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Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a “don't eat me” signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N‐terminal pyro‐glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell‐mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab‐mediated direct growth inhibition, complement‐dependent cytotoxicity, or Ab‐dependent cell‐mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα‐Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose‐dependent manner, suggesting that pyro‐glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab‐dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte‐mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell‐mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors., Inhibition of CD47/signal regulatory protein alpha (SIRPα) interactions is of great interest in cancer immunotherapy. In this manuscript, we investigated the impact of glutaminyl cyclase inhibition by small molecules to interfere with CD47/SIRPa interactions in epidermal growth factor receptor Ab‐mediated tumor immunotherapy in vitro and in vivo.

Background MAP2K1 mutations, otherwise known as MEK mutations, are rare oncogenic alterations that have been implicated in MAPK pathway activation. The impact of MAP2K1 mutations in colorectal cancer on EGFR antibody response has not been characterized. Patients and Methods Antitumor activity was assessed in mouse xenograft models with SW48 cell lines harboring MAP2K1 mutation, and protein expression of the RAS signaling pathway was studied by Western blot analysis. We retrospectively identified patients with MAP2K1-mutated metastatic colorectal cancer patients treated at City of Hope Comprehensive Cancer Center between 2015 and 2020 using next-generation sequencing. Patients’ tumor characteristics, treatment response, and outcome are described. Additional patients with the MAP2K1 mutation were identified from The Cancer Genome Atlas and Memorial Sloan Kettering Cancer Center oncogenomic databases. Results Antitumor activity in mouse xenograft models demonstrated efficacy with combination therapy with EGFR and MEK inhibition with either BRAF or ERK inhibitors. Five patients treated at City of Hope between 2015 and 2020 harbored a MAP2K1 mutation at a frequency of 1%. APC and TP53 were common coalterations. All disease was RAS and BRAF wild type, except 1 case that harbored a concurrent KRAS mutation. Four RAS/BRAF wild-type MAP2K1-mutated patients was treated with anti-EGFR, anti-EGFR + MEK and BRAF inhibitors, and anti-EGFR + ERK inhibitors. All 4 patients experienced disease progression. Conclusion MAP2K1 mutation in colorectal cancer is associated with poor response to EGFR inhibition. EGFR inhibition with or without MEK, BRAF, or ERK inhibitors did not result in any clinical benefit in our limited experience.

Purpose: While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy. Experimental Design: We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3). Results: Forty patients with oncogenic non-V600 BRAF–mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14). Conclusions: Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment. See related commentary by Fontana and Valeri, p. 6896

Recent clinical data have linked KRAS/TP53 co-mutation (mut) to resistance to radiotherapy (RT), but supporting laboratory in vivo evidence is lacking. In addition, the ability of different radiation doses, with/without epidermal growth factor receptor (EGFR)-directed treatment, to achieve local tumour control as a function of KRAS status is unknown. Here, we assessed clonogenic radiation survival of a panel of annotated lung cancer cell lines. KRASmut/TP53mut was associated with the highest radioresistance in non-isogenic and isogenic comparisons. To validate these findings, isogenic TP53mut NCI-H1703 models, KRASmut or wild-type (wt), were grown as heterotopic xenografts in nude mice. A clinical RT schedule of 30 fractions over 6 weeks was employed. The dose that controlled 50% of tumours (TCD(50)) was calculated. The TCD(50) for KRASwt/TP53mut xenografts was 43.1 Gy whereas KRASmut/TP53mut tumours required a 1.9-fold higher TCD(50) of 81.4 Gy. The EGFR inhibitor erlotinib radiosensitized KRASmut but not KRASwt cells and xenografts. The TCD(50) associated with adding erlotinib to RT was 58.8 Gy for KRASmut, i.e., a ~1.4-fold dose enhancement. However, the EGFR antibody cetuximab did not have a radiosensitizing effect. In conclusion, we demonstrate for the first time that KRASmut in a TP53mut background confers radioresistance when studying a clinical RT schedule and local control rather than tumour growth delay. Despite the known unresponsiveness of KRASmut tumours to EGFR inhibitors, erlotinib radiosensitized KRASmut tumours. Our data highlight KRAS/TP53 co-mutation as a candidate biomarker of radioresistance that can be at least partially reversed by dose escalation or the addition of a targeted agent.

Objectives To establish a circulating tumor cell (CTC) enrichment system for non-small cell lung cancer (NSCLC) patients who received first-line treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKI), using EGFR magnetic liposomes (EGFR-ML). Materials and methods An inverted evaporation method was used to develop antibody modified EGFR-ML. Peripheral blood was collected from NSCLC patients who underwent first-line EGFR-TKI treatment for CTC enumeration. Results Protein electrophoresis, magnetic saturation curve, and ultraviolet absorption spectrum showed successful incorporation of the EGFR antibody on the surface of the magnetic microspheres, and the development of EGFR-ML was ascertained based on cell morphology and particle size. Using EGFR-ML, CTC were successfully enriched from blood samples and were identified in 77.3% (99/128) of the cohort. When compared to the 21L858R variant, EGFR-19del showed lower CTC counts by EGFR-ML (CTCEGFR). At one month after EGFR-TKI, a lower CTCEGFR was associated with partial response (PR) during treatment (CTCEGFR Conclusion We showed that establishing a CTC enrichment system by antibody modified EGFR-ML in NSCLC is feasible. CTC enumeration by EGFR-ML may have the potential to supplement RECIST in dynamically monitoring the response of NSCLC patients’ to first-line EGFR-TKI.

Epidermal growth factor receptor (EGFR) ectodomain variants mediating primary resistance or secondary treatment failure in cancer patients treated with cetuximab or panitumumab support the need for more resistance-preventive or personalized ways of targeting this essential pathway. Here, we tested the hypothesis that the EGFR nanobody 7D12 fused to an IgG1 Fc portion (7D12-hcAb) would overcome EGFR ectodomain–mediated resistance because it targets a very small binding epitope within domain III of EGFR. Indeed, we found that 7D12-hcAb bound and inhibited all tested cell lines expressing common resistance-mediating EGFR ectodomain variants. Moreover, we assessed receptor functionality and binding properties in synthetic mutants of the 7D12-hcAb epitope to model resistance to 7D12-hcAb. Because the 7D12-hcAb epitope almost completely overlaps with the EGF-binding site, only position R377 could be mutated without simultaneous loss of receptor functionality, suggesting a low risk of developing secondary resistance toward 7D12-hcAb. Our binding data indicated that if 7D12-hcAb resistance mutations occurred in position R377, which is located within the cetuximab and panitumumab epitope, cells expressing these receptor variants would retain sensitivity to these antibodies. However, 7D12-hcAb was equally ineffective as cetuximab in killing cells expressing the cetuximab/panitumumab-resistant aberrantly N-glycosylated EGFR R521K variant. Yet, this resistance could be overcome by introducing mutations into the Fc portion of 7D12-hcAb, which enhanced immune effector functions and thereby allowed killing of cells expressing this variant. Taken together, our data demonstrate a broad range of activity of 7D12-hcAb across cells expressing different EGFR variants involved in primary and secondary EGFR antibody resistance.

Background RAS short variant (SV) mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR) monoclonal antibody (EGFRmAb). However, the clinical implications for RAS amplification (RASa) as a biomarker for anti-EGFR therapy in CRC remain ill defined. Methods Genomic analysis was performed using the Foundation Medicine (FM) comprehensive genomic profiling database of 37,233 CRC cases. Clinical outcomes were assessed using two independent cohorts: the City of Hope (COH) cohort of 338 patients with metastatic CRC (mCRC) and the Flatiron Health–FM real-world clinicogenomic database (CGDB) of 3,904 patients with mCRC. Results RASa was detected in 1.6% (614/37,233) of primarily mCRC. RASa 6–9 (n = 241, 39%), 10–19 (n = 165, 27%), and ≥ 20 (n = 209, 34%) copy number subsets had co-RAS SV/BRAF V600E in 63%/3%, 31%/0.6%, and 4.8%/0% of cases, respectively. In the COH cohort, six patients with RASa (13–54 copies) received EGFRmAb, four of six had progressive disease, two had stable disease, and median time to treatment discontinuation (TTD) was 2.5 months. Of the CGDB EGFRmAb-treated patients, those with RASa (n = 9) had median TTD of 4.7 months and overall survival (OS) of 11.4 months, those with RAS SV (n = 101) had median TTD and OS of 5.3 and 9.4 months, and those with RAS/BRAF wild-type (n = 608) had median TTD and OS of 7.6 and 13.7 months. Conclusion Patients with RASa without RAS mutations (1.1% of mCRC) may have poor outcomes on EGFRmAb, although numbers herein were small, and interpretation is confounded by combination chemotherapy. Larger independent studies are warranted to determine if RASa, including degree of amplification, may act similarly to RAS mutation as a resistance mechanism to EGFRmAb therapies. Implications for Practice Genomic data suggest that RAS amplification occurs as the sole RAS/RAF alteration in >1% of colorectal cancer cases and that degree of amplification inversely correlates with co-occurring MAPK pathway alterations. Preliminary clinical evidence suggests that RAS amplification may function similarly to RAS mutation as a negative predictor of benefit from anti-epidermal growth factor receptor therapies in colorectal cancer. More clinical data are needed, and comprehensive genomic profiling, including detection of RAS amplification, should be used in trial design to inform therapy selection.

