Cetuximab Treatment Research Articles

Gastrointestinal Disasters of Cetuximab in the Treatment of Metastatic Colorectal Cancer: Mechanism and its Effect on Prognosis

Colorectal cancer (CRC) is among the top three cancers worldwide in terms of incidence and associated mortality. Colorectal cancer (CRC) is responsible for more than 880,000 deaths annually. The number of of CRC cases worldwide continues to increase, posing a serious threat to human health. Surgery and chemotherapy are the first treatments for CRC patients. The majority of CRC patients are diagnosed at an advanced stage, as symptoms are usually not apparent and difficult to diagnose in the early stage. The prognosis of metastatic CRC (mCRC) has long been unsatisfactory. Targeted drugs therapy, which targeting at specific genes and proteins, is a new treatment approach to CRC. Cetuximab is one of the most widely studied targeted drugs. By competitively binding to the epithelial growth factor receptor (EGFR), cetuximab inhibits the EGF and binding of the EGF ligand to the EGFR, thereby inhibiting tumor cell growth, invasion, and metastasis and inducing tumor cell apoptosis. The curative effect of cetuximab as a treatment for many kinds of tumors, especially mCRC, has been confirmed. Cetuximab combined with chemotherapy or monotherapy is used as first-line treatment in patients with RAS(rat sarcoma,Ras) wild-type mCRC. However, adverse drug reactions (ADRs) associated with the clinical application of cetuximab are attracting increasing attention, with numerous studies reporting adverse effects of cetuximab on the gastrointestinal system, with these effects having adverse consequences for the prognosis of CRC. In this review, we focus mainly on gastrointestinal disasters on cetuximab treatment for mCRC from three areas: the intestinal mucosal barrier (IMB), gut microbiota (GM)-host immune balance, and bacterial metabolites short chain fatty acids (SCFAs), and the impact of these effects on the prognosis of mCRC. We also make suggestions aimed at aiding oncological understanding of cetuximab as a treatment for mCRC. Keywords: Bacterial metabolites, cetuximab, colorectal cancer, gut microbiota-host immune balance, gastrointestinal disasters

Cetuximab-induced natural killer cell cytotoxicity in head and neck squamous cell carcinoma cell lines: investigation of the role of cetuximab sensitivity and HPV status

BackgroundThe epidermal growth factor receptor (EGFR) is overexpressed by 80–90% of squamous cell carcinoma of head and neck (HNSCC). In addition to inhibiting EGFR signal transduction, cetuximab, a monoclonal antibody targeting EGFR can also bind to fragment crystallisable domain of immunoglobulins G1 present on natural killer (NK), causing antibody-dependent cellular cytotoxicity (ADCC). However, presence of cetuximab resistance limits effective clinical management of HNSCC.MethodsIn this study, differences in induction of ADCC were investigated in a panel of ten HNSCC cell lines. Tumour cells were co-cultured with NK cells and monitored using the xCELLigence RTCA.ResultsWhile ADCC was not influenced by HPV status, hypoxia and cetuximab resistance did affect ADCC differentially. Intrinsic cetuximab-resistant cell lines showed an increased ADCC induction, whereas exposure to hypoxia reduced ADCC. Baseline EGFR expression was not correlated with ADCC. In contrast, EGFR internalisation following cetuximab treatment was positively correlated with ADCC.ConclusionThese findings support the possibility that resistance against cetuximab can be overcome by NK cell-based immune reactions. As such, it provides an incentive to combine cetuximab with immunotherapeutic approaches, thereby possibly enhancing the anti-tumoural immune responses and achieving greater clinical effectiveness of EGFR-targeting agents.

