BackgroundAbout half of Americans 50 to 75 years old do not follow recommended colorectal cancer (CRC) screening guidelines, leaving 40 million individuals unscreened. A simple blood test would increase screening compliance, promoting early detection and better patient outcomes. The objective of this study is to demonstrate the performance of an improved sensitivity blood-based Septin 9 (SEPT9) methylated DNA test for colorectal cancer. Study variables include clinical stage, tumor location and histologic grade.MethodsPlasma samples were collected from 50 untreated CRC patients at 3 institutions; 94 control samples were collected at 4 US institutions; samples were collected from 300 colonoscopy patients at 1 US clinic prior to endoscopy. SEPT9 methylated DNA concentration was tested in analytical specimens, plasma of known CRC cases, healthy control subjects, and plasma collected from colonoscopy patients.ResultsThe improved SEPT9 methylated DNA test was more sensitive than previously described methods; the test had an overall sensitivity for CRC of 90% (95% CI, 77.4% to 96.3%) and specificity of 88% (95% CI, 79.6% to 93.7%), detecting CRC in patients of all stages. For early stage cancer (I and II) the test was 87% (95% CI, 71.1% to 95.1%) sensitive. The test identified CRC from all regions, including proximal colon (for example, the cecum) and had a 12% false-positive rate. In a small prospective study, the SEPT9 test detected 12% of adenomas with a false-positive rate of 3%.ConclusionsA sensitive blood-based CRC screening test using the SEPT9 biomarker specifically detects a majority of CRCs of all stages and colorectal locations. The test could be offered to individuals of average risk for CRC who are unwilling or unable to undergo colonscopy.

Mutational analysis of KRAS codons 12 and 13 is standard for patients with metastatic colorectal cancer since mutations in these codons predict lack of response to anti-EGFR therapies. However, even among patients whose tumors are wildtype for KRAS codons 12 and 13, only a subset respond to therapy. Since additional activating mutations downstream of EGFR may also play a role in treatment resistance, we sought to establish the frequency of these mutations. We evaluated 2121 colorectal tumors for mutations in codons 12 and 13 of the KRAS gene. A subset of these samples, comprised of 513 samples wildtype for KRAS codons 12 and 13, were tested for mutations in codons 61 and 146 of KRAS, codon 600 of BRAF, and codons 12, 13, and 61 of NRAS. Mutation status was determined by targeted pyrosequencing. Mutations in KRAS codon 12 or 13 were identified in 900/2121 (42.4%) samples. Of the 513 wildtype samples tested for additional mutations, 78 samples were mutant for BRAF, 19 for KRAS codon 61, 17 for KRAS codon 146, and 26 for NRAS. In total, 140/513 (27.3%) tumors wildtype for KRAS codons 12 and 13 harbored a mutation in another of the RAS pathway genes. While further study is needed to determine the full therapeutic implications of mutations in these codons, mutational testing of these codons may be useful for identifying a significant proportion of patients who may also be resistant to anti-EGFR therapies.

There exists a need for robust approaches for tandem mass spectrometry (MS/MS)-based identification of proteins in formalin-fixed paraffin-embedded (FFPE) material. We demonstrate herein the identification of proteins in FFPE material using enzymatic cleavage for extraction of peptides from the FFPE specimen and liquid chromatography (LC) followed by MS/MS. We identified 324 proteins from a 3-year-old FFPE cell-block of a human lymphoma cell line. The identified proteins were assigned to the membrane, cytosol and nucleus, with diverse cellular functions. The results were comparable to those obtained with lysates from a fresh specimen of the lymphoma cell line. Western blotting analysis and immunofluorescence microscopy confirmed the expression of selected proteins. The functional significance of one protein (PKC g) was validated using a PKC inhibitory peptide which resulted in lymphoma cell death in vitro. The ability to identify proteins from FFPE specimens has significant implications for MS/MS-based proteomics of vast repositories of archival primary tissue samples for disease-related discovery research. Laboratory Investigation ( Keywords: bottom-up proteomics; formalin fixed; paraffin embedded; tandem mass spectrometry Formalin fixation and tissue embedding in paraffin wax is a universal approach for tissue processing prior to light microscopic evaluation. A major advantage afforded by formalin-fixed paraffin-embedded (FFPE) specimens is the preservation of cellular and architectural morphologic detail in tissue sections. 1 The standard buffered formalin fixative in which biopsy specimens are processed is an aqueous solution containing 37% formaldehyde and 10-15% methyl alcohol. Formaldehyde is a highly reactive dipolar compound that results in the formation of protein-nucleic acid and proteinprotein crosslinks in vitro. [1][2][3][4][5] FFPE specimens have not been routinely used for global mass spectrometry-based proteomic studies. This relates to the fact that formaldehyde-induced crosslinking also renders proteins relatively insoluble and unsuitable for routine biochemical extraction and analysis. This crosslinking effect has precluded protein extraction from FFPE for routine Western blotting analysis of protein expression. 2 Thus, while recently developed enzymatic and heatinduced antigen retrieval methods have facilitated the immunohistologic detection of proteins in tissue, many proteins are still not detectable using these approaches. 6,7 In addition, proteins lacking widely available antibodies to their formalin-resistant epitopes are largely undetectable by immunohistochemistry. Although recent advances in molecular biotechnology have permitted the global analysis of proteins expressed in various cellular and tissue systems, the diminished capacity to extract intact proteins has largely precluded the proteomic analysis of FFPE samples with definitive identification of the proteins present within cells and tissues in biopsy material.The development of strategies to permit utilization of the universa...

Germline mutation detection in PMS2, one of four mismatch repair genes associated with Lynch syndrome, is greatly complicated by the presence of numerous pseudogenes. We used a modification of a long-range PCR method to evaluate PMS2 in 145 clinical samples. This modification avoids potential interference from the pseudogene PMS2CL by utilizing a long-range product spanning exons 11-15, with the forward primer anchored in exon 10, an exon not shared by PMS2CL. Large deletions were identified by MLPA. Pathogenic PMS2 mutations were identified in 22 of 59 patients whose tumors showed isolated loss of PMS2 by immunohistochemistry (IHC), the IHC profile most commonly associated with a germline PMS2 mutation. Three additional patients with pathogenic mutations were identified from 53 samples without IHC data. Thirty-seven percent of the identified mutations were large deletions encompassing one or more exons. In 27 patients whose tumors showed absence of either another protein or combination of proteins, no pathogenic mutations were identified. We conclude that modified long-range PCR can be used to preferentially amplify the PMS2 gene and avoid pseudogene interference, thus providing a clinically useful germline analysis of PMS2. Our data also support the use of IHC screening to direct germline testing of PMS2.