Said Abdullah Khelwatty

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), cetuximab 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-fluorouracil, 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 association 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.

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.

Acquired resistance to anti-EGFR mAb ICR62 in cancer cells is accompanied by an increased EGFR expression, HER-2/HER-3 signalling and sensitivity to pan HER blockers

Background:The human epidermal growth factor receptor (EGFR) is an important target for cancer treatment. Currently, only the EGFR antibodies cetuximab and panitumumab are approved for the treatment of patients with colorectal cancer. However, a major clinical challenge is a short-term response owing to development of acquired resistance during the course of the treatment.Methods:In this study, we investigated the molecular mechanisms underlying development of acquired resistance in DiFi colorectal cancer cells to the anti-EGFR mAb ICR62 (termed DiFi62) and to the small molecule tyrosine kinase inhibitor (TKI) gefitinib (termed DiFiG) using a range of techniques.Results:Compared with the findings from parental DiFi and DiFiG cells, development of acquired resistance to anti-EGFR mAb ICR62 in DiFi62 cells was accompanied by an increase in cell surface EGFR and increased phosphorylation of HER-2 and HER-3. Interestingly, DiFi62 cells also acquired resistance to treatment with anti-EGFR mAbs cetuximab and ICR61, which bind to other distinct epitopes on the extracellular domain of EGFR, but these cells remained equally sensitive as the parental cells to treatment with pan-HER inhibitors such as afatinib.Conclusions:Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed.

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.

Immunohistochemical discrimination of wild-type EGFR from EGFRvIII in fixed tumour specimens using anti-EGFR mAbs ICR9 and ICR10.

Background:The human epidermal growth factor receptor (EGFR) is an important therapeutic target in oncology, and three different types of EGFR inhibitors have been approved for the treatment of cancer patients. However, there has been no clear association between the expression levels of EGFR protein in the tumours determined by the FDA-approved EGFR PharmDx kit (Dako) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors.Method:In this study, we investigated the potential of our anti-EGFR monoclonal antibodies (mAbs; ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild-type EGFR and/or the type-III deletion mutant form of EGFR (EGFRvIII) in formalin-fixed, paraffin-embedded human tumour specimens.Results:We found that the anti-EGFR mAb in the EGFR PharmDx kit stained both wild-type and EGFRvIII-expressing cells in formalin-fixed, paraffin-embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR10 and ICR9 were specific for the wild-type EGFR.Conclusion:We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild-type EGFR protein in tumour specimens using immunohistochemistry, and to determine their prognostic significance, as well as predictive value for response to therapy with EGFR antibodies.

The impact of co-expression of wild-type EGFR and its ligands determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer

Anti-EGFR mAbs cetuximab and panitumumab are routinely used for the treatment of patients with KRAS-wild type metastatic colorectal cancer (mCRC). However, in some patients their efficacy remains modest and with no clear association between the EGFR protein expression determined by PharmDx™ kit, and response to anti-EGFR therapies. Therefore, we investigated the relative expression and predictive value of wild-type EGFR (wtEGFR), mutated EGFRvIII and EGFR ligand proteins in mCRC patients treated with cetuximab. The expression levels of wtEGFR, EGFRvIII, and EGFR ligand were determined by immunohistochemistry (IHC) in 60 tumour specimens using specific antibodies. Sections were scored according to the percentage of positive tumour cells, intensity and cellular location of staining, and these were associated with response, overall survival (OS) and progression-free survival (PFS). At cut-off value > 5%, wtEGFR, and EGFRvIII were present in 44%, and 41%, betacellulin (BTC) in 72%, followed by epigen (67%), TGFα (58%), amphiregulin (34%), EGF (31%) of the cases, respectively and 96% of the wtEGFR positive cases had co-expression of at least one ligand. We found a significant association between the expression of wtEGFR and poor response to cetuximab. In addition, the co-expression of wtEGFR with one ligand at a cut-off value of > 5% and > 10% was associated with worse response to cetuximab (P = 0.021, and P = 0.005 respectively). We found a 3-fold and 5-fold increased risk of shorter OS with expression of BTC and epigen. Interestingly, the expression of wtEGFR and its co-expression with one or two ligands was associated with shorter PFS but not with OS. The relative expression of wtEGFR and its competing ligands, which is the target for therapeutic interventions with anti-EGFR antibodies, could serve as a more reliable predictive biomarker of response to therapy with anti-EGFR mAbs in mCRC patients and warrants further investigation in large prospective studies.

