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Using a newly developed method dubbed SILVER-Seq—enabling extracellular RNA sequencing (exRNA-seq) directly from a small volume of human serum or plasma— Yan et al. recently reported in Current Biology a potential exRNA biomarker for the early diagnosis of Alzheimer’s disease [1]. After the publication of the initial paper describing the SILVER-Seq method [2], we reported our concern regarding potential DNA contamination in their datasets [3]. Although the authors replied they were able to successfully treat RNA samples with DNase to avoid such contamination, they did not address our observations of the majority of reads without evidence of being derived from RNA, nor documented verified absence of DNA after DNase treatment [4]. To assess whether the newly data generated may suffer from DNA contamination, we downloaded the publicly available sequencing data and evaluated two quality control metrics (i.e., fraction of exonic and splice reads), which were not reported in the paper. We found that both quality metrics were much lower than expected for RNA-seq data (6.28% exonic and 0.478% splice reads), in line with our previous findings on the first SILVER-Seq paper. These observations suggest the data and results presented by Yan et al. are affected by DNA contamination, an issue that may be inherent to the SILVER-Seq technology.

Purpose To compare the abilities of three pulsed focused ultrasound regimes (that cause tissue liquefaction, permeabilization, or mild heating) to release tumor-derived microRNA into the circulation in vivo and to evaluate release dynamics. Materials and Methods All rat experiments were approved by the University of Washington Institutional Animal Care and Use Committee. Reverse-transcription quantitative polymerase chain reaction array profiling was used to identify candidate microRNA biomarkers in a rat solid tumor cell line. Rats subcutaneously grafted with these cells were randomly assigned among three pulsed focused ultrasound treatment groups: (a) local tissue liquefaction via boiling histotripsy, (b) tissue permeabilization via inertial cavitation, and (c) mild (

Extracellular RNA s (exRNA s) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNA s in blood plasma are extensively characterized, extracellular messenger RNA (mRNA ) and long non‐coding RNA (lncRNA ) studies are limited. We report that plasma contains fragmented mRNA s and lncRNA s that are missed by standard small RNA ‐seq protocols due to lack of 5′ phosphate or presence of 3′ phosphate. These fragments were revealed using a modified protocol (“phospho‐RNA ‐seq”) incorporating RNA treatment with T4‐polynucleotide kinase, which we compared with standard small RNA ‐seq for sequencing synthetic RNA s with varied 5′ and 3′ ends, as well as human plasma exRNA . Analyzing phospho‐RNA ‐seq data using a custom, high‐stringency bioinformatic pipeline, we identified mRNA /lncRNA transcriptome fingerprints in plasma, including tissue‐specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow‐ and liver‐enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof‐of‐concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho‐RNA ‐seq opens up new possibilities for plasma transcriptomic biomarker development., We acknowledge funding support from the NIH Extracellular RNA Communication Common Fund grants: U01 grants HL126499 to M. Tewari and HL126496 to D.J.G., and from the A. Alfred Taubman Medical Research Institute (Grand Challenge Award) to M. Tewari and S.W.C. Research reported in this publication was also supported by the National Cancer Institute of the NIH under Award Number P30CA046592 by the use of the following Cancer Center Shared Resource at the University of Michigan: DNA Sequencing. M.D.G. acknowledges support from a Precision Health Scholar Award from the University of Michigan Precision Health Center and a Juan Rodes contract (JR18/00026) funded by the Spanish Institute of Health Carlos III from the Ministry of Economy and Competitiveness (co‐funded by European Social Fund (ESF)). D.J.G. also acknowledges a special technology support award and partial funding from the Pacific Northwest Research Institute to his laboratory.

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Whereas extracellular microRNAs (miRNAs) in blood plasma are extensively characterized, extracellular messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are less well-studied. We report that plasma contains fragmented mRNAs and lncRNAs that are largely missed by standard small RNA-seq protocols due to lack of 5’ phosphate or presence of 3’ phosphate. These fragments were revealed using a modified protocol (“phospho-sRNA-seq”) incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5’ and 3’ ends, as well as human plasma exRNA. Analyzing phospho-sRNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets. In a longitudinal study of bone marrow transplant patients, bone marrow-and liver-enriched exRNA genes tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho-sRNA-seq opens up new possibilities for plasma transcriptomic biomarker development.