Multicenter Single-Arm, Two-Stage Phase 2 Study of Panitumumab in Patients With Cetuximab-Refractory Metastatic Colorectal Cancer: The PACER Trial

PurposeTo assess whether panitumumab is active in patients with cetuximab-refractory metastatic colorectal cancer (mCRC). Patients and MethodsEligible patients had pretreated RAS (renin–angiotensin system) wild-type mCRC that progressed after cetuximab treatment, after having shown either objective response or stable disease. A minimax two-stage design was applied, with progression-free rate at 2 months as the primary end point. At least 12 of 28 and 21 of 41 successes at the first and second stage, respectively, were required for a positive result. Panitumumab 6 mg/kg was provided every 2 weeks, until progression or unacceptable toxicity. ResultsOverall, 52 patients with KRAS (Kirsten rat sarcoma viral oncogene) wild-type disease were enrolled, but 11 were found to have mutated disease after all-RAS retesting. Among 41 eligible patients, median time since diagnosis was 38 months, and 71% experienced an objective response to previous cetuximab. First stage was passed with 12 of 28 patients alive without progression at 2 months. At the second stage, 17 of 41 patients were alive without progression at 2 months. At a median follow-up of 21.8 months, 35 patients experienced disease progression, and 26 died. Median progression-free survival was 2.1 months (95% confidence interval, 1.8-3.6) and median overall survival 6.8 months (95% confidence interval, 4.6-16.6). Most of the patients experienced no adverse reactions; 25% of patients had grade 3 rash. ConclusionAccording to our study design, panitumumab was not effective in patients with cetuximab-refractory RAS wild-type mCRC.

Anti-EGFR Therapy Induces EGF Secretion by Cancer-Associated Fibroblasts to Confer Colorectal Cancer Chemoresistance.

Targeted agents have improved the efficacy of chemotherapy for cancer patients, however, there remains a lack of understanding of how these therapies affect the unsuspecting bystanders of the stromal microenvironment. Cetuximab, a monoclonal antibody therapy targeting the epidermal growth factor receptor (EGFR), is given in combination with chemotherapy as the standard of care for a subset of metastatic colorectal cancer patients. The overall response to this treatment is underwhelming and, while genetic mutations that confer resistance have been identified, it is still not known why this drug is ineffective for some patients. We discovered that cancer-associated fibroblasts (CAFs), a major cellular subset of the tumor stroma, can provide a source of cancer cell resistance. Specifically, we observed that upon treatment with cetuximab, CAFs increased their secretion of EGF, which was sufficient to render neighboring cancer cells resistant to cetuximab treatment through sustained mitogen-activated protein kinases (MAPK) signaling. Furthermore, we show the cetuximab-induced EGF secretion to be specific to CAFs and not to cancer cells or normal fibroblasts. Altogether, this work emphasizes the importance of the tumor microenvironment and considering the potential unintended consequences of therapeutically targeting cancer-driving proteins on non-tumorigenic cell types.

Impact of cetuximab sequence on progress-free survival (PFS) and overall survival (OS) in patients with RAS wild-type metastatic colorectal cancer (mCRC): A real-world study.

e16042 Background: Cetuximab provides a clear clinical benefit in the treatment in patients with RAS wild-type mCRC irrespective of treatment line, but the best sequence is still under investigation. Methods: Patients with RAS wild-type mCRC (2011-2019) who received cetuximab therapy were retrospectively analyzed. They were stratified based on the cetuximab treatment sequence, into the 1st, 2nd, 3rd or later-lines groups. The primary objective was to investigate the impact on Cetuximab sequence (2nd vs. 3rd and later-line) in PFS and OS. As for patients received the 3rd or later-line cetuximab with irinotecan therapy after refractory to the prior 1st and 2nd-line combined chemotherapy with fluoropyrimidine, oxaliplatin, and irinotecan, they will get another PFS of 2nd-line chemotherapy (PFSchemo) besides the PFS of 3rd or later-line cetuximab compared with the cetuximab 2nd-line used patients. We combined the PFSchemo to the PFS of 3rd or later-line cetuximab, then compared with PFS of 2nd -line cetuximab to evaluate the primary objective in PFS. As for the OS of primary objective, we calculated it from start of the 2nd-line of treatment. The secondary objective was to compare the efficacy of cetuximab sequence (1st vs. 2nd and later-lines) in OS calculated from start of the 1st-line of treatment. Results: In total, 193 patients were included: 106 in the 1st, 41 in the 2nd, and 46 in the 3rd-line groups. No difference was observed in baseline characteristics as sex,age,site of primary tumour,number of metastatic sites in the three groups. The median PFS of the 2nd-line and 3rd or later-line groups were 7.1 (95% CI 6.39-7.80) and 13.87 months(95% CI 11.44-16.29) respectively. PFS of the 3rd or later-line group was significantly longer than that of the 2nd-line group (hazard ratio[HR], 0.552; 95% CI, 0.349 to 0.871; P = 0.01). Median OS was 17.8 months (95% CI 13.5-22.1) in the 2nd-line and 27.4 months (95% CI 20.69-34.16) in the 3rd or later-line group (HR, 0.597; 95% CI 0.341 to 1.043; P = 0.07) from start of 2nd-line therapy. The median OS was 28.17 months (95% CI 22.11-34.22) in the 1st-line group and 33.10 months (95% CI 26.88-39.31) in the 2nd and later-lines group (HR, 0.724; 95% CI 0.507 to 1.304; P = 0.075) calculated from the 1st-line of therapy. Conclusions: In a real-world cohort we found that later-line especially 3rd or later-line therapy of cetuximab, may be more benefit for patients with RAS wild type mCRC, as 3rd or later-line use of cetuximab give one more line therapy chance.

