Co-expression and prognostic significance of putative CSC markers CD44, CD133, wild-type EGFR and EGFRvIII in metastatic colorectal cancer

The presence of colorectal cancer stem cells (CSCs) have been associated with tumour initiation and resistance to therapy. This study investigated the co-expression and prognostic significance of the CSCs biomarkers CD44 and CD133 with wild-type EGFR (wtEGFR) and EGFRvIII in colorectal cancer (CRC). The expression of these biomarkers were determined in tumours from 70 patients with metastatic CRC by immunohistochemistry, and in a panel of human CRC cell lines, and their variants with acquired-resistance to EGFR inhibitors, by flow cytometry. The expression of CD44, CD133, wtEGFR and EGFRvIII were present in 17%, 23%, 26% and 13% of cases and the co-expression of CD44/CD133 with wtEGFR and EGFRvIII were present in 9% and 3% of the cases respectively. Only co-expression of CSCs/EGFRvIII (P = 0.037), and amphiregulin (P = 0.017) were associated with worse overall survival. Interestingly, disease-free survival was improved in BTC expressing patients (P = 0.025). In vitro CD133 expression and its co-expression with CD44 were associated with primary-resistance to irinotecan and acquired-resistance to anti-EGFR inhibitors respectively. Our results suggest co-expression of CSCs and EGFRvIII could be potential biomarkers of worse overall survival and resistance to therapy in patients with mCRC and warrants further validation in a larger cohort

The biological and clinical significance of HER family members and cancer stem cells in colorectal cancer and response to therapeutic interventions

Despite the approval of the anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) cetuximab and panitumumab for the treatment of colorectal cancer patients, there is currently no reliable predictive marker for response to therapy. EGFR is the prototype of human epidermal growth factor receptor (HER) family, which includes three additional members namely HER-2, HER-3 and HER-4. The aim of this study was to investigate the sensitivity of a panel of human colorectal cancer cell lines to treatment with afatinib, an irreversible pan-HER blocker, the EGFR tyrosine kinase inhibitors (TKls) gefitinib and erlotinib, anti-EGFR mAb ICR62 and cytotoxic drugs and to determine whether there was any association between the expression levels of HER family members, the putative cancer stem cell (CSC) markers CD44 and CD133 and ABC transporters (e.g. p-glycoprotein) and response to these agents. The expression pattern and clinical significance of these markers in tumour specimens from 86 Dukes' C and D colorectal cancer patients was determined. Overexpression of the HER family members were found to be uncommon in this panel of human colorectal cancer cell lines, except for EGFR overexpression in DiFi cells. Of the HER inhibitors, afatinib was the most effective agent for inhibiting the growth in vitro of human colorectal cancer cell lines and the expression of HER -4 alone and the co-expression of all the HER family members were associated with response to treatment with afatinib. Acquired resistance of the EGFR overexpressing colorectal cancer cells to treatment with afatinib, ICR62 or gefitinib was accompanied by the up-regulation of EGFR. However, tumour cells that acquired resistance to anti-EGFR mAb ICR62 remained sensitive to treatment with afatinib and vice versa, suggesting that mechanisms underlying the antitumor activity of ICR62 and afatinib are different. The expression ofEGFR, HER-2, HER-3, HER-4 and co-expression of all the HER family members was found in 42.5%, 77%, 52%, 91 % and 18% of the tumour specimens from 86 Dukes' C and D colorectal cancer patients respectively. While all the patients expressed COX-2 and Ki-67, the expression ofCD44 and CD133 was found in 58.6% and 81.9% of the cases, respectively. The expression of EGFR and low expression of Ki-67 were found to be significantly associated with poor disease free survival. The co-expression of HER family members in colorectal cancer patients provides a rationale for investigating the pattern, prognostic significance and predictive value of the HER family members for response to therapy with anti-EGFR mAbs. Further investigations in vivo on the therapeutic potential of pan-HER blockers, such as afatinib, in colorectal cancer patients whose tumours co-express more than one member of the HER family should also be considered

Prognostic significance and targeting of HER family in colorectal cancer

Colorectal cancer is one of the leading causes of cancer deaths. At present, anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) cetuximab and panitumumab and anti-vascular endothelial growth factor (VEGF) mAb bevacizumab have been incorporated into treatment paradigms for patients with refractory metastatic colorectal cancer. However, many patients simply do not respond to these treatments or eventually acquire resistance following a short course therapy. In this article, we review the literature for studies on the expression patterns, prognostic significance and predictive value of HER (also called erbB) family members and other factors for response to therapy with the HER inhibitors in colorectal cancer. We discuss some of the advances, challenges as well as future opportunities for more effective targeting of the HER receptors using a cocktail of HER inhibitors (e.g. dual and pan HER TKIs, monospecific or bispecific antibodies) in combination with other therapeutic interventions

