DOI: 10.5061/dryad.h35p4
title: Data from: High sensitivity isoelectric focusing to establish a signaling biomarker for the diagnosis of human colorectal cancer

description: Background: The progression of colorectal cancer (CRC) involves recurrent
 amplifications/mutations in the epidermal growth factor receptor (EGFR)
 and downstream signal transducers of the Ras pathway, KRAS and BRAF.
 Whether genetic events predicted to result in increased and constitutive
 signaling indeed lead to enhanced biological activity is often unclear
 and, due to technical challenges, unexplored. Here, we investigated
 proliferative signaling in CRC using a highly sensitive method for protein
 detection. The aim of the study was to determine whether multiple changes
 in proliferative signaling in CRC could be combined and exploited as a
 “complex biomarker” for diagnostic purposes. Methods: We used robotized
 capillary isoelectric focusing as well as conventional immunoblotting for
 the comprehensive analysis of epidermal growth factor receptor signaling
 pathways converging on extracellular regulated kinase 1/2 (ERK1/2), AKT,
 phospholipase Cγ1 (PLCγ1) and c-SRC in normal mucosa compared with CRC
 stage II and IV. Computational analyses were used to test different
 activity patterns for the analyzed signal transducers. Results: Signaling
 pathways implicated in cell proliferation were differently dysregulated in
 CRC and, unexpectedly, several were downregulated in disease. Thus, levels
 of activated ERK1 (pERK1), but not pERK2, decreased in stage II and IV
 while total ERK1/2 expression remained unaffected. In addition, c-SRC
 expression was lower in CRC compared with normal tissues and
 phosphorylation on the activating residue Y418 was not detected. In
 contrast, PLCγ1 and AKT expression levels were elevated in disease.
 Immunoblotting of the different signal transducers, run in parallel to
 capillary isoelectric focusing, showed higher variability and lower
 sensitivity and resolution. Computational analyses showed that, while
 individual signaling changes lacked predictive power, using the
 combination of changes in three signaling components to create a “complex
 biomarker” allowed with very high accuracy, the correct diagnosis of
 tissues as either normal or cancerous. Conclusions: We present techniques
 that allow rapid and sensitive determination of cancer signaling that can
 be used to differentiate colorectal cancer from normal tissue.
description: Raw Protein
 RPAsThe relative peak area
 (RPA), i.e. peak area value of the 23 different activity levels of the 7
 signal transducers after normalization to the HSP70 level analyzed in
 parallel in each sample (columns 2-24). This file contains one sample per
 row and one protein per column with the sample name in the first column
 and the protein name in the first row. Columns 25-27 contain the result of
 the mutation analysis of KRAS and BRAF. One in column 25 (MutationKRAS)
 indicate that KRAS is mutated in the sample, one in column 26
 (MutationBRAF) indicates that BRAF is mutated, while one in column 27
 (Wildtype) indicate that neither KRAS nor BRAF is mutated. A one in the
 binary variables in column 28-31 indicate the classification of each
 sample as normal mucosa, colorectal cancer (CRC) stage II, CRC stage IV,
 or metastasis. NaN is used to indicate that no measurement was
 done.Padhan
 BMC Cancer 2016 Raw Protein
 RPAs.csvRaw Protein RPAs
 Constructed features replicate correctedFile shows the relative
 peak area (RPA), i.e. peak area value of the measured protein after
 normalization to the HSP70 level analyzed in parallel in each sample
 (columns 2-24, 28-42). This file contains one sample per row and one
 protein per column with the sample name in the first column and the
 protein name in the first row. Column 25-27 contain the result of the
 mutation analysis of KRAS and BRAF. One in column 25 (MutationKRAS)
 indicate that KRAS is mutated in the sample, one in column 26
 (MutationBRAF) indicates that BRAF is mutated, while one in column 27
 (Wildtype) indicate that neither KRAS nor BRAF is mutated. Columns 28 to
 42 contain the RPA values of the constructed features, i.e. features that
 are calculated based on the 23 different activity levels of the 7 signal
 transducers in column 2-24. The four replicates of each constructed
 feature contains the minimum, maximum, mean, and median value based on all
 possible ways to combine the replicates of the proteins used to construct
 the feature. A one in the binary variables in column 43-46 indicate the
 classification of each sample as normal mucosa, colorectal cancer (CRC)
 stage II, CRC stage IV, or metastasis. In the last column the
 classification is 1 = normal mucosa, 2 = colorectal cancer (CRC) stage II,
 3 = CRC stage IV, or 5 = metastasis. NaN is used to indicate that no
 measurement was done.Padhan BMC Cancer 2016 Raw
 Protein RPAs Constructed features replicate
 corrected.csvPadhan BMC Cancer 2016
 Supplemental ResultsFile contains the test
 statistic (T) for each possible combination of 1-3 features, including the
 constructed features (column 5 and 6). One combination is shown per row
 with the name of the feature combination in the first column and the
 header explaining the value in each column in the first
 row.