High amphiregulin mRNA expression is a strong prognostic biomarker with response to cetuximab in FIRE-1, CIOX, and FIRE-3.

4026 Background: Amphiregulin ( AREG) and epiregulin ( EREG) were discussed as biomarkers for treatment of metastatic colorectal cancer (mCRC). Data from randomized controlled trials (RCT) are limited. Methods: AREG and EREG mRNA expression by RTqPCR in relation to housekeeping genes were available from 688 patients of three RCT (FIRE-1, n = 192, FUFIRI vs. mIrOx; CIOX, n = 113, cetuximab + CAPIRI/CAPOX; FIRE-3, n = 383, FOLFIRI+cetuximab/bevacizumab) and were normalized to their respective range of each trial with median and 3rd quartile as threshold values. Kaplan-Meier estimated overall survival (OS) and progression-free survival (PFS). Cox regression analysis calculated hazard ratio (HR) and 95% confidence interval (95% CI). Overall response rate (ORR) was compared by chi square test. Results: Across all trials, high AREG mRNA expression appeared as strong prognostic biomarker for OS, PFS and ORR for all threshold values. In RAS wildtype patients, high AREG expression was associated with better OS and PFS for cetuximab but not bevacizumab treatment. (Table) No effects were seen for epiregulin when all trials were analysed together. Conclusions: High AREG mRNA expression appeared as strong prognostic biomarker in mCRC. Positive predictive information might exist for cetuximab treatment. [Table: see text]

Whole exome and transcriptome sequencing of colorectal and pancreatic cancer.

e15666 Background: Integration of Whole Exome Sequencing (WES) into clinical cancer therapeutics has revolutionized medicine in recent years. DNA sequencing alone may miss clinically actionable variants or identify aberrations that are not being transcribed. In this study we investigated the utility of integrating DNA and RNA sequencing in clinical samples. Methods: A cohort of 32 patient samples were analyzed by WES and RNA sequencing. Differential expression analysis was performed using a cohort of controls. Pathway analysis was performed using Ingenuity Pathway Analysis. WES and RNA analysis detected alterations including SNVs, indels, copy number events, fusions, alternate transcripts, TMB, MSI status and differential expression. Results: A total of 25 CRC (39-78yrs) and 7 pancreatic cancers (PCs) (51-91 yrs) were profiled by WES and RNA seq. A 66 yr old pt with neoplasm of rectosigmoid junction tumor was found to be KRAS wildtype and was treated with cetuximab plus FOLFIRI. Patient failed therapy after 2 yrs and sequencing revealed MET amplification which is a known mechanism of resistance to cetuximab treatment. Further, RNA expression analysis showed 44-fold increase in MET expression along with overexpression of AREG and EREG. Out of the 7 PCs, 3 cases that did not harbor KRAS mutation were found to harbor VTCN1/NRG1 fusion, FGFR1/G3BP2 fusion and BRAF V600E mutation respectively. A 50 year-old stage IV metastatic (met) PC pt was treated with standard of care regimens. Upon relapse the sample was found to harbor VTCN1/NRG1 fusion along with a TERT promoter mutation. RNA expression analysis revealed 54-fold increased expression of NRG1 which may lead to clinical trial enrollment. A 52 year-old male met Stage IV PC, was treated with rucaparib and irinotecan based on prior sequencing data. Upon relapse, the pt went on ATR inhibitor (BAY1895344) and progressed very quickly. Sequencing of the met lesion was found to harbor FGFR1/G3BP2 fusion which was also confirmed by RNA expression. A 55 year-old met pt with PC was treated with standard chemotherapy. Upon disease progression pt was sequenced and based on presence of BRAF V600E, pt was treated with trametinib and dabrafenib. Following disease progression on BRAF/MEKi, met sample was resequenced and RNA expression analysis found increased expression of MET, MACC1 and SMAD7 and 4-fold decrease in PTEN which could potentially cause resistance to BRAF/MEKi therapy. Conclusions: Our study highlights the utility of comprehensive testing integrating genomic and transcriptomic data, in identifying targeted therapy options for cancer patients.