Measuring disease biomarkers in biofluids to detect and monitor pathophysiologic events in tissues has enormous appeal. While detecting specific DNA sequences in blood plasma has been successful in informing about the presence of cancers, viruses, and a range of pathogens, this approach does not provide gene expression information, which could reveal more detailed information about disease activity. Measuring cell-free RNA in plasma would provide such information and has the potential to be more sensitive, given that one DNA molecule in a cell gives rise often to thousands or more RNA molecules. However, with the exception of microRNAs, broad sequencing of cell-free messenger RNA (mRNA) profiles in plasma has been generally challenging. We discovered that a major barrier to cell-free RNA sequencing from plasma is that the large majority of mRNAs are both fragmented and have modified phosphorylation states at their 5’ and 3’ ends that make them invisible to standard small RNA-seq methods. We present a modified RNA-seq methodology, called phospho-RNA-seq, which revealed thousands of mRNA in blood plasma. The key to this approach is (i) the incorporation of T4-polynucleotide kinase treatment to change the phosphorylation states at the RNA ends, and (ii) a stringent bioinformatics analysis pipeline to reduce false positive alignments. Phospho-RNA-seq identified cohorts of gene transcripts in plasma, including ones expressed in a tissue-specific manner. As proof-of-concept validation of the approach for biomarker identification, we used phospho-RNA-seq to longitudinally profile plasma specimens collected from patients undergoing hematopoietic stem cell transplantation. We detected bone marrow-enriched and liver-enriched transcript sets in plasma, which tracked with bone marrow recovery and hepatic injury, respectively. By providing expanded access to the transcriptome in plasma and potentially other biofluids, phospho-RNA-seq enables the discovery of new cell-free RNA biomarker signatures for a wide variety of liquid biopsy applications. Citation Format: Maria D. Giraldez, Ryan M. Spengler, Alton Etheridge, Annika J. Goicochea, Missy Tuck, Sung W. Choi, David J. Galas, Muneesh Tewari. Phospho-RNA-seq: A liquid biopsy approach for cell-free mRNA/lncRNA profiling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA23.

Droplet-based digital PCR provides high-precision, absolute quantification of nucleic acid target sequences with wide-ranging applications for both research and clinical diagnostic applications. Droplet-based digital PCR enables absolute quantification by counting nucleic acid molecules encapsulated in discrete, volumetrically defined water-in-oil droplet partitions. The current available systems overcome the previous lack of scalable and practical technologies for digital PCR implementation. Extracellular microRNAs in biofluids (plasma, serum, urine, cerebrospinal fluid, etc.) are promising noninvasive biomarkers in multiple diseases and different clinical settings (e.g., diagnosis, early diagnosis, prediction of recurrence, and prognosis). Here we describe a protocol that enables highly precise and reproducible absolute quantification of extracellular microRNAs using droplet digital PCR.

The EPICOLON consortium was initiated in 1999 by the Gastrointestinal Oncology Group of the Spanish Gastroenterology Association. It recruited consecutive, unselected, population-based colorectal cancer (CRC) cases and control subjects matched by age and gender without personal or familial history of cancer all over Spain with the main goal of gaining knowledge in Lynch syndrome and familial CRC. This epidemiological, prospective and multicentre study collected extensive clinical data and biological samples from ∼2000 CRC cases and 2000 controls in Phases 1 and 2 involving 25 and 14 participating hospitals, respectively. Genetic susceptibility projects in EPICOLON have included candidate-gene approaches evaluating single-nucleotide polymorphisms/genes from the historical category (linked to CRC risk by previous studies), from human syntenic CRC susceptibility regions identified in mouse, from the CRC carcinogenesis-related pathways Wnt and BMP, from regions 9q22 and 3q22 with positive linkage in CRC families, and from the mucin gene family. This consortium has also participated actively in the identification 5 of the 16 common, low-penetrance CRC genetic variants identified so far by genome-wide association studies. Finishing their own pangenomic study and performing whole-exome sequencing in selected CRC samples are among EPICOLON future research prospects. © 2012 The Author.

Resumen Las formas hereditarias y familiares de cancer colorrectal (CCR) representan aproximadamente un 30–35% de los casos de CCR. Su identificacion y el correcto manejo de estas familias son fundamentales, ya que constituyen un grupo de alto riesgo de CCR que se beneficia de medidas preventivas especificas para evitar el desarrollo de esta neoplasia. Ademas, en algunas de estas formas de CCR, principalmente en el CCR familiar, las alteraciones responsables de la susceptibilidad genetica no estan bien caracterizadas, lo cual resulta prioritario para su adecuado diagnostico y manejo. En esta revision se discuten las novedades mas importantes, presentadas en el congreso de la American Gastroenterological Association, en cuanto a etiopatogenia, identificacion y manejo clinico de las formas hereditarias y familiares de CCR.