Purpose To investigate the efficacy and toxicity of cetuximab when added to radiochemotherapy for unresectable esophageal cancer. Methods This randomized phase 2 trial (clinicaltrials.gov, identifier NCT01787006) compared radiochemotherapy plus cetuximab (arm A) to radiochemotherapy (arm B) for unresectable esophageal cancer. Primary objective was 2‑year overall survival (OS). Arm A was considered insufficiently active if 2‑year OS was ≤40% (null hypothesis = H0), and promising if the lower limit of the 95% confidence interval was >45%. If that lower limit was >40%, H0 was rejected. Secondary objectives included progression-free survival (PFS), locoregional control (LC), metastases-free survival (MFS), response, and toxicity. The study was terminated early after 74 patients; 68 patients were evaluable. Results Two-year OS was 71% in arm A (95% CI: 55–87%) vs. 53% in arm B (95% CI: 36–71%); H0 was rejected. Median OS was 49.1 vs. 24.1 months (p = 0.147). Hazard ratio (HR) for death was 0.60 (95% CI: 0.30–1.21). At 2 years, PFS was 56% vs. 44%, LC 84% vs. 72%, and MFS 74% vs. 54%. HRs were 0.51 (0.25–1.04) for progression, 0.43 (0.13–1.40) for locoregional failure, and 0.43 (0.17–1.05) for distant metastasis. Overall response was 81% vs. 69% (p = 0.262). Twenty-six and 27 patients, respectively, experienced at least one toxicity grade ≥3 (p = 0.573). A significant difference was found for grade ≥3 allergic reactions (12.5% vs. 0%, p = 0.044). Conclusion Given the limitations of this trial, radiochemotherapy plus cetuximab was feasible. There was a trend towards improved PFS and MFS. Larger studies are required to better define the role of cetuximab for unresectable esophageal cancer.

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521–31. ©2017 AACR.

The benefit of EGFR–TKI in non–small cell lung cancer has been demonstrated in mutant EGFR tumors as first-line treatment but the benefit in wild-type EGFR tumors is marginal as well as restricted to maintenance therapy in pretreated patients. This work aimed at questioning the effects of cisplatin initial treatment on the EGFR pathway in non–small cell lung cancer and the functional consequences in vitro and in in vivo animal models of patient-derived xenografts (PDX). We establish here that cisplatin pretreatment specifically sensitizes wild-type EGFR-expressing cells to erlotinib, contrary to what happens in mutant EGFR cells and with a blocking EGFR antibody, both in vitro and in vivo. The sensitization entails the activation of the kinase Src upstream of EGFR, thereafter transactivating EGFR through a ligand-independent activation. We propose a combination of markers that enable to discriminate between the tumors sensitized to erlotinib or not in PDX models, which should be worth testing in patients. These markers might be useful for the selection of patients who would benefit from erlotinib as a maintenance therapy. Mol Cancer Ther; 16(8); 1634–44. ©2017 AACR.

ABT-414 is an antibody-drug conjugate (ADC) being developed for the treatment of tumors harboring amplification of the epidermal growth factor receptor (EGFR). This study evaluated the potential of ABT-414 to prolong the QT interval as part of the initial phase 1 study (NCT01741727). Data from patients who received ABT-414 monotherapy at a dose of 1–4 mg/kg once every 3 weeks or 1 or 1.5 mg/kg weekly for 2 out of every 3 weeks (alternate schedule) by intravenous infusion were included in the analysis of triplicate 12-lead ECGs obtained before dosing and through 168 h after dosing. Data from time-matched pharmacokinetic samples and QT interval assessments were evaluated using linear mixed-effects modeling to determine the effects of ABT-414, total ABT-806, and cysteine-maleimidocaproyl monomethyl auristatin F (Cys-mcMMAF) on the QT interval corrected using Fridericia’s formula (QTcF). Fifty-one patients were included in the analyses. ABT-414 had no clinically meaningful effect on QTcF. Using pooled data from doses ≥2 mg/kg, the estimated mean ∆QTcF reached a maximum of 4.30 ms after dosing, with a one-sided 95% upper confidence bound of 8.32 ms. The exposure–response analysis showed no statistically significant relationship between ΔQTcF and the concentration of any analyte (P > 0.05). No patient had a QTcF value >480 ms or a ∆QTcF value >30 ms. ABT-414 had no clinically meaningful effect on the QTcF interval at doses being evaluated for treatment of patients with solid tumors.

Therapeutic targeting of ASCT2, a glutamine transporter that plays a major role in glutamine uptake in cancer cells, is challenging because ASCT2 also has a biological role in normal tissues. In this study, we report our novel finding that ASCT2 is physically associated in a molecular complex with epidermal growth factor receptor (EGFR), which is often overexpressed in human head and neck squamous cell carcinoma (HNSCC). Furthermore, we found that ASCT2 can be co-targeted by cetuximab, an EGFR antibody approved for treating metastatic HNSCC. We demonstrated that cetuximab downregulated ASCT2 in an EGFR expression-dependent manner via cetuximab-mediated EGFR endocytosis. Downregulation of ASCT2 by cetuximab led to decreased intracellular uptake of glutamine and subsequently a decreased glutathione level. Cetuximab thereby sensitized HNSCC cells to reactive oxygen species (ROS)-induced apoptosis and, importantly, it is independent of effective inhibition of EGFR downstream signaling by cetuximab. In contrast, knockdown of EGFR by siRNA or inhibition of EGFR kinase with gefitinib, an EGFR kinase inhibitor, failed to sensitize HNSCC cells to ROS-induced apoptosis. Our findings support a novel therapeutic strategy for EGFR-overexpressing and cetuximab-resistant cancers by combining cetuximab with an oxidative therapy.

PURPOSE: Although EGFR exon20 insertion (ex20ins) mutations account for 4~12 % of EGFR mutant NSCLC patients, there is no effective and selectable anticancer drugs targeting ex20ins mutations so far due to various variant mutations in ex20ins mutations. Moreover, most EGFR ex20ins mutants show primary resistance to EGFR TKIs. JNJ-61186372 (JNJ-372), a bispecific antibody that targets the EGFR and cMet receptors, is currently being explored in a first-in-human study in patients with NSCLC. To better understand the mechanism of JNJ-372 activity in this patient population, we conducted preclinical studies exploring the activity of JNJ-372 in different EGFR ex20ins models. METHODS: To elucidate whether JNJ-372 has antitumor effect in EGFR ex20ins mutants via EGFR and c-MET inhibition, cell viability, western blot, cell cycle, colony formation assay, FACS analysis were performed in JNJ-372 treated BaF3 cells, PDCs, PDOs, and PDX expressing EGFR ex20ins mutation. For mouse tumor models, JNJ-372 was administered i.p. twice a week at 10 mg/kg or 30 mg/kg. Antibody dependent cellular cytotoxicity (ADCC) assay was assessed to figure out whether JNJ-372 had ADCC effects. Referenced patients with ex20ins disease were administered 1050 mg JNJ-372 i.v. weekly for the first 4-week cycle, then biweekly for each subsequent cycle RESULTS: JNJ-372 inhibited the growth of BaF3 cells, PDCs, and a PDO harboring a range of ex20ins, which were resistant to osimertinib and gefitinib. Mechanistic assays revealed the reduction of EGFR and cMet receptor levels and decreases in phospho-EGFR and c-Met, as well as inhibition of their downstream signaling pathways. Cleaved caspase-3 and BIMEL were upregulated at anti-proliferative doses, suggesting caspase-mediated cell death. JNJ-372 demonstrated corresponding antitumor activity in PDC and PDX models harboring different ex20ins; inhibition of signaling and engagement of the apoptotic pathway was confirmed in tumors of JNJ-372-treated mice. In PDCs with EGFR ex20ins mutation, we verified that JNJ-372 had a significant ADCC effect compared to EGFR antibody drug cetuximab. In the first-in-human trial, CT scans from two patients treated with JNJ-372 revealed reductions in tumor burden. A 58-year patient harboring H773delinsNPY showed -63% tumor reduction with progression-free survival of > 20 months and a 48-year patient harboring S768_D770dup showed -38.9% tumor reduction. CONCLUSION: JNJ-372 drives antitumor activity in preclinical models of EGFR ex20ins, which have no therapeutic options in clinic, by decreasing EGFR and cMet receptor levels, inhibiting downstream signaling cascades, activating apoptotic signaling as well as ADCC. These results provide a promising therapeutic option to patients with EGFR ex20ins mutations and an understanding of the activity of JNJ-372 being observed in the first-in-human study. Citation Format: Jiyeon Yun, Han Na Kang, Soo-Hwan Lee, Seo-Yoon Jeong, Chae Won Park, Jae-Hwan Kim, Kyoung-Ho Pyo, Ji Min Lee, Seok-Young Kim, Min Hee Hong, Hye Ryun Kim, Meena Thayu, Joshua Curtin, Roland Knoblauch, Matthew Lorenzi, Byoung Chul Cho. JNJ-61186372, an EGFR-cMet bispecific antibody, in EGFR Exon 20 insertion-driven advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5199.