Dynamic alterations of genome and transcriptome in KRAS G13D mutant CRC PDX model treated with cetuximab

BackgroundKRAS mutations have been characterized as the major predictive biomarkers for resistance to cetuximab treatment. However, studies indicate that not all KRAS mutations are associated with equivalent treatment outcomes. KRAS G13D mutations were observed to account for approximately 16% of all KRAS mutations in advanced colorectal cancer patients, and whether these patients can benefit from cetuximab has not been determined.MethodsAn established KRAS G13D mutant colorectal cancer (CRC) patient-derived xenograft (PDX) model was treated with cetuximab. After repeated use of cetuximab, treatment-resistant PDX models were established. Tissue samples were collected before and during treatment, and multiomics data were subsequently sequenced and processed, including whole-exome, mRNA and miRNA data, to explore potential dynamic changes.ResultsCetuximab treatment initially slowed tumor growth, but resistance developed not long after treatment. WES (whole-exome sequencing) and RNA sequencing found that 145 genes had low P values (< 0.01) when analyzed between the locus genotype and its related gene expression level. Among these genes, SWAP70 was believed to be a probable cause of acquired resistance. JAK2, PRKAA1, FGFR2 and RALBP1, as well as 10 filtered immune-related genes, also exhibited dynamic changes during the treatment.ConclusionsCetuximab may be effective in KRAS G13D mutation patients. Dynamic changes in transcription, as determined by WES and RNA sequencing, occurred after repeated drug exposure, and these changes were believed to be the most likely cause of drug resistance.

Abstract B43: A systems biology approach to elucidate the mechanism of EGFR inhibitor sensitivity in mutant KRAS-driven colorectal cancer

Abstract Standard protocol to treat colorectal cancer (CRC) includes surgery, chemotherapy and radiotherapy as first-line treatment options. When applicable, targeting agents such as anti-EGFR therapies (i.e., cetuximab and panitumumab) have become important long-term neoadjuvant therapies that increase survival in some patients. In recent years it has become common practice to sequence patients and their tumor specimens’ DNA. This recent push in genomic medicine has led to the identification that 30-40% of CRC patient harbor an activating KRAS mutation. These mutations are thought to be an early driver in the cancer’s progression. KRAS is also considered to be the key player in conferring resistance to the anti-EGFR treatments cetuximab and panitumumab. That Ras mutations confer resistance to EGFR inhibitors is intuitive. In recent years, clinical guidelines have extended the list of contraindicated genotypes to many mutations in both the KRAS and NRAS genes. In contrast with the current guidelines for managing CRC with EGFR inhibitors, it has been shown that patients harboring a KRAS G13D mutation are sensitive to cetuximab. This has been reproducibly shown in both in vitro and in vivo model systems and is supported by retrospective analysis of initial clinical trial data. The current mutant-RAS restrictive guidelines are supported by a method that relied upon the grouping of all common Ras mutations as “equal,” which potentially washes out less common yet sensitive mutations. Whether KRAS G13D mutations are sensitive to cetuximab has remained a controversial topic for many years due to the lack of understanding of a mechanism, as it is counterintuitive to the current understanding of EGFR signaling cascade. We here identify a novel mechanism by which G13D RAS mutant is sensitive to EGFR inhibition by a nonintuitive process of reliance upon WT RAS molecules. We utilized a computational model of RAS signaling previously developed by our laboratory to explore mutant Ras signaling and thereby investigated the controversial response of KRAS G13D to anti-EGFR agents. Our computational studies of the historically reported biochemical processes that regulate Ras signals reveal a nonintuitive, mutant-specific dependency of wild-type RAS activation on EGFR. The model also reveals this dependency is determined by the interaction strength between a KRAS mutant and tumor suppressor neurofibromin. Our prospective experiments confirm this mechanism that arises from the systems-level regulation of Ras pathway signaling. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine. Citation Format: Thomas McFall, Jolene Diedrich, Stacy L. Littlechild, Laura Sisk-Hackworth, James Moresco, Meron Mengistu1, Andrey Shaw, Ed Stites. A systems biology approach to elucidate the mechanism of EGFR inhibitor sensitivity in mutant KRAS-driven colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B43.