Colorectal cancer (CRC) risk associated with germline monoallelic MUTYH mutations remains controversial, although a slightly increased risk for this disease has been suggested. MUTYH and MSH6 proteins act in cooperation during the DNA repair process. Based on this interaction, it was hypothesized that the combination of heterozygote germline mutations in both genes could result in an increased CRC risk. To further clarify the interaction between MUTYH and MSH6, we analyzed the prevalence of MSH6 mutations in a cohort of CRC patients and controls previously tested for MUTYH mutations: CRC patients with and without a monoallelic MUTYH mutation (group I, n = 26; group II, n = 50, respectively), and healthy carriers with a monoallelic MUTYH mutation (group III, n = 21). In group I, we found three patients (11.5%) with MSH6 mutations, a missense mutation (p.R635G), a change in the 3′UTR region (c.*4098A > C) and a nonsense mutation (p.Q982X). In group II and III, no mutations were detected. In CRC patients, MSH6 mutations were more frequently found in MUTYH mutation carriers than in noncarriers (11.5% vs. 0%, P = 0.037). CRC patients carrying monoallelic MUTYH mutations harbor more frequently concomitant MSH6 mutations than patients without them, thus suggesting that both genes could act cooperatively and confer together an increased CRC risk.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. Death rates have been declining, largely as a result of early detection through mammography and improved treatment, but mammographic screening is controversial because of over-diagnosis of breast disease that might not require treatment, and under-diagnosis of cancer in women with dense breasts. Breast cancer screening could be improved by pairing mammography with a tumor circulating marker, of which there are currently none. Given genomic similarities between the basal breast cancer subtype and serous ovarian cancer, and given our success in identifying circulating markers for ovarian cancer, we investigated the performance in hormone receptor-negative breast cancer detection of both previously identified ovarian serum markers and circulating markers associated with transcripts that were differentially expressed in breast cancer tissue compared to healthy breast tissue from reduction mammaplasties.We evaluated a total of 15 analytes (13 proteins, 1 miRNA, 1 autoantibody) in sera drawn at or before breast cancer surgery from 43 breast cancer cases (28 triple-negative-TN-and 15 hormone receptor-negative-HRN-/ HER2-positive) and 87 matched controls.In the analysis of our whole cohort of breast cancer cases, autoantibodies to TP53 performed significantly better than the other selected 14 analytes showing 25.6% and 34.9% sensitivity at 95% and 90% specificity respectively with AUC: 0.7 (p0.001). The subset of 28 TN cancers showed very similar results. We observed no correlation between anti-TP53 and the 14 other markers; however, anti-TP53 expression correlated with Body-Mass-Index. It did not correlate with tumor size, positive lymph nodes, tumor stage, the presence of metastases or recurrence.None of the 13 serum proteins nor miRNA 135b identified women with HRN or TN breast cancer. TP53 autoantibodies identified women with HRN breast cancer and may have potential for early detection, confirming earlier reports. TP53 autoantibodies are long lasting in serum but may be affected by storage duration. Autoantibodies to TP53 might correlate with Body-Mass-Index.

Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers. We introduce a kinetic fingerprinting approach called Single Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS) for the amplification-free counting of single unlabeled miRNA molecules, which circumvents thermodynamic limits of specificity and virtually eliminates false positives. We demonstrate high-confidence single-molecule detection of synthetic and endogenous miRNAs in both buffer and minimally treated biological liquids, as well as >500-fold discrimination between single nucleotide polymorphisms.

8 p. Background: Colorectal cancer (CRC) is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool. -- Aim: Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC. -- Methods: In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7), urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF), cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2), and CD44) and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA) and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results. -- Results: Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79 +/- 0.25 in the CRC group vs 1.27 +/- 0.25 in the control group, P < 0.0005). The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79-0.96). The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81) and tetranectin (0.80), COX-2 (0.78), uPAR (0.78) and carbonic anhydrase (0.77). The markers which identified early stage CRC (Stages I and II) were alpha 1-antitrypsin, uPAR, COX-2 and MMP7. -- Conclusions: Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages. Centro de Investigación Biomédica en Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by the Instituto de Salud Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The clinical potential of circulating tumor cells (CTCs) in managing cancer metastasis is significant. However, low CTC isolation purities from patient blood have hindered sensitive molecular assays of these rare cells. Described herein is the ultra‐pure isolation of CTCs from patient blood samples and how this platform has enabled highly specific molecular (mRNA and miRNA) profiling of patient CTCs.