Three FDA-approved epidermal growth factor receptor (EGFR) antibodies (cetuximab, panitumumab, necitumumab) are clinically available to treat patients with different types of cancers. Interestingly, panitumumab is of human IgG2 isotype, which is often considered to have limited immune effector functions. Unexpectedly, our studies unraveled that human IgG2 antibodies against EGFR mediated effective CDC when combined with another noncross-blocking EGFR antibody. This second antibody could be of human IgG1 or IgG2 isotype. Furthermore, EGFR antibodies of human IgG2 isotype were highly potent in recruiting myeloid effector cells such as M1 macrophages and PMN for tumor cell killing by ADCC. Tumor cell killing by PMN was more effective with IgG2 than with IgG1 antibodies if tumor cells expressed lower levels of EGFR. Additionally, lower expression levels of the “don′t eat me” molecule CD47 on tumor cells enabled ADCC also by M2 macrophages, and improved PMN and macrophage-mediated ADCC. A TCGA enquiry revealed broadly varying CD47 expression levels across different solid tumor types. Together, these results demonstrate that human IgG2 antibodies against EGFR can promote significant Fc-mediated effector functions, which may contribute to their clinical efficacy. The future challenge will be to identify clinical situations in which myeloid effector cells can optimally contribute to antibody efficacy.

Epidermal growth factor receptor (EGFR) is a clinically validated target and often overexpressed in some solid tumors. Both EGFR tyrosine kinase inhibitors and ligand-blocking antibodies have been approved for treatment of NSCLC, head and neck cancers and colorectal cancers. However, clinical response is limited and often accompanied by significant toxicities due to normal tissue expression. To improve the effectiveness of targeting EGFR while minimizing the toxicities on normal tissues, we developed a low-affinity anti-EGFR antibody drug conjugate (ADC), RN765C. Potent in vitro cytotoxicity of RN765C, with nanomolar to subnanomolar EC50, was observed on a panel of cancer cell lines expressing moderate to high level of EGFR. In contrast, RN765C was less effective in killing normal human keratinocytes, presumably due to its lower receptor expression. Mechanistically, RN765C has multiple modes of action: inducing payload mediated mitotic arrest and cell death, blocking EGFR pathway signal and mediating antibody dependent cell cytotoxicity. In preclinical studies, a single dose of RN765C at 1.5-3 mg/kg was generally sufficient to induce tumor regression in multiple cell line and patient-derived xenograft models, including those that are resistant to EGFR-directed tyrosine kinase inhibitors. Our data support further investigation of RN765C in the clinic to treat EGFR expressing solid tumors.

Anti-epidermal growth factor receptor (EGFR) antibody therapy is used in EGFR expressing cancers including lung, colon, head and neck, and bladder cancers, however results have been modest. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate which is activated by NIR light. NIR-PIT is in clinical trials in patients with recurrent head and neck cancers using cetuximab-IR700 as the conjugate. However, its use has otherwise been restricted to mouse models. This is an effort to explore larger animal models with NIR-PIT. We describe the use of a recombinant canine anti-EGFR monoclonal antibody (mAb), can225IgG, conjugated to the photo-absorber, IR700DX, in three EGFR expressing canine transitional cell carcinoma (TCC) cell lines as a prelude to possible canine clinical studies. Can225-IR700 conjugate showed specific binding and cell-specific killing after NIR-PIT on EGFR expressing cells in vitro. In the in vivo study, can225-IR700 conjugate demonstrated accumulation of the fluorescent conjugate with high tumor-to-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 µg of can225-IR700 i.v. only; (3) NIR light exposure only; (4) 100 µg of can225-IR700 i.v., NIR light exposure. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups (p < 0.001), and significantly prolonged survival was achieved (p < 0.001 vs. other groups) in the treatment groups. In conclusion, NIR-PIT with can225-IR700 is a promising treatment for canine EGFR-expressing cancers, including invasive transitional cell carcinoma in pet dogs, that could provide a pathway to translation to humans.

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.

// Friederike Braig 1 , Manuela Marz 1 , Aneta Schieferdecker 1 , Alexander Schulte 2 , Mareike Voigt 1 , Alexander Stein 1 , Tobias Grob 3 , Malik Alawi 4 , Daniela Indenbirken 5 , Malte Kriegs 6 , Erik Engel 7 , Udo Vanhoefer 8 , Adam Grundhoff 5 , Sonja Loges 1,9 , Kristoffer Riecken 10 , Boris Fehse 10 , Carsten Bokemeyer 1 and Mascha Binder 1 1 Department of Oncology and Hematology, BMT with section Pneumology, Hubertus Wald Tumorzentrum / UCCH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 Department of Neurosurgery, Laboratory for Brain Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Department of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4 Bioinformatics Service Facility, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 5 Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology (HPI), Hamburg, Germany 6 Radiation Biology and Radio-Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 7 Hamatologisch-onkologische Praxis Altona (HOPA), Hamburg, Germany 8 Marienkrankenhaus, Zentrum fur Innere Medizin, Hamburg, Germany 9 Institute for Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 10 Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to: Mascha Binder, email: // Keywords : panitumumab, cetuximab, EGFR antibody resistance, mutation, circulating tumor DNA Received : January 30, 2015 Accepted : February 20, 2015 Published : March 14, 2015 Abstract Acquired resistance to epidermal growth factor receptor (EGFR) targeted antibodies represents a clinical challenge in the treatment of gastrointestinal tumors such as metastatic colorectal cancer, but its molecular mechanisms are incompletely understood. We scanned KRAS exon 2/3/4, NRAS exon 2/3/4 and the overlapping epitopes of the EGFR antibodies cetuximab and panitumumab for mutations in pre- and post-treatment tumor tissue of 21 patients with gastrointestinal cancer treated with chemotherapy +/- EGFR antibodies by next-generation sequencing (“tumor tissue” cohort). We describe a novel EGFR exon 12 mutation acquired in tumors of 1 out of 3 patients treated with panitumumab. The EGFR G465R mutation introduces a positive charge within the overlap of the panitumumab and cetuximab epitopes. It abrogates antibody binding and mediates cross-resistance to both antibodies in EGFR G465R-transfected Ba/F3 cells. In circulating tumor DNA from an independent “liquid biopsy” cohort of 27 patients, we found this novel mutation in 1 out of 6 panitumumab-treated cases while about one third of patients show acquired RAS mutations. We show that acquired resistance by epitope-changing mutations also emerges during panitumumab treatment, which can be easily detected by a liquid biopsy approach even before clinical resistance occurs and this may help in tailoring EGFR-targeted therapies.

BACKGROUND: There is increasing evidence that metastatic colorectal cancer (mCRC) is a genetically heterogeneous disease and that tumours arising from different sides of the colon (left versus right) have different clinical outcomes. Furthermore, previous analyses comparing the activity of different classes of targeted agents in patients with KRAS wild-type (wt) or RAS wt mCRC suggest that primary tumour location (side), might be both prognostic and predictive for clinical outcome. METHODS: This retrospective analysis investigated the prognostic and predictive influence of the localization of the primary tumour in patients with unresectable RAS wt mCRC included in six randomized trials (CRYSTAL, FIRE-3, CALGB 80405, PRIME, PEAK and 20050181), comparing chemotherapy plus EGFR antibody therapy (experimental arm) with chemotherapy or chemotherapy and bevacizumab (control arms). Hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival (OS) and progression-free survival (PFS) for patients with left-sided versus right-sided tumours, and odds ratios (ORs) for objective response rate (ORR) were estimated by pooling individual study HRs/ORs. The predictive value was evaluated by pooling study interaction between treatment effect and tumour side. RESULTS: Primary tumour location and RAS mutation status were available for 2159 of the 5760 patients (37.5%) randomized across the 6 trials, 515 right-sided and 1644 left-sided. A significantly worse prognosis was observed for patients with right-sided tumours compared with those with left-sided tumours in both the pooled control and experimental arms for OS [HRs = 2.03 (95% CI: 1.69-2.42) and 1.38 (1.17-1.63), respectively], PFS [HRs = 1.59 (1.34-1.88) and 1.25 (1.06-1.47)], and ORR [ORs = 0.38 (0.28-0.50) and 0.56 (0.43-0.73)]. In terms of a predictive effect, a significant benefit for chemotherapy plus EGFR antibody therapy was observed in patients with left-sided tumours [HRs = 0.75 (0.67-0.84) and 0.78 (0.70-0.87) for OS and PFS, respectively] compared with no significant benefit for those with right-sided tumours [HRs = 1.12 (0.87-1.45) and 1.12 (0.87-1.44) for OS and PFS, respectively; P value for interaction