Co-expression and prognostic significance of the HER family members, EGFRvIII, c-MET, CD44 in patients with ovarian cancer

EGFR and HER-2 are important targets but none of the monoclonal antibodies or small molecule tyrosine kinase inhibitors specific for the HER members has been approved for the treatment of patients with ovarian cancers. In some studies, co-expression of other growth factor receptors has been associated with resistance to therapy with the HER inhibitors. The aim of the present study was to determine the relative expression, cellular location, and prognostic significance of HER-family members, the EGFR mutant (EGFRvIII) c-MET, IGF-1R and the cancer stem cell biomarker CD44 in 60 patients with FIGO stage III and IV ovarian cancer. At cut off >5% of tumour cells with positive staining, 62%, 59%, 65% and 45% of the cases were EGFR, HER-2, HER-3 and HER-4 positive, and 3%, 22% and 48.3% of the cases were positive for EGFRvIII, c-MET, and CD44 respectively. Interestingly, 23% co-expressed all four members of the HER family. On univariate analysis, only EGFR staining at >50% of tumour cells (HR = 3.57, p = 0.038) and CD44 staining at 3+ intensity (HR = 7.99, p = 0.004) were associated with a poorer overall survival. EGFR expression (HR = 2.83, p = 0.019) and its co-expression with HER-2, HER-3, HER-2/HER-3, and c-MET were all associated with poorer disease-free survival. Our results suggest co-expression of the HER-family members is common in Stage III and IV ovarian cancer patients. Further studies on the prognostic significance and predictive value of all HER family member proteins for the response to treatment with various forms of the HER inhibitors are warranted.

Abstract 5014: Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer

The aberrant expression of the epidermal growth factor receptor (EGFR) has been reported in patients with colorectal cancer, and EGFR is an important therapeutic target in such patients. To date, of the anti-EGFR monoclonal antibodies (mAbs), only cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer (mCRC). While treatment with a combination of antibodies and cytotoxic drugs improves response rate and median time to progression in some colorectal cancer patients, the duration of response is often limited. In addition, no clear association has been found between the expression level of the EGFR protein in the tumours, determined by the FDA-approved EGFR PharmDx kit, or other standard anti-EGFR antibodies, and the response to the EGFR inhibitors. We have previously shown that kits such as the EGFR PharmDx™ kit, used to determine the expression of EGFR, does not discriminate between the wild-type EGFR (wtEGFR) and the type III deletion mutant EGFR (EGFRvIII), and as such could have contributed to the lack of association between the expression of EGFR and response to anti-EGFR antibodies. Therefore, the aim of this study was to determine the predictive value of wtEGFR for response to therapy with cetuximab. The expression levels of wtEGFR, mutated EGFRvIII, and phosphorylated EGFR (pEGFR Tyr1068 & Tyr1173) were determined using immunohistochemistry and specific antibodies in 61 FFPE tumour specimens from patients with mCRC treated with cetuximab. Sections were scored by the percentage of positive tumour cells, location and intensity of staining. Their associations with response and progression free survival were evaluated using Chi-squared and Kaplan-Meier survival curves and log-rank test respectively. Overall 11% and 46% of the cases were found to express membranous and cytoplasmic EGFR, using mAb specific for wtEGFR (DAK-H1-WT). On the other hand using anti EGFR.113 clone antibody, which does not discriminate between the wtEGFR and EGFRvIII, 11.5% and 18% of the cases were found to express the cytoplasmic and membranous EGFR respectively. While all cases were negative for the pEGFR, the expression of EGFRvIII was mainly cytoplasmic, occurring in 41% of the cases examined. Of these, only the cytoplasmic expression of wtEGFR was found to be significantly associated with poor response to treatment (P = 0.002) and progression free survival (95% CI, 0-3 months vs 4.5-17 months, P = 0.001). Taken together, our results suggest that cytoplasmic expression of wtEGFR is associated with a significantly shorter progression free survival, and is a predictive marker for poor response to treatment with the anti-EGFR mAb cetuximab. These findings underlie the need for further investigations, in a larger population of patients, to validate the predictive value of wtEGFR for response to therapy with anti-EGFR antibodies in patients with mCRC. Citation Format: Said Abdullah Khelwatty, Sharadah Essapen, Izhar Bagwan, Margaret Green, Alan Seddon, Helmout Modjtahedi. Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5014.

