Developing single molecule methods for measuring the pathway proteins ERK, AKT, cyclin d and p70s6k in localized colon cancer in relation to mutation status

Background The aim of this study was to quantify the intracellular pathway proteins ERK, AKT, cyclin d and p70s6k in localized colon cancer tissue to investigate the possible prognostic values and the ability to be used as screening markers for upstream mutations. Methods Colon cancer tissue and autologous reference tissue were collected from 176 patients who underwent surgery for colon cancer. Assays for quantifying ERK, AKT, cyclin d and p70s6k proteins were developed using single molecule array (Simoa). KRAS/BRAF/PIK3CA mutation status was determined using droplet digital PCR. Results Patients with BRAF mutations had decreased concentrations of ERK (p=0.0002), AKT (p=0.00004) and cyclin d (p=0.001) while no significant differences were found between patients with KRAS mutations and Wild type (Wt) patients. None of the investigated protein concentrations were associated with disease free survival or overall survival, if including all patients. However, when stratifying according to mutation status, significant correlations to overall survival were seen for patients with BRAF mutations and AKT (p=0.003) or ERK (p=0.046) and for patients with KRAS mutations and p70s6k (p=0.04). Furthermore, the combination of genetic mutations, stage 2 disease, and all of the investigated pathway proteins showed significant correlations to overall survival. Conclusions There is a strong correlation between pathway protein concentrations and mutational BRAF status. Overall survival in colon cancer patients depend both on gene mutation status and pathway protein concentrations. As significant correlations were found between BRAF mutations and ERK, AKT and cyclin d, concentration measurements of these pathway proteins might be useful as screening for upstream mutations.


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The intracellular signaling network of the epidermal growth factor receptor (EGFr) consists of two

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The use of inhibitors against growth factor receptors and tyrosine kinase activators has become 79 standard anti-cancer therapy during the last 10 to 15 years. Some of these monoclonal antibodies 80 used in the treatment are Cetuximab as a blocker to EGFr in colorectal cancer and the Trastuzumab 81 HER2 receptor blocker in breast cancer. Mutations in the receptor proteins or in the pathway 82 proteins result in resistance to treatment using these monoclonal antibodies. It is therefore important 83 to detect such mutations at an early phase before treating the patients as it can be predicted whether 84 the treatment will be without effect and thus the patients will only experience side effects of the 85 treatment.

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Usually known mutations are diagnosed using PCR or sequencing which are laborious, time 87 consuming, expensive and causes a delay of several days for reporting. In the future, the number of 88 mutations in the pathway proteins will increase and hence will the expense for sequencing or 89 detecting mutations by PCR. Alternative methodology to detect activation of the intracellular 90 pathway proteins could gain importance especially if such methods would be both faster and 91 considerable cheaper. 92 We therefore aimed to see whether quantification of pathway proteins in colon cancer tissue might 93 reflect upstream mutations and to detect whether changes in concentrations might be correlated to 94 effect of treatment or clinical outcome. We developed quantitative protein assays for measuring 95 phosphorylated ERK (pERK) as a marker of MAPK pathway activation, phosphorylated AKT 96 (pAKT) for PI13K/AKT pathway activation and phosphorylated p70s6k (pp70s6k) for mTOR 97 activation. Also the total protein levels of ERK (tERK), AKT (tAKT), and cyclin d were measured.     The six analyses were developed as single-plex assays using a Simoa 2-step assay for tAKT and 141 pAKT and a 3-step assay for tERK, pERK, pp70s6k and cyclin d. Before running the following      (Table 1). For determining the intra-assay CV%, controls 11 212 were analyzed in replicates of at least 6 in one assay and the total CV% was calculated from runs 213 from different days (Table 1). Autologous reference tissue and cancer tissue. 219 tERK, pERK, tAKT, pAKT, cyclin d and pp70s6k were measured in both autologous reference 220 tissue and colon cancer tissue (Fig 3 and   The median and range are shown for each pathway protein.  Correlations between the pathway proteins.

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Associations between the pathway proteins in autologous reference tissue or cancer tissue were 234 investigated. The Spearman's rank correlation coefficients are shown in Table 3. In the autologous 235 reference tissue the correlation between tAKT and pp70s6k was statistically significant (p= 0.0003).

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All other cases also showed statistically significant correlations (p<0.000001). Furthermore 237 significant correlations were found between cancer tissue and autologous reference tissue regarding 238 tAKT (p=0.003) and cyclin d (p=0.03).      tumors and differences in prognostic value in the two groups may be possible. Also method 308 differences, mutation data, sample sizes are some of the factors that might explain the divergent 309 results.

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Cancer tissues with genetic mutations result in a constitutive activation of the intracellular 311 pathways. It is therefore plausible, that an increased activation and turn-over may lead to an over-312 production or consumption of native proteins as seen in the complement and coagulation pathway 313 cascades. In this study there was an overall statistically significant correlation between pathway 314 protein concentrations and mutational status. However, the change in pathway protein 315 concentrations is too small to be used as screening indicators for mutations in practical medical use 316 and our hypothesis could therefore not be confirmed. As in the complement and coagulation 317 pathways the correct way to detect an increase in activity is to quantify not the native proteins but 318 degradation or split products from the single intracellular pathway proteins. Therefore, we now aim 319 to develop specific antibodies and methods to measure these degradation products, as previously 320 done for complement C3d [28;29]. We believe this approach will increase both the prediction of 321 mutations and survival.