// Dominik Paul Modest 1, 2 , Sebastian Stintzing 1, 2 , Ludwig Fischer von Weikersthal 3 , Thomas Decker 4 , Alexander Kiani 5 , Ursula Vehling-Kaiser 6 , Salah-Eddin Al-Batran 7 , Tobias Heintges 8 , Christoph Kahl 9 , Gernot Seipelt 10 , Frank Kullmann 11 , Werner Scheithauer 12 , Markus Moehler 13, 14 , Julian Walter Holch 1, 2 , Jobst Christian von Einem 1, 2 , Swantje Held 15 and Volker Heinemann 1, 2 1 Department of Medicine III, University Hospital, LMU Munich, Munich, Germany 2 German Cancer Consortium (DKTK), German Cancer Research Centre (DKFZ), Heidelberg, Germany 3 Gesundheitszentrum St. Marien, Amberg, Germany 4 Oncological Practice, Ravensburg, Germany 5 Medizinische Klinik IV, Klinikum Bayreuth, Bayreuth, Germany 6 Oncological Practice, Landshut, Germany 7 Department of Hematology and Oncology, Krankenhaus Nordwest Frankfurt/Main, Frankfurt, Germany 8 Department of Medicine II, Stadtisches Klinikum Neuss, Neuss, Germany 9 Haematology and Oncology, Staedtisches Klinikum Magdeburg, Magdeburg, Germany 10 Oncological Practice, Bad Soden, Germany 11 Department of Medicine I, Klinikum Weiden, Weiden in der Oberpfalz, Germany 12 Department of Internal Medicine I & Comprehensive Cancer Center, Medical University Vienna, Vienna, Austria 13 Medical Department 1, Johannes-Gutenberg Universitat Mainz, Mainz, Germany 14 University Cancer Center Frankfurt/Mainz and German Cancer Consortium (DKTK), Heidelberg, Germany 15 ClinAssess GmbH, Leverkusen, Germany Correspondence to: Dominik Paul Modest, email: dominik.modest@med.uni-muenchen.de Keywords: colorectal cancer; tumor sidedness; EGFR antibody; bevacizumab; sequential therapy Received: July 31, 2017 Accepted: October 13, 2017 Published: November 11, 2017 ABSTRACT Purpose: To assess the impact of primary tumor sidedness on outcome of patients with metastatic colorectal cancer (mCRC) across treatment lines. Patients and Methods: Patients of the FIRE-3 trial (initial FOLFIRI plus either cetuximab or bevacizumab) were separately evaluated according to primary tumor site differentiating left-sided (LPT) from right-sided primary tumors (RPT). Efficacy (i.e. progression-free survival (PFS2nd) and overall survival (OS2nd) of second-line therapy) was evaluated by Kaplan-Meier method and compared by log rank test as well as Cox regression analyses. All analyses were also reported according to drug sequences. Results: 411 of 592 patients (69%) with KRAS exon 2 wild-type tumors received 2nd-line therapy has and had available information on primary tumor location, of those 309 patients (75%) presented with LPT. In patients with LPT, PFS2nd was markedly longer than in patients with RPT (6.0 months [95% CI 5.5-6.5] versus 3.8 months [95% CI 2.5-5.2], hazard ratio: 0.61 [95% CI 0.47-0.78], P

Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC-A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ-H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC-A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ-H2AX and 53BP1 foci. IMC-A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. Addition of IMC-A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC-A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non–small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (CtxR cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152–64. ©2014 AACR.

Preclinical studies demonstrate that epidermal growth factor receptor (EGFR) signals through both kinase-dependent and independent pathways and that combining a small-molecule EGFR inhibitor, EGFR antibody, and/or anti-angiogenic agent is synergistic. We conducted a dose-escalation, phase I study combining erlotinib, cetuximab, and bevacizumab. The subset of patients with metastatic colorectal cancer was analyzed for safety and antitumor activity. Forty-one patients with heavily pretreated metastatic colorectal cancer received treatment on a range of dose levels. The most common treatment-related grade ≥2 adverse events were rash (68%), hypomagnesemia (37%), and fatigue (15%). Thirty of 34 patients (88%) treated at the full FDA-approved doses of all three drugs tolerated treatment without drug-related dose-limiting effects. Eleven patients (27%) achieved stable disease (SD) ≥6 months and three (7%) achieved a partial response (PR) (total SD>6 months/PR= 14 (34%)). Of the 14 patients with SD≥6 months/PR, eight (57%) had received prior sequential bevacizumab and cetuximab, two (5%) had received bevacizumab and cetuximab concurrently, and four (29%) had received prior bevacizumab but not cetuximab or erlotinib (though three had received prior panitumumab). The combination of bevacizumab, cetuximab, and erlotinib was well tolerated and demonstrated antitumor activity in heavily pretreated patients with metastatic colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer death globally. Significant improvements in survival have been made in patients with metastasis by new therapies. For example, Cetuximab and Panitumumab are monoclonal antibodies that inhibit the epidermal growth receptor (EGFR). KRAS mutations in codon 12 and 13 are the recognized biomarkers that are analyzed in clinics before the administration of anti-EGFR therapy. Genetic analyses have revealed that mutations in KRAS predict a lack of response to Panitumumab and Cetuximab in patients with metastatic CRC (mCRC). Notably, it is estimated that 35–45% of CRC patients harbor KRAS mutations. Therefore, KRAS mutation testing should be performed in all individuals with the advanced CRC in order to identify the patients who will not respond to the monoclonal EGFR antibody inhibitors. New techniques for KRAS testing have arisen rapidly, and each technique has advantages and disadvantages. Herein, we review the latest published literature specific to KRAS mutation testing techniques. Since reliability and feasibility are important issues in clinical analyses. Therefore, this review also summarizes the effectiveness and limitations of numerous KRAS mutation testing techniques.

Mutation status of Kirsten rat sarcoma viral oncogene homolog (KRAS) may serve as a negative predictive marker of Cetuximab in treating colorectal cancer. The present study was to determine the role of KRAS status in EGFR antibody treatment for gastric cancer. KRAS status was clarified in SGC-7901 (wild type) and YCC-2 (G→A mutation) gastric cancer cell lines. Anti-proliferative and pro-apoptotic effects of Cetuximab were tested both in vitro and in vivo, the expression of phosphorylated (p) ERK, a downstream protein in EGFR-RAS-MEK pathway, was also analyzed. No significant in vitro anti-proliferative or pro-apoptotic effects of Cetuximab were observed in both cell lines. The growth of either SGC-7901 or YCC-2 gastric cancer xenograft was significantly inhibited by Cetuximab. Apoptosis was induced in SGC-7901 but not in YCC-2 xenografts after Cetuximab treatment. The expression of pERK was up-regulated in YCC-2 but not SGC-7901 xenografts after Cetuximab treatment. In conclusion, KRAS (G→A) mutation does not affect in vivo anti-cancer efficacy of Cetuximab.

Lessons LearnedGC1118 is a novel fully human anti-epidermal growth factor receptor (EGFR) antibody with unique binding epitopes and different ligand-binding inhibitory activity compared with cetuximab or panitumumab. GC1118 showed promising antitumor activity, especially in patients with colorectal cancer resistant to prior EGFR antibody. Skin toxicities were more common and diarrhea was less frequent compared with other anti-EGFR antibodies.BackgroundGC1118 is a novel monoclonal antibody targeting epidermal growth factor receptor (EGFR) with more potent ligand inhibition than cetuximab or panitumumab. We conducted a first-in-human, phase I study of GC118 in patients with refractory solid tumors.MethodsIn the dose escalation part, GC1118 was administered on days 1, 8, 15, and 22, followed by a 2-week rest, during which dose-limiting toxicities (DLTs) were evaluated. In the expansion part, patients were enrolled into three cohorts (Cohort 1 [C1], patients with colorectal cancer [CRC] without prior anti-EGFR treatment; Cohort 2 [C2], patients with CRC with tumors resistant to anti-EGFR therapy; Cohort 3 [C3], EGFR-overexpressing gastric cancer).ResultsIn the dose escalation part, 24 patients were treated at five dose levels: 0.3, 1.0, 3.0, 4.0, and 5.0 mg/kg. In the 5.0 mg/kg cohort, two patients experienced DLTs (skin toxicities). The maximum-tolerated dose (MTD) was 4.0 mg/kg. Common adverse events were skin toxicities. In the expansion part, 39 patients were enrolled. In Cohort 1, stable disease (SD) was observed in 58%; in Cohort 2, partial response (PR) 17% and SD 8%; in Cohort 3, PR 8% and SD 17%.ConclusionGC1118 showed promising antitumor activity and was well tolerated. Infrequent diarrhea compared with other anti-EGFR antibodies might be advantageous for further development.

Purpose: Epidermal growth factor receptor (EGFR) represents an important target for treatment of locally advanced breast cancer (LABC), since about 30% of patients with LABC have triple negative breast tumor, and these tumors are often EGFR-positive. Our aim was to synthesize and characterize a novel form of “gold nanobomb” (GNB) for localized targeted radioimmunotherapy of LABC. GNB uses gold nanoparticles (AuNPs) as a carrier to conjugate both EGFR targeting monoclonal antibody (panitumumab) and the β-particle emitting radionuclide 177Lu. Ultimately, these GNB would be administered locally in LABC for tumor treatment while minimizing normal tissue exposure. Methods: The basic idea for construction of this new-targeted agent is to use different length of poly(ethylene glycol) (PEG) spacers to conjugate antibody and DOTA metal chelators for 177Lu. First, OPSS-PEG-NH2 was used to synthesize OPSS-PEG-DOTA for complexing 177Lu and OPSS-PEG-SVA was used to link antibody. Then these PEG spacers were linked to 30 nm AuNPs to form “GNB”. OPSS-PEG-antibody was analyzed by SDS-PAGE and trinitrobenzenesulfonic acid (TNBSA) assay to determine the degree of amine substitution after PEGylation. A radiometric assay was used to determine the number of PEGylated antibody and DOTA-PEG-OPSS per AuNPs. Specificity of GNB for EGFR and internalization into MBA-MD-468 (1×106 EGFR/cell), MBA-MD-231 (1×105 EGFR/cell) and MCF-7 (1×104 EGFR/cell) was confirmed by confocal and darkfield fluorescence microscopy. Binding assay and cell fractionation study were used to determine the binding affinity to EGFR and the total amount of GNB internalized. Results: Reacting panitumumab with increasing ratios of PEG resulted in an increase in molecular weight from 147 to 250 kDa. This result was in accordance with TNBSA assay where increasing ratio of PEG was associated with increasing lysine substitution (1.4 to 50.9 PEGylated lysine per antibody). PEGylation of 1.4 ± 1.7 lysine per antibody resulted in retention of immunoreactivity and when the immunoconjugates were linked to AuNPs, 8.09 ± 0.38 antibodies were attached to one AuNP. The total number of OPSS-PEG-DOTA per AuNP was estimated to be 812 ± 53. The total size of GNB was 67.3 ± 0.6 nm and there was no particle precipitation or aggregation after incubation in PBS. Competitive binding assay revealed that GNB was targeted strongly to EGFR (Kd antibody: 2.7 ± 0.3 nM; Kd GNB: 5.0 ± 4.9 nM) and darkfield microscopy showed that low PEG to antibody ratio increased the targeting efficiency when compared to high PEG to antibody ratio. Confocal microscopy revealed specific GNB uptake in MDA-MB-468 cells and cell fractionation study demonstrated receptor-mediated internalization of GNB, where the total amount of GNB internalized was about 3.1 and 22.2 fold higher in MDA-MB-468 cells than in MDA-MB-231 or MCF-7 cells. Conclusion: This GNB is very effective in targeting and in internalizing into EGFR, thus the GNB labeled with 177Lu emitting β-particles may be a promising new localized treatment for LABC. We plan to now examine the cytotoxicity of GNB both in vitro and in vivo will be explored for treatment of EGFR positive tumors. Supported by a grant from the Canadian Breast Cancer Foundation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-04.