Abstract 5631: Acquired resistance to anti-EGFR mAb ICR62 or afatinib is accompanied by upregulation of the EGFR in colorectal cancer .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The aberrant expression and activation of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (also called HER or erbB family) have been implicated in a wide range of cancers. These receptors have emerged as important therapeutic targets in human cancers. To date, of the two classes of HER inhibitors (i.e. monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors (TKIs), cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer. However, many patients develop resistance to the EGFR inhibitors and consequently the duration of response to these treatments is often short. In this study on colorectal cancer cells, we evaluated whether there was any association between the expression levels of the HER family members, putative colorectal cancer stem cell (CSC) markers CD133 and CD44, and ABC transporter and the development of resistance to anti-EGFR mAb ICR62 or afatinib, an irreversible erbB blocker. A wide range of techniques were employed in this study including FACS analysis, Western blot analysis and SRB assay on the EGFR over-expressing human colorectal tumour cell line DiFi, which is highly sensitive to treatment with mAb ICR62 (IC50=1.2 nM) and afatinib (IC50=10 nM). Continuous exposure of DiFi cells to increasing doses of mAb ICR62 or afatinib for 6-10 months resulted in the emergence of ICR62 variant DiFi cells (DiFi62) and afatinib-variant DiFi cells (DiFiAf) with IC50 values of 209 nM and 35 nM respectively. Interestingly, we found that ICR62 resistant DiFi cells (DiFi62) also become less sensitive to treatment with 5FU, oxaloplatin and irinotecan, but remained highly sensitive to treatment with afatinib (IC50=8 nM). In contrast to DiFi62 cells, afatinib-resistant DiFiAf cells were less sensitive to treatment with 5FU and irinotecan, but like parental DiFi cells remained highly sensitive to treatment with ICR62 and oxaliplatin. Of the HER family members, there was only upregulation of EGFR in both DiFi62 and DiFiAf cells, but no changes in the expression levels of other members of the HER family or CD133. In contrast, the expression of CD44, the other putative colorectal CSC marker, was found to be downregulated by around 38% in DiFiAf cells. Of the ABC transporters, there was an increase in the expression of p-glycoprotein in both DiFi62 and DiFiAf cells and an increased expression of both MRP2 and ABCG2 in DiFi62, but not in DiFiAf cells. Taken together, our results suggest that colorectal tumour cells which acquire resistance to anti-EGFR mAb ICR62 remain sensitive to treatment with the irreversible erbB blocker afatinib and vice versa, suggesting that mechanisms underlying the antitumor activity of ICR62 and afatinib are different in these cells. In addition, upregulation of the EGFR upon treatment with these inhibitors may contribute to resistance. Citation Format: Said Abdullah Khelwatty, Sharadah Essapen, Alan M. Seddon, Zhen Fan, Helmout Modjtahedi. Acquired resistance to anti-EGFR mAb ICR62 or afatinib is accompanied by upregulation of the EGFR in colorectal cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5631. doi:10.1158/1538-7445.AM2013-5631

Abstract 694: Immunohistochemical discrimination of wild type EGFR from EGFRvIII in fixed tumor specimens using anti-EGFR mAbs ICR9 and ICR10

Abnormal expression of the epidermal growth factor receptor (EGFR) has been reported in a wide range of human epithelial malignancies and in some studies has been associated with poor prognosis and treatment resistance. Currently, three different types of EGFR inhibitors have been approved by the FDA for the treatment of patients with head and neck, metastatic colorectal, pancreatic or breast cancers: anti-EGFR monoclonal antibodies [(mAbs), cetuximab and panitumumab], small molecule EGFR tyrosine kinase inhibitors [(TKIs) gefitinib, erlotinib] or a dual EGFR and HER-2 tyrosine kinase inhibitor (lapatinib) However, there has been no clear association between the expression levels of EGFR in the tumours determined by the FDA approved EGFR PharmDx™ kit (DakoCytomation) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors. One reason could be that the antibody used in the diagnosis of EGFR is not specific to the wild type EGFR and can also bind to the type-III mutant form of EGFR (EGFRvIII). We have extensively profiled our unique panel of high affinity rat anti-EGFR mAbs for use in the diagnosis and therapy of human cancers. The aim of the present investigation was to evaluate the potential of several of these antibodies (ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild type EGFR and/or EGFRvIII in formalin fixed paraffin embedded tumour specimens. We prepared 10% (v/v) neutral buffered formalin paraffin embedded sections of HN5 and HC2 20d2/c pelleted cells, which overexpress wild type EGFR and EGFRvIII respectively. Tumour sections were incubated with primary anti-EGFR mAbs followed by HRP linked secondary antibody (ABD Serotec Ltd., UK). The sections were then treated with DAB+ substrate-chromogen solution (Dako), counterstained with haematoxylin and mounted. The EGFR PharmDx™ kit, containing positive and negative control cell lines was purchased from Dako (UK) and the immunostaining of sections was conducted according to the manufacturer9s protocol. We found that the mouse anti-EGFR mAb in the EGFR pharmDx™ kit stained both wild type and EGFRvIII expressing cells in the formalin fixed paraffin embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR9 and ICR10, which are directed against two distinct epitopes on the external domain of the EGFR, were specific for wild-type EGFR in formalin-fixed paraffin embedded tumour specimens. We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild type EGFR (membranous, nuclear, and cytoplasmic) in tumour specimens using immunohistochemistry and to determine their prognostic significance as well as predictive value for response to therapy with EGFR antibodies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 694.