Qualitative Ras pathway signature for cetuximab therapy reveals resistant mechanism in colorectal cancer.

Cetuximab therapy, which heavily relies on the activation of Ras pathway, has been used in KRAS, NRAS, BRAF, and PIK3CA wild-type colorectal cancer (CRC) (Ras-normal). However, the response rate only reached 60%, due to false-negative mutation detection and mutation-like transcriptome features in wild-type patients. Herein, by integrating RNA-seq, microarray, and mutation data, we developed a Ras pathway signature by characterizing KRAS/NRAS/BRAF/PIK3CA mutations to identify the hidden nonresponders from the Ras-normal patients by mutation detection. Using public and in-house data of CRC patients treated with cetuximab, discovery of the signature could identify cetuximab-resistant samples from the Ras-normal samples. Cetuximab resistance-related genes, such as PTEN, were significantly and frequently mutated in the identified Ras-activated samples, whereas two cetuximab sensitivity-related genes, APC and TP53, showed comutation and significantly higher mutation frequencies in the remaining Ras-normal samples. Furthermore, all the NF1- and BCL2L1-mutated samples were identified as Ras-activated from the Ras-normal samples by the Ras pathway signature with significantly under-regulated expression. Genes co-expressed with the two genes were both involved in Ras signaling pathway, the out-of-control of which could be attributed by the genes' loss-of-function mutations. To improve the treatment of cetuximab in CRC, NF1 and BCL2L1 could be used as complementary detection technique to those applied in clinical. In conclusion, the proposed Ras pathway signature could identify the hidden CRC patients resistant to cetuximab therapy and help to reveal resistance mechanisms.

Metabolic Imaging Using Hyperpolarized Pyruvate-Lactate Exchange Assesses Response or Resistance to the EGFR Inhibitor Cetuximab in Patient-Derived HNSCC Xenografts.

Optimal head and neck squamous cell carcinoma (HNSCC) patient selection for anti-EGFR-based therapy remains an unmet need since only a minority of patients derive long-term benefit from cetuximab treatment. We assessed the ability of state-of-the-art noninvasive in vivo metabolic imaging to probe metabolic shift in cetuximab-sensitive and -resistant HNSCC patient-derived tumor xenografts (PDTXs). Three models selected based on their known sensitivity to cetuximab in patients (cetuximab-sensitive or acquired-resistant HNC007 PDTXs, cetuximab-naïve UCLHN4 PDTXs, and cetuximab-resistant HNC010 PDTXs) were inoculated in athymic nude mice. Cetuximab induced tumor size stabilization in mice for 4 weeks in cetuximab-sensitive and -naïve models treated with weekly injections (30 mg/kg) of cetuximab. Hyperpolarized 13C-pyruvate-13C-lactate exchange was significantly decreased in vivo in cetuximab-sensitive xenograft models 8 days after treatment initiation, whereas it was not modified in cetuximab-resistant xenografts. Ex vivo analysis of sensitive tumors resected at day 8 after treatment highlighted specific metabolic changes, likely to participate in the decrease in the lactate to pyruvate ratio in vivo. Diffusion MRI showed a decrease in tumor cellularity in the HNC007-sensitive tumors, but failed to show sensitivity to cetuximab in the UCLHN4 model. This study constitutes the first in vivo demonstration of cetuximab-induced metabolic changes in cetuximab-sensitive HNSCC PDTXs that were not present in resistant tumors. Using metabolic imaging, we were able to identify hyperpolarized 13C-pyruvate as a potential marker for response and resistance to the EGFR inhibitor in HNSCC.