// Martin Pool 1 , Arjan Kol 1 , Marjolijn N. Lub-de Hooge 2, 3 , Christian A. Gerdes 4 , Steven de Jong 1 , Elisabeth G.E. de Vries 1 , Anton G.T. Terwisscha van Scheltinga 2 1 Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 3 Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 4 Department of Roche Pharma Research and Early Development, Roche Innovation Center Zurich, Schlieren, Switzerland Correspondence to: Anton G.T. Terwisscha van Scheltinga, email: a.g.t.terwisscha.van.scheltinga@umcg.nl Keywords: imgatuzumab, EGFR, 89 Zr, immunoPET, shedding Received: January 06, 2016 Accepted: August 27, 2016 Published: September 02, 2016 ABSTRACT Preclinical positron emission tomography (PET) imaging revealed a mismatch between in vivo epidermal growth factor receptor (EGFR) expression and EGFR antibody tracer tumor uptake. Shed EGFR ectodomain (sEGFR), which is present in cancer patient sera, can potentially bind tracer and therefore influence tracer kinetics. To optimize EGFR-PET, we examined the influence of sEGFR levels on tracer kinetics and tumor uptake of EGFR monoclonal antibody 89 Zr-imgatuzumab in varying xenograft models. Human cancer cell lines A431 (EGFR overexpressing, epidermoid), A549 and H441 (both EGFR medium expressing, non-small cell lung cancer) were xenografted in mice. Xenografted mice received 10, 25 or 160 μg 89 Zr-imgatuzumab, co-injected with equal doses 111 In-IgG control. MicroPET scans were made 24, 72 and 144 h post injection, followed by biodistribution analysis. sEGFR levels in liver and plasma samples were determined by ELISA. 89 Zr-imgatuzumab uptake in A431 tumors was highest (29.8 ± 5.4 %ID/g) in the 160 μg dose group. Contrary, highest uptake in A549 and H441 tumors was found at the lowest (10 μg) 89 Zr-imgatuzumab dose. High 89 Zr-imgatuzumab liver accumulation was found in A431 xenografted mice, which decreased with antibody dose increments. 89 Zr-imgatuzumab liver uptake in A549 and H441 xenografted mice was low at all doses. sEGFR levels in liver and plasma of A431 bearing mice were up to 1000-fold higher than levels found in A549, H441 and non-tumor xenografted mice. 89 Zr-imgatuzumab effectively visualizes EGFR-expressing tumors. High sEGFR levels can redirect 89 Zr-imgatuzumab to the liver, in which case tumor visualization can be improved by increasing tracer antibody dose.

KRAS mutations are proved to confer dramatic resistance to EGFR target therapy of cancer patients. The aim of this study was to establish a convenient and accurate method to screen plasma KRAS mutations of cancer patients since tumor specimens were not always available in clinical practice. A modified PNA-PCR method was established and evaluated in plasma of 19 pancreatic cancer patients. Our results showed that the modified PNA-PCR assay was a sensitive (87.5%, 14/16) and accurate (92.9%, 13/14) method to screen plasma KRAS mutations of pancreatic cancer patients and there was a high consistency of KRAS mutation status between plasma samples and tumor specimens. The modified protocol could not only screen plasma KRAS mutations rapidly and accurately but also had potential to quantify KRAS mutant DNA to predict treatment response of cancer patients and monitor disease progression. It's should be indicated that this modified assay was only confirmed in the pancreatic cancer patients in this study and need to be verified in other cancer patients.

Squamous cell carcinoma of head and neck (SCCHN) is the most common neoplasm of the upper aerodigestive tract. In this paper, we attempt to summarize the role and applications of the epidermal growth factor receptor (EGFR) inhibitors monoclonal antibodies (moAbs) and tyrosine kinase inhibitors (TKIs) locally advanced as well as metastatic SCCHN. Targeted therapy in SCCHN is now incorporated in the first-line regimes for advanced disease. Novel targeted agents, including the EGFR antibody, cetuximab, have been approved for use as single agents or in combination with radiation therapy or chemotherapy in treatment of recurrent metastatic or locally advanced SCCHN. Refractory mechanisms that bypass the pathway of EGFR inhibitors activity are identified explaining resistance to targeted therapy. Strategies of cotargeting EGFR and other pathways are under investigation. Examples of targeted therapy being used include mammalian target of rapamycin (mtor) inhibitors, antivascular endothelial growth factor (VEGF) moAb, and other inhibitors. We will be focusing our paper on the preclinical and clinical aspects of EGFR inhibition in SCCHN and touch upon other targeted therapies in application.

Necitumumab (EGFR inhibitor) in combination with chemotherapy provides a modest, yet a significant improvement in overall survival over chemotherapy alone in patients with advanced squamous non-small cell lung carcinoma (NSCLC). However, combination therapies targeting EGFR and PD-1 pathway blockers may represent a better way to extend clinical benefit to more cancer patients given that PD-(L)1 antibodies have emerged as a standard of care in NSCLC. Antibodies targeting EGFR have the potential to promote an inflamed tumor microenvironment through engagement of Fc-gamma receptors (FcγR) on innate immune cells resulting in an improved antigen presentation and T cell priming. Therefore, the present study was initiated to understand the combinatorial effect of immune checkpoint (PD-(L)1) inhibitors with necitumumab. Preclinical modeling of EGFR/PD-(L)1 mAb combination in mice is challenging due to the lack of cross-reactivity of necitumumab with mouse EGFR. Syngeneic mouse tumor models widely used to study effects of immunomodulatory agents express low or no EGFR. To overcome this limitation, we used two immunocompetent model systems to study the combination effect of EGFR mAb with PD-1 (RMP1-14) or PD-L1 (178G7) mAbs: 1) genetically engineered mouse model of lung adenocarcinoma (TD model) driven by mutant forms of human EGFR (exon 19 deletion and T790M mutation) and 2) CT26 syngeneic mouse tumor model with ectopic expression of human EGFR (CT26-hEGFR). To engage mouse immune cells more efficiently, a murinized version of necitumumab was generated through antibody engineering, with human EGFR binding Fabs and a mouse Fc backbone. Intratumor immune response was evaluated by immunohistochemistry and a custom-made immune profiling Quantigene Plex (QGP) gene expression panel. In both models, targeting EGFR and PD-1 pathway resulted in the combinatorial antitumor efficacy exemplified by decreased tumor burden compared to the monotherapy groups. QGP analysis of CT26-hEGFR tumor tissue revealed that the combination treatment enhanced intra-tumor immune response exemplified by an upregulation of immune-related genes indicative of T cell infiltration (Cd3e, Cd4, Cd8b1), T cell activation (Ifng, Cd274, Pdcd1lg2, Icos, Tnfrsf4, Tnfrsf18, Cd69, Ido1, Havcr2, Lag3), myeloid cell infiltration (Cd86, Timd4, Vista, Cd68, Mpo, Nos2). Histopathological analysis confirmed an increase in T cell infiltration indicating an improved immune response in the combination therapy group. Taken together, these results provide a rationale for further evaluation of EGFR and PD-(L)1 mAbs in clinical setting. Citation Format: Veena Kandaswamy, Amelie Forest, Marianne Deroose, David A. Schaer, Ting Chen, Shengwu Liu, David Surguladze, Yung-mae Yao, Thompson Doman, Gerald Hall, Kwok-Kin Wong, Michael Kalos, Ruslan D. Novosiadly. Combination of EGFR antibody with PD-1 pathway inhibitors improves anti-tumor efficacy and enhances intra-tumor immune response in preclinical mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3632.