Colorectal cancer (CRC) is the second most common cancer in Western countries. Hereditary forms only correspond to 5% of CRC burden. Recently, genome-wide association studies have identified common low-penetrant CRC genetic susceptibility loci. Early-onset CRC (CRC50 years old) is especially suggestive of hereditary predisposition although 85-90% of heritability still remains unidentified. CRC50 patients (n = 191) were compared with a late-onset CRC group (CRC65 years old) (n = 1264). CRC susceptibility variants at 8q23.3 (rs16892766), 8q24.21 (rs6983267), 10p14 (rs10795668), 11q23.1 (rs3802842), 15q13.3 (rs4779584), 18q21 (rs4939827), 14q22.2 (rs4444235), 16q22.1 (rs9929218), 19q13.1 (rs10411210) and 20p12.3 (rs961253) were genotyped in all DNA samples. A genotype-phenotype correlation with clinical and pathological characteristics in both groups was performed. Risk allele carriers for rs3802842 [Odds ratio (OR) = 1.5, 95% confidence interval (CI) 1.1-2.05, P = 0.0096, dominant model) and rs4779584 (OR = 1.39, 95% CI 1.02-1.9, P = 0.0396, dominant model) were more frequent in the CRC50 group, whereas homozygotes for rs10795668 risk allele were also more frequent in the early-onset CRC (P = 0.02, codominant model). Regarding early-onset cases, 14q22 (rs4444235), 11q23 (rs3802842) and 20p12 (rs961253) variants were more associated with family history of CRC or tumors of the Lynch syndrome spectrum excluding CRC. In our entire cohort, sum of risk alleles was significantly higher in patients with a CRC family history (OR = 1.40, 95% CI 1.06-1.85, P = 0.01). In conclusion, variants at 10p14 (rs10795668), 11q23.1 (rs3802842) and 15q13.3 (rs4779584) may have a predominant role in predisposition to early-onset CRC. Association of CRC susceptibility variants with some patient's familiar and personal features could be relevant for screening and surveillance strategies in this high-risk group and it should be explored in further studies.

BACKGROUND: Colorectal cancer (CRC) is the second cause of cancer-related death in the Western world. Much of the CRC genetic risk remains unidentified and may be attributable to a large number of common, low-penetrance genetic variants. Genetic linkage studies in CRC families have reported additional association with regions 9q22-31, 3q21-24, 7q31, 11q, 14q and 22q. There are several plausible candidate genes for CRC susceptibility within the aforementioned linkage regions including PTCH1, XPA and TGFBR1 in 9q22-31, and EPHB1 and MRAS in 3q21-q24. METHODS: CRC cases and matched controls were from EPICOLON, a prospective, multicentre, nationwide Spanish initiative, composed of two independent phases. Phase 1 corresponded to 515 CRC cases and 515 controls, whereas phase 2 consisted of 901 CRC cases and 909 controls. Genotyping was performed for 172 single-nucleotide polymorphisms (SNPs) in 84 genes located within regions 9q22-31 and 3q21-q24. RESULTS: None of the 172 SNPs analysed in our study could be formally associated with CRC risk. However, rs1444601 (TOPBP1) and rs13088006 (CDV3) in region 3q22 showed interesting results and may have an effect on CRC risk. CONCLUSIONS: TOPBP1 and CDV3 genetic variants on region 3q22 may modulate CRC risk. Further validation and meta-analysis should be undertaken in larger CRC cohorts. British Journal of Cancer (2011) 105, 870-875. doi: 10.1038/bjc.2011.296 www.bjcancer.com Published online 2 August 2011, We are sincerely grateful to all patients participating in this study who were recruited in 25 (EPICOLON 1) and 14 (EPICOLON 2) Spanish hospitals as part of the EPICOLON project. We are also grateful to the Spanish National Genotyping Center (CEGEN-ISCIII)-USC and UPF nodes, and the Genome Analysis Platform of the CIC-BioGUNE. The work was carried out (in part) at the Esther Koplowitz Centre, Barcelona. SC-B, CF-R and MDG are supported by contracts from the Fondo de Investigacion Sanitaria (CP 03-0070 to SC-B, CM10/00084 to MDG, and PS09/02368 to CF-R). JM is supported by a contract from the CIBERehd. Both CIBERehd and CIBERER are funded by the Instituto de Salud Carlos III. This work was supported by grants from the Fondo de Investigacion Sanitaria/FEDER (06/1384, 08/0024, 08/1276, PS09/02368, 10/00918), Instituto de Salud Carlos III (Accion Transversal de Cancer), Xunta de Galicia (07PXIB9101209PR), Ministerio de Ciencia e Innovacion (SAF2010-19273), Asociacion Espanola contra el Cancer (Fundacion Cientifica y Junta de Barcelona), Fundacio Olga Torres (SCB and CRP) and FP7 CHIBCHA Consortium (LC-C, ACar and SC-B).

Although receiving adjuvant chemotherapy after radical surgery, a disappointing proportion of patients with colorectal cancer will develop tumor recurrence. Probability of relapse is currently predicted from pathological staging, there being a need for additional markers to further select high-risk patients. This study was aimed to identify a gene-expression signature to predict tumor recurrence in patients with Stages II and III colon cancer treated with 5'fluoruracil (5FU)-based adjuvant chemotherapy. Two-hundred and twenty-eight patients diagnosed with Stages II-III colon cancer and treated with surgical resection and 5FU-based adjuvant chemotherapy were included. RNA was extracted from formalin-fixed, paraffin-embedded tissue samples and expression of 27 selected candidate genes was analyzed by RT-qPCR. A tumor recurrence predicting model, including clinico-pathological variables and gene-expression profiling, was developed by Cox regression analysis and validated by bootstrapping. The regression analysis identified tumor stage and S100A2 and S100A10 gene expression as independently associated with tumor recurrence. The risk score derived from this model was able to discriminate two groups with a highly significant different probability of tumor recurrence (HR, 2.75; 95%CI, 1.71-4.39; p = 0.0001), which it was maintained when patients were stratified according to tumor stage. The algorithm was also able to distinguish two groups with different overall survival (HR, 2.68; 95%CI, 1.12-6.42; p = 0.03). Identification of a new gene-expression signature associated with a high probability of tumor recurrence in patients with Stages II and III colon cancer receiving adjuvant 5FU-based chemotherapy, and its combination in a robust, easy-to-use and reliable algorithm may contribute to tailor treatment and surveillance strategies.