The novel long noncoding RNA CRART16 confers cetuximab resistance in colorectal cancer cells by enhancing ERBB3 expression via miR-371a-5p

BackgroundLong noncoding RNAs (lncRNAs) have been shown to participate in multiple biological processes and confer drug resistance. However, it remains unclear whether lncRNAs are involved in conferring cetuximab resistance in colorectal cancer (CRC) cells.MethodsCell Counting Kit-8 (CCK-8) assays were performed to assess the sensitivity of CRC cell lines to cetuximab treatment. We incubated Caco-2 cells, which are partially responsive to cetuximab, with increasing concentrations of cetuximab for approximately 6 months to generate Caco-2 cetuximab-resistant (Caco-2 CR) cells. Microarray analysis comparing Caco-2 CR with Caco-2 cells was used to identify lncRNAs that were potentially related to cetuximab resistance. Caco-2 cells were stably transduced with cetuximab resistance-associated RNA transcript 16 (CRART16) or an empty vector using lentiviral infection; the cells were designated Caco-2-CRART16 and Caco-2-NC, respectively, and were analyzed with RNA sequencing (RNA-seq). Quantitative real-time PCR (qRT-PCR) was performed to investigate RNA expression. Flow cytometry and TUNEL assays were used to assess apoptosis levels induced by cetuximab. The cell cycle, stemness biomarkers and membrane proteins of CRC cells were assessed via flow cytometry. RNA fluorescence in situ hybridization (FISH) was used to examine CRART16 localization and expression. Bioinformatics analysis was performed to predict the potential mechanism of CRART16, which was further validated by a dual-luciferase reporter assay. Differences in measurement data were compared using Student’s t test, one-way ANOVA followed by Dunnett’s test and two-way ANOVA.ResultsThe novel lncRNA CRART16 was upregulated in Caco-2 CR cells. CRART16 overexpression reversed the effects of cetuximab on cell viability and reduced cetuximab-induced apoptosis. Meanwhile, CRART16 overexpression led to increases in the proportion of CD44+/CD133+ cells. In addition, CRART16 acts as a competing endogenous RNA (ceRNA) for miR-371a-5p to regulate V-Erb-B2 Erythroblastic Leukemia Viral Oncogene Homolog 3 (ERBB3) expression. MiR-371a-5p mimics counteracted the cetuximab resistance induced by CRART16 overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that after CRART16 was overexpressed, the resulting differentially expressed mRNAs were mainly enriched in the MAPK signaling pathway.ConclusionsCRART16 overexpression may contribute to cetuximab resistance through the miR-371a-5p/ERBB3/MAPK pathway. Additionally, CRART16 contributes to the acquisition of stemness properties.

Model-based analysis of response and resistance factors of cetuximab treatment in gastric cancer cell lines.

Targeted cancer therapies are powerful alternatives to chemotherapies or can be used complementary to these. Yet, the response to targeted treatments depends on a variety of factors, including mutations and expression levels, and therefore their outcome is difficult to predict. Here, we develop a mechanistic model of gastric cancer to study response and resistance factors for cetuximab treatment. The model captures the EGFR, ERK and AKT signaling pathways in two gastric cancer cell lines with different mutation patterns. We train the model using a comprehensive selection of time and dose response measurements, and provide an assessment of parameter and prediction uncertainties. We demonstrate that the proposed model facilitates the identification of causal differences between the cell lines. Furthermore, our study shows that the model provides predictions for the responses to different perturbations, such as knockdown and knockout experiments. Among other results, the model predicted the effect of MET mutations on cetuximab sensitivity. These predictive capabilities render the model a basis for the assessment of gastric cancer signaling and possibly for the development and discovery of predictive biomarkers.