Background: Circulating tumor cells (CTCs) are not only a surrogate of distant metastasis, but also useful for material of liquid biopsy. "Universal" CTC-chip is a polymeric microfluidic device that can capture CTCs according to their surface marker from whole blood. We have examined the capture efficiency of lung cancer and mesothelioma cells in the blood with CTC-chip coated with antibodies against various surface marker. Methods: Lung cancer cell lines (PC-9) or mesothelioma cell lines (ACC-MESO-4, MSTO-211H) were used in this study. Cells were CFSE-labeled and added in the blood of healthy donor. A polymeric CTC-chip was coated with two steps: base- and capture-antibody. As capture antibody, we used anti-EpCAM, podoplanin, and EGFR antibody. After flowing these samples, capture efficiency was calculated (number of captured cells/number of flowed cells). Results: Capture efficiency of PC-9, MESO-4, and METO-211H was 100.0%, 9.5% and 9.1% (EpCAM-chip; anti-EpCAM antibody clone HEA125); 5.8%, 85.9% and 9.0 % (podoplanin-chip; anti-podoplanin antibody clone E1); 38.6, 112.5%, 11.7% (podoplanin-chip; anti-podoplanin antibody clone NZ-1); 30.2%, 21.8%, 38.5% (EGFR-chip; anti-EGFR antibody clone 528), respectively. Conclusion: Using "Universal CTC-chip" with various antibodies, it was possible to capture nonepithelial tumor cells such as mesothelioma cells. Citation Format: Kazue Yoneda, Taiji Kuwata, Fumihiro Tanaka. Capture of tumor cells in blood with "Universal CTC-chip" [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1599.

(1) To determine the safety of the epidermal growth factor receptor (EGFR) antibody cetuximab with concurrent gemcitabine and abdominal radiation in the treatment of patients with locally advanced adenocarcinoma of the pancreas. (2) To evaluate the feasibility of pancreatic cancer cell epithelial-mesenchymal transition (EMT) molecular profiling as a potential predictor of response to anti-EGFR treatment.Patients with non-metastatic, locally advanced pancreatic cancer were treated in this dose escalation study with gemcitabine (0-300 mg/m(2)/week) given concurrently with cetuximab (400 mg/m(2) loading dose, 250 mg/m(2) weekly maintenance dose) and abdominal irradiation (50.4 Gy). Expression of E-cadherin and vimentin was assessed by immunohistochemistry in diagnostic endoscopic ultrasound fine-needle aspiration (EUS-FNA) specimens.Sixteen patients were enrolled in 4 treatment cohorts with escalating doses of gemcitabine. Incidence of grade 1-2 adverse events was 96%, and incidence of 3-4 adverse events was 9%. There were no treatment-related mortalities. Two patients who exhibited favorable treatment response underwent surgical exploration and were intraoperatively confirmed to have unresectable tumors. Median overall survival was 10.5 months. Pancreatic cancer cell expression of E-cadherin and vimentin was successfully determined in EUS-FNA specimens from 4 patients.Cetuximab can be safely administered with abdominal radiation and concurrent gemcitabine (up to 300 mg/m(2)/week) in patients with locally advanced adenocarcinoma of the pancreas. This combined therapy modality exhibited limited activity. Diagnostic EUS-FNA specimens could be analyzed for molecular markers of EMT in a minority of patients with pancreatic cancer.

3555Background: MET amp is a well described driver of acquired EGFR antibody (Ab) resistance. Blood-based genomic profiling of cell free (cf)DNA is a safe and efficient means to identify pts with a...

Background: The validation of KRAS mutations as a negative marker of response to anti-epidermal growth factor receptor (EGFR) antibodies has meant a seminal advance towards treatment individualisation of colorectal cancer (CRC) patients. However, as a KRAS wild-type status does not guarantee a response to anti-EGFR antibodies, a current challenge is the identification of other biomarkers of response. On the basis of pre-clinical evidence, we hypothesised that mitogen-activated protein kinase phosphatase-1 (MKP-1), a phosphatase that inactivates MAPKs, could be a mediator of resistance to anti-EGFR antibodies. Methods: Tumour specimens from 48 metastatic CRC patients treated with cetuximab-based chemotherapy were evaluated for KRAS and BRAF mutational status and MKP-1 expression as assessed by immunohistochemistry. Results: As expected, clinical benefit was confined to wild-type KRAS and BRAF patients. Mitogen-activated protein kinase phosphatase-1 was overexpressed in 16 patients (33%) and was not associated with patient baseline clinicopathological characteristics and KRAS mutational status. All patients with BRAF mutations (n=3) had MKP-1 overexpression. Among KRAS wild-type patients, MKP-1 overexpressors had a 7% response rate (RR), whereas patients not overexpressing MKP-1 had a 44% RR (P=0.03). Moreover, median time to progression was significantly longer in MKP-1 non-overexpressing patients (32 vs 13 weeks, P=0.009). Conclusion: These results support the concept of MKP-1 as a promising negative marker of response to cetuximab-based treatment in CRC patients with wild-type KRAS.

Purpose: Epidermal growth factor receptor (EGFR) antibody therapy is established in patients with wild-type KRAS colorectal carcinoma; however, up to 50% of these patients do not respond to this therapy. To identify the possible causes of this therapy failure, we searched for mutations in different EGFR-dependent signaling proteins and analyzed their distribution patterns in primary tumors and corresponding metastases. Experimental Design: Tumor tissues, macrodissected from tumor centers, invasion fronts (n = 100), lymph nodes (n = 55), and distant metastases (n = 20), respectively, were subjected to DNA extraction and mutation analysis of KRAS, BRAF, and PIK3CA. Results: Activating mutations were detected in 41% (KRAS), 7% (BRAF), and 21% (PIK3CA) of the primary tumors. By comparing tumor centers and invasion fronts, the intratumoral heterogeneity of KRAS, BRAF, and PIK3CA mutations was observed in 8%, 1%, and 5% of primary tumors, respectively. Heterogeneity between primary tumors and lymph node metastases was found in 31% (KRAS), 4% (BRAF), and 13% (PIK3CA) of the cases. Heterogeneity between primary tumors and distant metastases was present in two patients (10%) for KRAS and one patient for PIK3CA (5%), but not for BRAF. Discordant results between primary tumors and metastases could markedly be reduced by testing the additional tumor samples. Conclusions: Failure of EGFR antibody therapy in patients with wild-type KRAS colorectal cancer may result from activating BRAF or PIK3CA mutations and false-negative sequencing results caused by intratumoral heterogeneity. Due to the particularly high rates of heterogeneity between primary tumors and lymph node metastases, the latter are least suitable for diagnostic mutation analysis. Clin Cancer Res; 16(3); 790–9

Immunohistochemistry is widely used to assess epidermal growth factor receptor (EGFR) expression on colorectal carcinomas to select patients for treatment with cetuximab, an anti-EGFR antibody. The data comparing different commercial EGFR antibodies is limited, and no cost comparisons have been made. We analyzed 65 advanced colorectal cancers from 36 patients using the EGFR pharmDx kit (DakoCytomation) and Clone 31G7 (Zymed Laboratories, Inc). EGFR expression was seen in 35 (53%) tumors (21 primary, 14 metastatic) with the Dako pharmDx kit. The Zymed antibody showed positive results in 41 (63%) tumors (25 primary, 16 metastatic). The cost per test was $40.00 with the pharmDx kit and $3.52 with the Zymed antibody. The Zymed antibody detects 10% more cases of colorectal cancer as EGFR positive, and is 10 times cheaper than the Dako pharmDx kit. There is little justification for the use of expensive kits for testing EGFR expression, when other available antibodies without the kit can give comparable or superior results.

Squamous cell cancer of the head and neck (HNSCC) would seem to be the ideal malignancy for epidermal growth factor receptor (EGFR) inhibition either with antibodies or small molecule tyrosine kinase (TK) inhibitors. EGFR protein is overexpressed in more than 90% of tumors relative to normal tissue, and high expression is associated with poor disease control. Data from preclinical models show at least additive effects of the two classes of EGFR inhibitors when combined with cisplatin, providing rationale for combination therapy. To date, cetuximab has been studied in recurrent HNSCC in combination with cisplatin and platinum plus fluorouracil (FU) as first-line treatment for recurrence and in the setting of platinum refractory disease, whereas the small molecule TK inhibitors have been tested as single agents in the secondor third-line setting. The optimal timing of EGFR inhibitors in the palliation of patients with disease recurrence has not been well defined. HNSCC has the theoretical advantage of providing easily accessible tumor for biopsy, which could facilitate study of the putative effects of molecularly targeted therapies on the complex signaling pathways downstream of the EGFR. Such correlative tissue studies are needed to elucidate EGFR pathway tumor biology and identify predictive markers for better patient selection for these costly therapies. This issue of the Journal of Clinical Oncology contains five articles that provide some insights into these questions and the results of an analysis of the impact of radiotherapy (RT) plus EGFR antibody therapy on quality of life (QoL). QoL is a complex measure, particularly for those not immersed in this field, seemingly with its own terminology, variety of tools, and myriad statistical tests. However, the importance of QoL cannot be underestimated for patients with head and neck cancer. As multimodality therapies are intensified, consideration of QoL in therapeutic decision-making, and in formulating standards of care, is of paramount importance. This issue of JCO contains a report of QoL assessments that were performed as part of the prospective comparison of RT alone to RT plus concurrent cetuximab in patients with locally advanced HNSCC published by Bonner et al. Significant improvements in overall survival and local-regional recurrence-free survival were observed for patients in the cetuximab treatment group without an apparent increase in toxicity. The current paper bolsters those findings through the use of two validated questionnaires (European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire C30 and Quality of Life Questionnaire, Head and Neck Module) that address symptom burden and its effect on physical and functional well-being, emotional, and social interactions. No significant differences were found between the two treatments in QoL at baseline or during the course of treatment out to 12 months. In both groups, QoL declined during treatment, with the greatest impact on function due to fatigue, loss of appetite, swallowing difficulty, and sensory problems, and then improved by 8 to 12 months. The authors interpreted this as a positive result. This pattern of decline and then return to baseline by about 12 months confirms data from other head and neck trials as does the observed correlation between global health/QoL and survival. Because a significant difference in local-regional control was reported in the primary paper, one might have postulated that the symptom burden of uncontrolled disease would result in a worse QOL for patients in the control group. In other words, the real goal and indication of a positive outcome should be an improvement in QOL for patients receiving the treatment that afforded them significantly longer survival and disease control, in this case RT plus cetuximab. It is not clear whether the occurrence of more dropouts over time among controls (RT alone) obscured this effect or the questionnaires used were not sensitive enough to detect differences between the treatment groups (not just within treatment groups). The conclusion that QOL was not worse for patients receiving RT plus cetuximab than for those treated with RT alone is, unfortunately, not very satisfying. As advances are made in multimodality therapies, we should seek improvements in QoL that parallel improvements in survival. Two therapeutic trials are reported in this issue. Vermorken et al tested single-agent cetuximab in a multicenter phase II trial enrolling 103 patients with recurrent/metastatic HNSCC that had progressed on platinum-based chemotherapy. In a two-part design, patients were first treated with cetuximab alone until disease progression, and then they were offered continuation of cetuximab with the addition of platinum. The objective response rate (complete plus partial response) for treatment with cetuximab alone was 13% (95% CI, 7 to 21), the disease control rate (complete plus partial plus stable response) was 46% (95% CI, 36 to 56), and the median time to progression (TTP) was 70 days. Of these 103 patients, 53 proceeded to the second part of the trial and received JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 16 JUNE 1 2007

