 , Fernando Franco 1 , Clara Perez-Barrios 2 , Miguel Barquin 2 , Ana Royuela 3 , Francisco Garcia-Garcia 4 , Coralia Bueno 5 , Aranzazu Garcia-Grande 6 , Carlos Camps 7 , Bartomeu Massuti 8 , Eduardo Sotomayor 9 and Atocha Romero 1, 2 1 Medical Oncology Department, Hospital Universitario Puerta de Hierro-Majadahonda, Majadahonda, Spain 2 Liquid Biopsy Laboratory, Biomedical Sciences Research Institute Puerta de Hierro-Majadahonda University Hospital, Majadahonda, Spain 3 Biostatistics Department, Biomedical Sciences Research Institute Puerta de Hierro-Majadahonda University Hospital, Majadahonda, Spain 4 Computational Genomics, Centro de Investigacion Principe Felipe, Valencia, Spain 5 Medical Oncology Department, Hospital Infanta Cristina, Parla, Spain 6 Cytometry Unit, Biomedical Sciences Research Institute Puerta de Hierro-Majadahonda, Spain 7 Medical Oncology Department, Hospital General de Valencia, CIBERONC Network, Valencia, Spain 8 Medical Oncology Department, Hospital Universitario de Alicante, Alicante, Spain 9 George Washington Cancer Center, Washington D.C., United States of America Correspondence to: Mariano Provencio, email: mariano.provencio@salud.madrid.org Atocha Romero, email: atocha10@hotmail.com Keywords: ctDNA, non-small cell lung cancer, tyrosine kinase inhibitor, EGFR Received: June 15, 2017 Accepted: September 30, 2017 Published: November 16, 2017 ABSTRACT Background: Circulating tumor DNA (ctDNA) levels correlate well with tumor bulk. In this paper we aim to estimate the prognostic value of the dynamic quantification of ctDNA levels. Materials and Methods: A total of 251 serial plasma samples from 41 non-small-cell lung cancer patients who carried an activating EGFR mutation were analysed by digital PCR. For survival analysis, ctDNA levels were computed as a time-dependent covariate. Results: Dynamic ctDNA measurements had prognostic significance (hazard ratio for overall survival and progression free survival according to p.T790M mutant allele frequency; 2.676 and 2.71 respectively; P < 0.05). In the same way, patients with p.T790M-negative or unchanging or decreasing plasma levels of sensitizing EGFR mutation were 12 and 4.8 times more likely to maintain response or stable disease, respectively, than patients in which the opposite occurred ( P < 0.05).On average, the p.T790M mutation was detected in plasma 51 days before the assessment of progression disease by CT-scan. Finally, ctDNA outperformed CTCs for assessing tumor progression ( P = 0.021). Conclusions: The appearance or increase in a unit of the p.T790M allele frequency almost triples the risk of death and progression. This information can be used to design clinical trials aiming to estimate whether T790M positive patients should start second line treatment based on molecular data rather than imaging data.

Background: Liquid biopsy has evolved from being a promising line to becoming a validated approach for biomarker testing. However, its utility for individualization of therapy has been scarcely reported. In this study, we show how monitoring levels of EGFR mutation in plasma can be useful for the individualization of treatment. Results: Longitudinal EGFR mutation levels in plasma always correlated with tumor response ascertained by RECIST criteria. Moreover, decreasing EGFR mutation levels were detected in all patients benefiting from locoregional radiotherapy, whereas the opposite occurred when a patient progressed soon after radiotherapy treatment. Similarly, increasing EGFR mutation levels anticipated disease progression after TKI dose reduction, discontinuation of treatment, or reduced bioavailability due to drug interactions. In addition, EGFR mutation levels were useful to monitor treatment outcome of new therapies and constituted a decisive factor when the clinical situation of the patient did not correlate with responses ascertained by radiologist. Finally, our results indicate that cancer associated body fluids (pleural, pericardial or cerebrospinal fluid) are certainly a suitable source for biomarker testing that can extend EGFR mutation detection to biofluids other than blood. Materials and Methods: A total of 180 serial plasma samples from 18 non-smallcell lung cancer patients who carried an activating EGFR mutation were investigated by digital PCR. Conclusions: Monitoring levels of EGFR mutation in plasma allows resolving doubts that frequently arise in daily clinical practice and constitutes a major step towards achieving personalized medicine, This study was supported by Carlos III Institute of Health, Spanish Ministry of Science and Innovation, and European Regional Development Fund (grant number: PI16/01818 and PIE14/00064), A Romero is supported by Joan Rodés fellowship (grant number: JR14/00017) and CP pre-doctoral studies are supported by Jose Luís Castaño Foundation

The implementation of liquid biopsy for biomarker testing and response to treatment monitoring in cancer patients would presumable increase laboratory throughput, requiring the development of automated methods for circulating free DNA (cfDNA) isolation.The present study compares the MagNA Pure Compact (MPC) Nucleic Acid Isolation Kit I and MaxwellFirstly, we observed that MPC method significantly extracted less cfDNA than MR (P0.0001). However, there were no significant differences in extraction yields of QCNA and MR kits. cfDNA isolation yield was also associated with tumor stage but not with tumor location. Secondly, an oligonucleosomal DNA ladder pattern was observed in 88% of the samples and significant differences in the recovery of mono-, di- and tri-nucleosomes DNA fragments were observed between MPC and MR methodologies. Finally, tumor mutation quantification on cfDNA was performed on 38 paired samples using digital PCR. Mutant allele fractions (MAFs) between paired samples were not significantly different.Methods for isolation of cfDNA can affect DNA yield and molecular weight fractions recovery. These observations should be taken into account for cfDNA analysis in routine clinical practice.