Novel EGFR ectodomain mutations associated with ligand-independent activation and cetuximab resistance in head and neck cancer.

Epidermal growth factor receptor (EGFR) is a pro-tumorigenic receptor tyrosine kinase that facilitates growth for cancer cells that overexpress the receptor. Monoclonal anti-EGFR antibody Cetuximab (CTX) provides significant clinical benefit in patients with head and neck squamous cell carcinoma (HNSCC). Missense mutations in the ectodomain (ECD) of EGFR can be acquired under CTX treatment and mimic the effect of large deletions on spontaneous untethering and activation of the receptor. Little is known about the contribution of EGFR ECD mutations to EGFR activation and CTX resistance in HNSCC. We identified two concurrent non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD in patient-derived HNSCC cells that were selected for CTX resistance through repeated exposure to the agent in an effort to mimic what may occur clinically. Structural modeling predicted that the G33S and N56K mutants would restrict adoption of a fully closed (tethered) and inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation, leading to persistent downstream AKT signaling. Our results demonstrate that HNSCC cells can select for EGFR ECD mutations under CTX exposure that converge to trap the receptor in an open, ligand-independent, constitutively activated state. These mutants impede the receptor’s competence to bind CTX possibly explaining certain cases of CTX treatment-induced or de novo resistance to CTX.

Radiomics Response Signature for Identification of Metastatic Colorectal Cancer Sensitive to Therapies Targeting EGFR Pathway.

The authors sought to forecast survival and enhance treatment decisions for patients with liver metastatic colorectal cancer by using on-treatment radiomics signature to predict tumor sensitiveness to irinotecan, 5-fluorouracil, and leucovorin (FOLFIRI) alone (F) or in combination with cetuximab (FC). We retrospectively analyzed 667 metastatic colorectal cancer patients treated with F or FC. Computed tomography quality was classified as high (HQ) or standard (SD). Four datasets were created using the nomenclature (treatment) - (quality). Patients were randomly assigned (2:1) to training or validation sets: FCHQ: 78:38, FCSD: 124:62, FHQ: 78:51, FSD: 158:78. Four tumor-imaging biomarkers measured quantitative radiomics changes between standard of care computed tomography scans at baseline and 8 weeks. Using machine learning, the performance of the signature to classify tumors as treatment sensitive or treatment insensitive was trained and validated using receiver operating characteristic (ROC) curves. Hazard ratio and Cox regression models evaluated association with overall survival (OS). The signature (area under the ROC curve [95% confidence interval (CI)]) used temporal decrease in tumor spatial heterogeneity plus boundary infiltration to successfully predict sensitivity to antiepidermal growth factor receptor therapy (FCHQ: 0.80 [95% CI = 0.69 to 0.94], FCSD: 0.72 [95% CI = 0.59 to 0.83]) but failed with chemotherapy (FHQ: 0.59 [95% CI = 0.44 to 0.72], FSD: 0.55 [95% CI = 0.43 to 0.66]). In cetuximab-containing sets, radiomics signature outperformed existing biomarkers (KRAS-mutational status, and tumor shrinkage by RECIST 1.1) for detection of treatment sensitivity and was strongly associated with OS (two-sided P < .005). Radiomics response signature can serve as an intermediate surrogate marker of OS. The signature outperformed known biomarkers in providing an early prediction of treatment sensitivity and could be used to guide cetuximab treatment continuation decisions.

TGF-Beta-Activated Cancer-Associated Fibroblasts Limit Cetuximab Efficacy in Preclinical Models of Head and Neck Cancer.