Purpose: Lynch syndrome accounts for 2-4% of all colorectal cancer, and is mainly caused by germline mutations in the DNA mismatch repair genes. Our aim was to characterize the genetic mutation responsible for Lynch syndrome in an extensive Colombian family and to study its prevalence in Antioquia. Methods: A Lynch syndrome family fulfilling Amsterdam criteria II was studied by immunohistochemistry and by multiplex ligation-dependent probe amplification (MLPA). Results were confirmed by additional independent MLPA, Southern blotting, and sequencing. Results: Index case tumor immunohistochemistry results were MLH1-, MSH2+, MSH6+, and PMS2-. MLPA analysis detected a duplication of exons 12 and 13 of MLH1. This mutation was confirmed and characterized precisely to span 4219 base pairs. Duplication screening in this family led to the identification of six additional carriers and 13 noncarriers. We also screened 123 early-onset independent colorectal cancer cases from the same area and identified an additional unrelated carrier. CONCLUSION:: A novel duplication of exons 12 and 13 of the MLH1 gene was detected in two independent Lynch syndrome families from Colombia. A putative founder effect and prescreening Lynch syndrome Antioquia families for this specific mutation before thorough mismatch repair mutational screening could be suggested. © 2011 Lippincott Williams and Wilkins.

Purpose: Early-onset colorectal cancer (CRC) is suggestive of a hereditary predisposition. Lynch syndrome is the most frequent CRC hereditary cause. The MUTYH gene has also been related to hereditary CRC. A systematic characterization of these two diseases has not been reported previously in this population. Experimental Design: We studied a retrospectively collected series of 140 patients ≤50 years old diagnosed with nonpolyposis CRC. Demographic, clinical, and familial features were obtained. Mismatch repair (MMR) deficiency was determined by microsatellite instability (MSI) analysis, and immunostaining for MLH1, MSH2, MSH6, and PMS2 proteins. Germline MMR mutations were evaluated in all MMR-deficient cases. Tumor samples with loss of MLH1 or MSH2 protein expression were analyzed for somatic methylation. Germline MUTYH mutations were evaluated in all cases. BRAF V600E and KRAS somatic mutational status was also determined. Results: Fifteen tumors (11.4%) were MSI, and 20 (14.3%) showed loss of protein expression (7 for MLH1/PMS2, 2 for isolated MLH1, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1 for MSH6/PMS2). We identified 11 (7.8%) germline MMR mutations, 4 in MLH1, 1 in MSH2, and 6 in MSH6. Methylation analysis revealed one case with somatic MLH1 methylation. Biallelic MUTYH mutations were detected in four (2.8%) cases. KRAS and BRAF V600E mutations were present in 39 (27.9%) and 5 (3.6%) cases, respectively. Conclusions: Loss of MSH6 expression is the predominant cause of MMR deficiency in early-onset CRC. Our findings prompt the inclusion of MSH6 and MUTYH screening as part of the genetic counseling of these patients and their relatives. Clin Cancer Res; 16(22); 5402–13. ©2010 AACR.

IntroductionBreast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. Death rates have been declining, largely as a result of early detection through mammography and improved treatment, but mammographic screening is controversial because of over-diagnosis of breast disease that might not require treatment, and under-diagnosis of cancer in women with dense breasts. Breast cancer screening could be improved by pairing mammography with a tumor circulating marker, of which there are currently none. Given genomic similarities between the basal breast cancer subtype and serous ovarian cancer, and given our success in identifying circulating markers for ovarian cancer, we investigated the performance in hormone receptor-negative breast cancer detection of both previously identified ovarian serum markers and circulating markers associated with transcripts that were differentially expressed in breast cancer tissue compared to healthy breast tissue from reduction mammaplasties.MethodsWe evaluated a total of 15 analytes (13 proteins, 1 miRNA, 1 autoantibody) in sera drawn at or before breast cancer surgery from 43 breast cancer cases (28 triple-negative-TN-and 15 hormone receptor-negative-HRN-/ HER2-positive) and 87 matched controls.ResultsIn the analysis of our whole cohort of breast cancer cases, autoantibodies to TP53 performed significantly better than the other selected 14 analytes showing 25.6% and 34.9% sensitivity at 95% and 90% specificity respectively with AUC: 0.7 (pConclusionNone of the 13 serum proteins nor miRNA 135b identified women with HRN or TN breast cancer. TP53 autoantibodies identified women with HRN breast cancer and may have potential for early detection, confirming earlier reports. TP53 autoantibodies are long lasting in serum but may be affected by storage duration. Autoantibodies to TP53 might correlate with Body-Mass-Index.