Purpose: To optimally integrate epidermal growth factor receptor (EGFR) inhibitors into the clinical treatment of head and neck cancer, two important questions must be answered: (a) does EGFR inhibition add to the effects of radiochemotherapy, and (b) if so, which method of inhibiting EGFR is superior (an EGFR antibody versus a small molecule tyrosine kinase inhibitor)? We designed an in vivo study to address these questions. Experimental Design: Nude mice with UMSCC-1 head and neck cancer xenografts received either single, double, or triple agent therapy with an EGFR inhibitor (either cetuximab or gefitinib), gemcitabine, and/or radiation for 3 weeks. Tumor volumes and animal weights were measured for up to 15 weeks. Immunoblotting and immunofluorescent staining were done on tumors treated with either cetuximab or gefitinib alone. Results: The addition of an EGFR inhibitor significantly delayed the tumor volume doubling time, from a median of 40 days with radiochemotherapy (gemcitabine and radiation) alone, to 106 days with cetuximab and 66 days with gefitinib (both P < 0.005). Cetuximab resulted in significantly less weight loss than gefitinib. Immunoblot analysis and immunofluorescent staining of tumors show that although levels of phosphorylated AKT and extracellular signal–regulated kinase were decreased similarly in response to cetuximab or gefitinib, cetuximab caused prolonged suppression of pEGFR, pSTAT3, and BclXL compared with gefitinib. Conclusions: EGFR inhibition, particularly with cetuximab, improves the effectiveness of radiochemotherapy in this model of head and neck cancer. The correlation of response with prolonged suppression of EGFR, STAT3, and BclXL offers the possibility that these may be candidate biomarkers for response.

Purpose: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the treatment of advanced EGFR-mutant lung adenocarcinoma. However, almost all patients who respond develop acquired resistance on average approximately 1 year after starting therapy. Resistance is commonly due to a secondary mutation in EGFR (EGFRT790M). We previously found that the combination of the EGFR TKI afatinib and the EGFR antibody cetuximab could overcome EGFRT790M-mediated resistance in preclinical models. This combination has shown a 29% response rate in a clinical trial in patients with acquired resistance to first-generation TKIs. An outstanding question is whether this regimen is beneficial when used as first-line therapy. Experimental Design: Using mouse models of EGFR-mutant lung cancer, we tested whether the combination of afatinib plus cetuximab delivered upfront to mice with TKI-naïve EGFRL858R-induced lung adenocarcinomas delayed tumor relapse and drug-resistance compared with single-agent TKIs. Results: Afatinib plus cetuximab markedly delayed the time to relapse and incidence of drug-resistant tumors, which occurred in only 63.6% of the mice, in contrast to erlotinib or afatinib treatment where 100% of mice developed resistance. Mechanisms of tumor escape observed in afatinib plus cetuximab resistant tumors include the EGFRT790M mutation and Kras mutations. Experiments in cell lines and xenografts confirmed that the afatinib plus cetuximab combination does not suppress the emergence of EGFRT790M. Conclusions: These results highlight the potential of afatinib plus cetuximab as an effective treatment strategy for patients with TKI-naïve EGFR-mutant lung cancer and indicate that clinical trial development in this area is warranted. Clin Cancer Res; 22(2); 426–35. ©2015 AACR.

Purpose: The novel dual-action humanized IgG1 antibody MEHD7945A targeting HER3 and EGFR inhibits ligand-dependent HER dimer signaling. This phase I study evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of MEHD7945A. Experimental Design: Patients with locally advanced or metastatic epithelial tumors received escalating doses of MEHD7945A (1–30 mg/kg) every 2 weeks (q2w) until disease progression or intolerable toxicity. An expansion cohort was enrolled at the recommended phase II dose (14 mg/kg, q2w). Plasma samples, tumor biopsies, FDG-PET were obtained for assessment of pharmacokinetics, and pharmacodynamic modulation downstream of EGFR and HER3. Results: No dose-limiting toxicities or MEHD7945A-related grade ≥ 4 adverse events (AE) were reported in dose-escalation (n = 30) or expansion (n = 36) cohorts. Related grade 3 AEs were limited to diarrhea and nausea in the same patient (30 mg/kg). Related AEs in ≥20% of patients ≤24 hours after the first infusion included grade 1/2 headache, fever, and chills, which were managed with premedication and/or symptomatic treatment. Pharmacodynamic data indicated target inhibition in 25% of evaluable patients. Best response by RECIST included 2 confirmed partial responses in squamous cell carcinomas of head and neck (SCCHN) patients with high tumor tissue levels of the HER3 ligand heregulin; 14 patients had stable disease ≥8 weeks, including SCCHN (n = 3), colorectal cancer (n = 6), and non–small cell lung cancer (n = 3). Conclusions: MEHD7945A was well-tolerated as single agent with evidence of tumor pharmacodynamic modulation and antitumor activity in SCCHN. Phase II studies were initiated with flat (nonweight-based) dosing at 1,100 mg q2w in SCCHN and colorectal cancer. Clin Cancer Res; 21(11); 2462–70. ©2015 AACR.

Epidermal growth factor receptor (EGFR), which plays an important role in tumorigenesis, is overexpressed in a high percentage of patients with late-stage colorectal cancer.1 EGFR can maintain cancer cell survival independent of its kinase activity,2 and preclinical studies have demonstrated that combining anti-EGFR antibodies and kinase inhibitors was synergistic in colon cancer cell lines.3 Angiogenesis, which plays an important role in tumor development and metastasis, is partly mediated by vascular endothelial growth factor (VEGF) and its receptor VEGFR.4 Resistance to EGFR inhibitors may be mediated, at least partly, by activating VEGF-dependent signaling, and strategies that combine anti-EGFR and anti-VEGF agents appear promising in preclinical and clinical studies.5 Preclinical studies demonstrate that EGFR signals through both kinase-dependent and independent pathways and that combining a small-molecule EGFR inhibitor, EGFR antibody, and/or anti-angiogenic agent may be synergistic.2,5 Supporting these preclinical observations, our recent report in Oncoscience demonstrates antitumor activity for the combination of cetuximab, erlotinib, and bevacizumab in heavily pretreated patients with metastatic colorectal cancer entered on a phase I trial of this triple-drug regimen.6 This is the first description of dual EGFR inhibition (erlotinib plus cetuximab) together with an anti-angiogenic agent (bevacizumab) in patients with advanced colorectal cancer. Cetuximab is a monoclonal antibody against EGFR that is approved by the Food and Drug Administration (FDA) for KRAS wild-type metastatic colorectal cancer. Erlotinib, an EGFR tyrosine kinase, is FDA-approved for non-small cell lung cancer and pancreatic adenocarcinoma. Bevacizumab, a monoclonal antibody to VEGF, is FDA-approved for metastatic colorectal cancer. The key findings of the report include description of the preliminary anti-tumor activity and safety profile of this 3-drug combination in patients with metastatic colorectal cancer. Forty-one patients were enrolled, and the study population had advanced disease, with a median of 5 prior systemic therapies. Almost all of the patients (n = 38, 93%) had previously received bevacizumab, and 33 patients (80%) had received prior cetuximab. In spite of the extensive prior treatment of the enrolled patients, stable disease (SD) of at least 6 months (n = 11) or partial response (PR) (n = 3) was achieved in 14 patients (34%), and was observed even in patients who had received prior bevacizumab and/or cetuximab or who had been treated at doses below the recommended phase 2 dose. Among the 31 patients who had received prior sequential cetuximab and bevacizumab, 10 (32%) achieved SD ≥ 6 months/PR. Of the 4 patients who had received prior concurrent bevacizumab and cetuximab, 2 achieved SD ≥ 6 months/PR. Recent investigations suggest that combining EGFR kinase inhibitors and anti-EGFR antibodies could be more effective than either alone, possibly because EGFR has the ability to maintain cancer cell survival independent of its kinase activity.1,2 A prior preclinical study combining erlotinib and cetuximab demonstrated synergistic antitumor activity in colorectal cancer, and a phase 2 trial of this combination achieved an overall response rate of 31%,3 which is similar to the rate of SD ≥ 6 months/PR observed in the present study. Prior clinical trials that combined cetuximab and bevacizumab with cytotoxic chemotherapy in patients with colorectal cancer had disappointing results, including shorter overall survival, shorter time to progression, and lower quality of life.7 It is conceivable that the chemotherapy included in such regimens may be the variable contributing to the negative outcomes in such trials, in contrast to regimens that combine anti-EGFR and anti-VEGF agents without cytotoxic chemotherapy, which may be promising and may deserve further investigation. Analysis of the side effects observed reveals that this 3-drug combination was well-tolerated in patients with metastatic colorectal cancer. The recommended phase 2 dose (RP2D) was determined to be the full FDA-approved doses of cetuximab, erlotinib, and bevacizumab. Among the 34 patients treated at the RP2D, 31 patients (91%) tolerated the treatment without drug-related dose-limiting effects. The most common treatment-related adverse events of grade 2 or higher occurring in at least 15% of patients included rash (68%) and hypomagnesemia (44%). Only two patients (5%) withdrew from the study because of toxicity, including one patient with grade 2 skin rash and another individual with grade 2 diarrhea and fatigue. Although previous studies have demonstrated correlation between rash and response to EGFR inhibitors, no such correlation or trend was observed in the current study. Overall, the findings of this study have demonstrated that dual inhibition of EGFR with erlotinib and cetuximab, combined with the VEGF antibody bevacizumab, was well-tolerated and was associated with significant antitumor activity in heavily pretreated patients with metastatic colorectal cancer. These results are promising and merit further investigation in future, larger studies.