Growth response of human colorectal tumour cell lines to treatment with afatinib (BIBW2992), an irreversible erbB family blocker, and its association with expression of HER family members

Despite the approval of the anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs), cetu-ximab and panitumumab, for the treatment of colorectal cancer patients, there is currently no reliable predictive marker for response to therapy. In addition, the duration of response is often limited. Therefore, this study aimed to investigate the effect of afatinib, an irreversible erbB family blocker, as a single agent or in combination with cytotoxic drugs (5-fluor-ouracil, irinotecan and oxaliplatin) or mAb ICR62 on the proliferation of a panel of human colorectal tumour cell lines and the association between the expression levels of the EGFR family members and response to treatment. Of the cells examined, EGFR-overexpressing DiFi cells were the most sensitive to treatment with both afatinib (IC50=45 nM) and ICR62 (IC50=4.33 nM). Afatinib also inhibited the growth of other tumour cell lines with IC50 values which ranged from 0.33 µM (CCL-221) to 1.62 µM (HCT-116). A significant asso-ciation was found between the co-expression of EGFR, human epidermal growth factor receptor (HER)-2 and HER-3 and response to treatment with afatinib (R=0.915, P=0.021). Treat-ment with afatinib and cytotoxic drugs was accompanied by an increase in the proportion of these cells in the sub-G0/G1 and in the S and G2/M phase of the cell cycle, respectively. We conclude that afatinib as monotherapy or in combination with other drugs shows activity in colorectal tumour cells and that determination of the co-expression of HER family members should be conducted in clinical trials using drugs targeting erbB signaling. This approach could lead to the identification of a specific subpopulation of cancer patients more likely to benefit from erbB-directed therapy

Co-expression of HER family members in patients with Dukes' C and D colon cancer and their impacts on patient prognosis and survival.

The human epidermal growth factor receptor (EGFR) is an important therapeutic target in patients with metastatic colorectal cancer and anti-EGFR antibodies cetuximab and panitumumab have been approved for the treatment of such patients. Despite these advances, the duration of response in some patients can be limited. Since, EGFR is capable of forming heterodimers with the other members of the HER (Human epidermal receptor) family, it is important to investigate the co-expression and prognostic significance of all members of the HER family in colorectal cancer patients. The expression of the HER family members were determined in tumour specimens from 86 patients with Dukes' C and D (metastatic) colon cancer using immunohistochemistry. Sections were scored by the percentage of positive tumour cells and intensity of staining. Their associations with clinicopathological parameters, and overall survival and disease free survival were evaluated using univariate and multivariate analysis. Overall, 43%, 77%, 52% and 92% of the cases were EGFR, HER-2, HER-3 and HER-4 positive respectively. Interestingly, 35%, 24%, 43%, and 18% of the cases had co-expression of EGFR/HER-2, EGFR/HER-3, EGFR/HER-4 and all four members of the HER family respectively. Of these, only the expression of EGFR and co-expression of EGFR/HER-4 were associated with poorer disease-free survival in both univariate and multivariate analysis. Co-expression of all members of the HER family in colon cancer supports the need for further investigations on their predictive value for response to therapy with anti-EGFR mAbs and whether such sub-population of patients may benefit from therapy with the new generation of pan-HER inhibitors

The association between the expression of the HER family members and disease-free survival in univariate and multivariate analysis.

<p>P-value of <i>≤0.05</i> was considered significant.</p

The association between EGFR and EGFR/HER-4 and disease-free survival in Dukes’ C and D colon cancer patients.

<p>Kaplan-Meier survival curves showing the impact on the disease-free survival of the patients with EGFR expression (A), membranous EGFR expression (B), EGFR 1+ immunostaining (C) and EGFR/HER-4 co-expression (D). A log-rank test value of <i>P≤0.05</i> was considered statistical significance.</p

The co-expression of all HER family members in a patient with Dukes’ C colon cancer.

<p>Co-expression of EGFR, HER-2, HER-3, and HER-4 in a formalin fixed paraffin embedded tumour sections stained immunohistochemically as described under methods and patients section. Magnification: ×200.</p

Clinicopathological parameters and survival of Dukes’ C and D colon cancer patients.

<p>Overall survival and disease-free survival relative to the indicated features was determined by Kaplan-Meier analysis and the log-rank test. P-value of <i>≤0.05</i> was considered significant.</p