Introduction and Objective: Our previous studies demonstrated that some circulation-bound tumor-derived factors are taken up and concentrated by circulating platelets, and are released upon platelet activation. MicroRNAs (miRNAs) are small, non-coding nucleic acid molecules involved in regulation of gene expression that have potential as biomarkers in prostate cancer. Prior descriptions of miRNA presence in platelets of healthy individuals indicates that uptake of tumor-derived miRNAs may be reflected by changes in platelet miRNA signature. To date, in the prostate cancer setting, the role of platelets as a source of miRNA-based biomarkers has not been investigated. The aim of this study was to ascertain the presence of prostate cancer-associated miRNAs in platelets from patients with aggressive disease and explore the potential use of platelets as a source of miRNA-based biomarkers. Methods Patients with aggressive prostate cancer (n = 12) were identified at a large, tertiary care medical center, informed of the study and consented to provide whole blood samples. Platelets were isolated from venous blood using citrate containing tubes and purified by serial centrifugations and washings. Platelet purification was confirmed at >99.9% by flow cytometry and platelet specific antibodies. Total RNA was extracted from platelets of men with aggressive prostate cancer (n = 2), low risk disease (n = 1) and age matched (50+) controls (n = 2). By performing a Real Time-quantitative PCR (RT-qPCR) profiling using the Human Panel I (Exiqon) (which encompasses 373 microRNAs representing the best-established and well-annotated human miRNAs) we identified several miRNAs as potential candidates of the aggressive disease. TaqMan miRNA Reverse Transcription kit and miRNA-specific stem loop primers were then used to validate the presence of miRNA candidates identified by the array in platelets from patients with aggressive disease (n = 10). All RT-qPCR reactions were performed in triplicates using TaqMan 2x Universal PCR Master Mix. Results Eleven of 12 patients in our initial study had castrate-resistant prostate cancer (the remaining one was characterized as having low risk prostate cancer). Patient ages ranged from 57 to 82. Upregulated expression of the following prostate cancer-specific miRNA molecules in platelets were identified: mir-141 (n = 7/12 patients), mir-134 (6/12), mir-181c (7/12), mir-193 (3/12),and mir-132 (7/12). Conclusions Our findings indicate that platelets may potentially represent a novel source of biomarkers in prostate cancer. Citation Format: Brady Miller, Mackenzie Adams, Harene Venghatakrishnan, Maria Giraldez, Muneesh Tewari, Ganesh Palapattu, Alexander Zaslavsky. Platelets possess prostate cancer specific miRNAs: Implications for a novel source of biomarkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2015-3966

Background & Aims Circulating microRNAs (miRNAs/miRs) might be used as biomarkers for the diagnosis of cancer and other diseases. Noninvasive approaches are needed to complement and improve upon current strategies for colorectal cancer (CRC) screening. We investigated whether plasma levels of miRNA can differentiate patients with CRC from healthy individuals. We also investigated whether plasma samples from patients with premalignant neoplastic lesions, such as advanced adenomas (AAs), also had a different expression pattern of miRNAs. Methods We analyzed 196 plasma samples from 123 patients newly diagnosed with sporadic colorectal neoplasia (63 with CRC and 60 with AAs) and 73 healthy individuals (controls) seen at 2 tertiary medical centers in Spain. An initial set of samples was analyzed using a genome-wide miRNA expression profiling assay (n = 61). Quantitative reverse-transcription PCR was used to validate the expression of selected miRNAs in an independent cohort (n = 135). Results Patients with CRC or AAs had plasma miRNA expression profiles that differed significantly from those of controls. We selected a group of 13 miRNAs for validation in an independent cohort of patients; 6 (miR18a, miR19a, miR19b, miR15b, miR29a, and miR335) were confirmed to be significantly up-regulated in patients with CRC, differentiating patients with CRC from controls with area under the receiver operating characteristic curve values ranging from 0.80 (95% confidence interval [CI], 0.71–0.89) to 0.70 (95% CI, 0.59–0.80). Only miR18a was confirmed to be significantly up-regulated in patients with AAs, compared with controls; the area under the receiver operating characteristic curve value was 0.64 (95% CI, 0.52–0.75). Conclusions Patients with CRC have significantly different patterns of miRNA expression than healthy individuals. These patterns might be developed as biomarkers for CRC, although they have limited value in identifying patients with premalignant neoplastic lesions.