We hypothesized that aberrations activating epidermal growth factor receptor (EGFR) via dimerization would be more sensitive to anti-dimerization agents (e.g., cetuximab). EGFR exon 19 abnormalities (L747_A750del; deletes amino acids LREA) respond to reversible EGFR kinase inhibitors (TKIs). Exon 20 in-frame insertions and/or duplications (codons 767 to 774) and T790M mutations are clinically resistant to reversible/some irreversible TKIs. Their impact on protein function/therapeutic actionability are not fully elucidated.In our study, the index patient with non-small cell lung cancer (NSCLC) harbored EGFR D770_P772del_insKG (exon 20). A twenty patient trial (NSCLC cohort) (cetuximab-based regimen) included two participants with EGFR TKI-resistant mutations ((i) exon 20 D770>GY; and (ii) exon 19 LREA plus exon 20 T790M mutations). Structural modeling predicted that EGFR exon 20 anomalies (D770_P772del_insKG and D770>GY), but not T790M mutations, stabilize the active dimer configuration by increasing the interaction between the kinase domains, hence sensitizing to an agent preventing dimerization. Consistent with predictions, the two patients harboring D770_P772del_insKG and D770>GY, respectively, responded to an EGFR antibody (cetuximab)-based regimen; the T790M-bearing patient showed no response to cetuximab combined with erlotinib. In silico modeling merits investigation of its ability to optimize therapeutic selection based on structural/functional implications of different aberrations within the same gene.

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

Background Epidermal growth factor receptor (EGFR) is overexpressed in 80%–90% of non-small-cell lung cancer (NSCLC). Matuzumab, a humanized immunoglobulin G1 (IgG1) anti-EGFR monoclonal antibody, blocks activation of EGFR. Paclitaxel and EGFR inhibitors have additive antitumour effects in vitro. This phase I study assessed the tolerability, pharmacokinetics and efficacy of the combination of matuzumab and paclitaxel in patients with advanced NSCLC. Materials and methods Eighteen chemotherapy-naive (n = 9) or pretreated (n = 9) patients with stage IIIB or IV EGFR-positive NSCLC received weekly doses of matuzumab (100, 200, 400 or 800 mg) followed by paclitaxel 175 mg/m2 every 3 weeks. Toxicity was evaluated weekly and pharmacokinetics were measured during cycles 1 and 2. Results The maximum planned matuzumab dose of 800 mg was achieved without reaching the maximum tolerated dose. Grade 4 neutropenia occurred in one of three patients at 800 mg but resolved within 1 week; five additional patients treated with 800 mg had no dose-limiting toxicity (DLT). Grade 1/2 acneiform skin rash in 14 patients was the most frequent matuzumab-related side-effect. There were no higher-grade adverse events. Grade 2 toxicities included pruritus (n = 2), bronchospasm (n = 1), fissures (n = 1), abdominal pain (n = 1) and hot flushes (n = 1). Paclitaxel was discontinued in four patients due to allergic reactions. Coadministration of paclitaxel did not alter matuzumab pharmacokinetics. Responses occurred in four of 18 patients and included one complete response. Conclusions Matuzumab doses up to 800 mg weekly with paclitaxel 175 mg/m2 every 3 weeks are well tolerated, with no apparent drug interactions and with evidence of antitumor activity.

Purpose: Targeting epidermal growth factor receptor (EGFR) overexpressed by many epithelial-derived cancer cells with anti-EGFR monoclonal antibodies (mAb) inhibits their growth. A limited number of clinical responses in patients treated with the anti-EGFR mAb, (cetuximab), may reflect variability in EGFR type or signaling in neoplastic cells. This study combines EGFR-targeting with the non-MHC–restricted cytotoxicity of anti-CD3 activated T cells (ATC) to enhance receptor-directed cytotoxicity. Experimental Design: ATC from normal and patient donors were expanded ex vivo. Specific cytolytic activity of ATC armed with anti-CD3 × anti-EGFR (EGFRBi) against EGFR-expressing cancer cells derived from lung, pancreas, colon, prostate, brain, skin, or EGFR-negative breast cancer cells was evaluated in 51Cr release assays. In vivo studies comparing tumor growth delay induced by EGFRBi-armed ATCs or cetuximab were done in severe combined immunodeficient/Beige mice (SCID-Beige) bearing COLO 356/FG pancreatic and LS174T colorectal tumors. Results: At effector/target ratios from 3.125 to 50, both EGFRBi-armed normal and patient ATC were significantly more cytotoxic, by 23% to 79%, against EGFR-positive cells over ATC, cetuximab, anti-CD3 alone, or ATC armed with irrelevant BiAb directed at CD20. EGFRBi-armed ATC also secreted significantly higher levels of some TH1/TH2 cytokines compared with ATC alone. In mice, i.v. infusions of EGFRBi-armed ATC (0.001 mg equivalent/infusion) were equally effective as cetuximab (1 mg/infusion) alone for significantly delaying growth of established COLO 356/FG but not LS174T tumors compared with mice that received ATC alone or vehicle (P < 0.001). Conclusions: Combining EGFR antibody targeting with T cell–mediated cytotoxicity may overcome some limitations associated with EGFR-targeting when using cetuximab alone.

The epidermal growth factor receptor (EGFR) plays an important role in the carcinogenesis of many human malignancies and is therefore an attractive target against which anticancer therapy may be effective. At present, there are two ways in which this may be achieved clinically: antibodies against EGFR and inhibitors of the EGFR tyrosine kinase. This review describes presently approved agents cetuximab (monoclonal EGFR antibody), gefitinib and erlotinib (EGFR tyrosine kinase inhibitors) in detail. Efficacy data for these agents in various human malignancies is presented. Various other agents that are in the early stages of development at present have also been mentioned.

Contains fulltext : 152192.pdf (Publisher’s version ) (Closed access) Molecular imaging of specific biomarkers can have prognostic, predictive or monitoring value in head and neck squamous cell carcinoma (HNSCC). The epidermal growth factor receptor (EGFR) is involved in various radiation resistance mechanisms as it steers the pathways related to DNA damage repair, proliferation, hypoxia and apoptosis. Radiolabeled labeled F(ab')2 fragments of the EGFR antibody cetuximab can be applied for non-invasive imaging of this receptor. Preclinical studies have shown that radioresistant tumors had a higher tracer uptake after irradiation, probably due to upregulation of membranous EGFR, thereby increasing target availability possibly as a compensation mechanism. Tumors with increased EGFR availability were also more responsive to the EGFR inhibitor cetuximab. Potentially, radionuclide imaging of the EGFR can be applied for monitoring treatment regimens in clinical practice.

Background Oral squamous cell carcinoma (OSCC) is a genetic disease with high prevalence and poor survival. Evaluation of recurrent tumor for a valid biomarker may serve as a predictive system for analyzing prognostic course of this disease. This study was designed to evaluate and compare the immunoscoring of epidermal growth factor receptor (EGFR) expression in recurrent cases with their matched regional/locoregional recurrence and non-recurrent cases of OSCC. Methodology A total of 64 cases of OSCC were studied and immunostained with polyclonal EGFR antibody. Of these, 33 cases recurred at the end of 5 years of follow-up. For positive evaluation of EGFR staining, immunscoring was performed by adding the extent score and intensity score. Results We observed significant increase in EGFR immunoscore in recurrent cases of OSCC (P-value 0.001). Immunoscoring of EGFR expression is an independent prognostic and/or predictive parameter for recurrence in OSCC (Odds Ratio: 6.52). Conclusion Immunoscore using EGFR can be incorporated into traditional classification, thus providing an essential prognostic and potentially predictive tool.