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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.

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.

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.