Introduccion La multicentricidad en el cancer colorrectal (CCR) se ha relacionado principalmente con los sindromes hereditarios poliposicos y no poliposicos. Sin embargo, hay escasa evidencia de la implicacion de otros factores moleculares en la multicentricidad del CCR, sobre todo de la metilacion genica aberrante. Objetivo Evaluar la posible implicacion de la hipermetilacion genica en la multicentricidad del CCR, determinando el grado de metilacion en la region promotora de genes supresores de tumor implicados en el CCR, como prueba de la existencia de un posible defecto de campo o “field defect” subyacente. Pacientes y metodos Se incluyeron 82 pacientes, 41 con CCR sincronico o metacronico y 41 con CCR unico, apareados por edad y sexo, unos pertenecientes al proyecto Epicolon I y otros recogidos de forma prospectiva en el Servicio de Endoscopia del Hospital Clinic. Los pacientes con sindromes poliposicos, sindrome de Lynch o enfermedad inflamatoria intestinal fueron excluidos. Se extrajo DNA a partir de tejido congelado de mucosa tumoral y mucosa aparentemente sana correspondiente de cada individuo. Tras realizar un primer tratamiento bisulfito, se cuantifico la presencia de metilacion en la region promotora de 7 genes supresores de tumor: MGMT1 (region promotora), MGMT2 (region potenciadora), p16, sFRP1, HPP1, 3OST2, RASSF1A y GATA-4 mediante PCR a tiempo real (tecnica “Methylight”). El analisis de los datos fue realizado de forma cuantitativa y cualitativa mediante analisis univariante y multivariante no parametricos, utilizando como punto de corte el valor de PMR de 4, relacionado con la silenciacion del gen afecto. Resultados El grado de metilacion de todos los genes estudiados fue mayor en el tumor de los pacientes con CCR multiple que en el de los pacientes con CCR unico, siendo esta diferencia estadisticamente significativa para los genes MGMT1 (p=0,026), MGMT2 (p=0,001) y RASSF1A (p=0,01), y permaneciendo significativa tras ajustar por edad y sexo como posibles factores de confusion. El analisis cualitativo de los datos (punto de corte PMR=4) tambien revelo una mayor frecuencia de hipermetilacion en el tumor de los pacientes con CCR multiple comparado con el tumor de aquellos con CCR unico por lo que respecta a MGMT2 (40% vs. 14%, respectivamente; p=0,007), RASSF1A (17% vs. 0%, respectivamente; p=0,006) y GATA-4 (81% vs. 61% respectivamente; p=0,04), permaneciendo significativo tras ajustar por edad y sexo. De forma similar, el grado de metilacion en la mucosa normal correspondiente al tumor de los pacientes con CCR multiple fue mayor que en la mucosa normal de los pacientes con CCR unico en MGMT1, RASSF1A y p16, aunque no alcanzo significacion estadistica. Conclusion Los resultados de este estudio indican que la hipermetilacion genica se asocia a la multicentricidad en el CCR. Este potencial defecto de campo podria tener notables implicaciones en la vigilancia de los pacientes con formas no hereditarias de CCR.

Introduccion Las protesis metalicas autoexpandibles son utilizadas cada vez con mas frecuencia en la resolucion de la obstruccion colorrectal maligna. Sin embargo, se han comunicado complicaciones hasta en el 50% de los pacientes. Objetivos Evaluar retrospectivamente el exito clinico a largo plazo de las protesis metalicas autoexpandibles en pacientes con obstruccion colorrectal maligna en un centro terciario e identificar posibles factores predictores de complicaciones. Pacientes y metodos Durante un periodo de 5 anos se realizaron 47 intentos de colocacion de protesis en 47 pacientes. Las protesis (de 9 cm de longitud y 22 o 25 mm de diametro) se colocaron bajo control endoscopico y radiologico. El correcto posicionamiento de las protesis se comprobo a las 24 h mediante radiografia abdominal. Todos los pacientes fueron seguidos posteriormente de forma ambulatoria. Resultados La colocacion de la protesis se consiguio en 45 casos (96%) y la descompresion colonica inicial fue adecuada en 43 de 47 intentos (91%) y en 43 de los 45 (96%) en los que la protesis se logro implantar con exito. La localizacion de la estenosis fue en recto (7 casos; 15%), sigma (33 casos; 70%), colon izquierdo (4 casos; 9%) y recurrencia anastomotica (3 casos; 6%). La mayoria de los pacientes presentaban un cancer colorrectal en estadio IV (40 casos; 85%). La protesis sirvio como puente a la cirugia en 10 de 47 casos (21%) y como tratamiento paliativo definitivo en 33 de 47 (70%). La incidencia de fallo clinico a largo plazo fue del 47% (22 casos) y se debio a complicaciones: perforacion (4; 8%), obstruccion (10; 21%) y tenesmo (1; 2%). La perforacion ocurrio durante la insercion de la protesis (n=1) y a los 3, 4 y 34 dias post-insercion y todos los casos fallecieron. La tasa de mortalidad acumulada fue del 25% (12 casos) con una mediana de seguimiento de 3 meses. En el grupo de puente a la cirugia, solo se consiguio la realizacion de anastomosis primaria en 4/10 pacientes (27%). El fallo clinico a largo plazo no se asocio con ningun factor relacionado con el tumor. Conclusiones 1. La eficacia de las protesis metalicas autoexpandibles se encuentra limitada por las complicaciones. 2. La ausencia de exito clinico a largo plazo no se asocian con ningun factor relacionado con el tumor. 3. La elevada tasa de complicaciones sugiere que en estos pacientes deberian valorarse otros tratamientos.

Introduccion El cancer colorrectal (CCR) de debut precoz (⩽50 anos) es sugestivo de predisposicion hereditaria. El sindrome de Lynch, causado por mutaciones germinales en los genes de reparacion del DNA ( MLH1 , MSH2 , MSH6 y PMS2 ), es la forma mas frecuente de CCR hereditario. Sin embargo, unicamente un pequeno subgrupo de pacientes con CCR de debut precoz corresponde a esta patologia. Por ello, es fundamental identificar las bases moleculares del CCR en esta poblacion y asi poder establecer estrategias preventivas adecuadas. Objetivos Evaluar las caracteristicas clinicas, histologicas y moleculares de los pacientes con CCR de debut precoz, y analizar la prevalencia de alteracion del sistema de reparacion del DNA en esta poblacion. Pacientes y metodos Se seleccionaron de forma retrospectiva todos los pacientes con diagnostico de CCR a una edad ⩽50 anos que fueron tratados quirurgicamente en 2 centros espanoles entre los anos 95-07. Se recogieron datos demograficos, clinicos y familiares. Se realizo extraccion de DNA de tejido normal y tumoral parafinado de cada paciente. Se analizo inestabilidad de microsatelites (MSI) con el panel de mononucleotidos (BAT25, BAT26, NR21, NR24 y MONO27). Se definio inestabilidad alta (MSI-H) como >2/5 marcadores inestables y estabilidad (MSS) como ⩽2 marcadores inestables. La expresion de las proteinas MLH1, MSH2 y MSH6 fue valorada mediante inmunohistoquimica. BRAFV600E y los hotspots del exon 2 de KRAS se analizaron mediante sondas TaqMan alelo-especificas y secuenciacion directa, respectivamente. Resultados Se incluyeron 83 pacientes con una edad media de 44 anos (rango 23–50); 46 (56%) hombres. El 86% de los tumores estaban localizados en colon izquierdo, incluyendo 39% en el recto. Nueve tumores (11%) mostraron MSI-H y 13 (16%) perdida de expresion proteica (4 para MLH1, 2 para MSH2/MSH6 y 7 para MSH6 de forma aislada). Globalmente, 13 (16%) pacientes mostraron alteracion del sistema de reparacion del DNA, definida por perdida de expresion proteica y/o MSI-H. Todos los tumores con perdida de MLH1 o MSH2 mostraron MSI-H y solo 4 de 7 (60%) de los tumores con perdida aislada de MSH6 mostraron MSI-H. Ademas, se detecto la presencia de mutaciones en KRAS y BRAF en 16 (19,5%) y 3 (3,7%) casos, respectivamente. Las mutaciones de BRAF no se asociaron con perdida de expresion de MLH1. Conclusiones La alteracion del sistema de reparacion del DNA justifica menos del 20% de los casos de CCR de debut precoz. Esta cohorte muestra un perfil molecular de alteracion del sistema de reparacion diferente, caracterizado por perdida aislada de MSH6, baja sensibilidad del analisis de MSI con marcadores mononucleotidos, y baja frecuencia de mutaciones en la via RAS/RAF/ERK. Estas observaciones sugieren una alta frecuencia de mutaciones germinales en MSH6 y cuestionan la realizacion de MSI con el panel de mononucleotidos como metodo de cribado en esta cohorte.

12 p. Objective: Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously. -- Design: We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed 50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated. -- Results: Mean LINE-1 methylation levels (+/- SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p= 65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test). -- Conclusions: LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC. The present work was supported by grants R01 CA72851 and CA129286 from the National Cancer Institute, National Institutes of Health; and funds from the Baylor Research Institute to CRB and AG. FB was supported by a grant from Fundación Alfonso Martín Escudero and from Instituto de Salud Carlos III (PI10/00384). MA was supported by a “Type I Postgraduate Scholarship” given by the CONICET (National Council of Scientific and Technical Investigations) dependent on the Argentine Ministry of Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.