Most head and neck cancer (HNC) patients are resistant to cetuximab, an antibody against the epidermal growth factor receptor. Such therapy resistance is known to be mediated, in part, by stromal cells surrounding the tumor cells; however, the mechanisms underlying such a resistance phenotype remain unclear. To identify the mechanisms of cetuximab resistance in an unbiased manner, RNA-sequencing (RNA-seq) of HNC patient-derived xenografts (PDXs) was performed. Comparing the gene expression of HNC-PDXs before and after treatment with cetuximab indicated that the transforming growth factor-beta (TGF-beta) signaling pathway was upregulated in the stromal cells of PDXs that progressed on cetuximab treatment (CetuximabProg-PDX). However, in PDXs that were extremely sensitive to cetuximab (CetuximabSen-PDX), the TGF-beta pathway was downregulated in the stromal compartment. Histopathological analysis of PDXs showed that TGF-beta-activation was detected in cancer-associated fibroblasts (CAFs) of CetuximabProg-PDX. These TGF-beta-activated CAFs were sufficient to limit cetuximab efficacy in vitro and in vivo. Moreover, blocking the TGF-beta pathway using the SMAD3 inhibitor, SIS3, enhanced cetuximab efficacy and prevented the progression of CetuximabProg-PDX. Altogether, our findings indicate that TGF-beta-activated CAFs play a role in limiting cetuximab efficacy in HNC.

Emergence of RAS mutations in patients with metastatic colorectal cancer receiving cetuximab-based treatment.

208 Background: In management of patients with RAS wild-type mCRC, anti-EGFR therapies have demonstrated clinical benefit. However, the correlation between emergence of RAS mutations in circulating tumor DNA (ctDNA) and secondary resistance to anti-EGFR therapies requires further elucidation. In this study, we aim to examine evolutionary changes in RAS mutations through liquid biopsy in patients with mCRC during and after anti-EGFR therapy (NCT03401957). Methods: A total of 39 intention-to-treat (ITT) patients diagnosed with RAS wild-type mCRC, from Cathay General Hospital (CGH) and Kaohsiung Medical University Hospital (KMUH), were analyzed in this study. Of them, 24 were male and 15 were female, with median age of 55 years. Patients received cetuximab-based FOLFIRI or FOLFOX regimen as first-line treatment. Blood samples from enrolled patients were collected before and every 3 months during cetuximab-based treatment and also at disease progression. These blood samples were evaluated for RAS resistance mutations using MassARRAY platform. The primary endpoint is percentage of RAS mutations detected in ctDNA from patients during cetuximab treatment. The correlation between the tumor response of these patients and the emergence of RAS mutations in ctDNA is further analyzed. Results: Between January 2018 and July 2019, 3 (8.3%) of 36 per-protocol (PP) patients subsequently with newly detected ctDNA KRAS/NRAS mutations in their blood samples during the treatment period, and all of three patients developed progressive disease (PD). Of 36 PP patients, 19 (52.8%) patients had complete/partial response (CR/PR), 13 (36.1%) patients had stable disease (SD) and 4 (11.1%) patients had PD. The disease control rate was 88.9% in the present study. Three of 4 PD patients were detected with ctDNA KRAS/NRAS mutations in their blood sample, with time to onset of newly detected ctDNA RAS at 3 and 6 months posttreatment, respectively. Conclusions: In current study, the frequency of RAS mutations in blood samples during the treatment period was 8.3% and all of these 3 patients developed PD. The results of this study will offer substantial, valuable information for anti-EGFR therapeutic strategy in patients with mCRC.

Efficacy of Oxaliplatin/5-Fluorouracil/Capecitabine-Cetuximab Combination Therapy and Its Effects on K-Ras Mutations in Advanced Colorectal Cancer.

BackgroundThe aim of this study was to perform an accurate exploration on the efficacy of oxaliplatin/5-fluorouracil/capecitabine-cetuximab combination therapy and its effects on K-Ras mutations in advanced colorectal cancer.Material/MethodsAmong 96 patients who suffered metastatic colorectal cancer without mutated K-Ras, 41 patients who were receiving treatment with oxaliplatin/5-fluorouracil/capecitabine and administered cetuximab as the initial treatment comprised the observation group; the remaining 55 patients receiving cetuximab as an alternative treatment comprised the control group.ResultsThe observation group experienced significantly higher objective response rates (ORRs), and disease control rates (DCRs), than the control group (P<0.05 for both). The median progression-free survival (PFS) rates of the observation group and the control groups were 11.2 months (95% confidence interval [CI]: 10.1–12.3 months) and 7.4 months (95% CI: 6.6–8.2 months). The median overall survival (OS) rates were 16.8 months (95% CI: 15.2–18.4 months) and 12.4 months (95% CI: 11.6–13.2 months), respectively. The observation group had significantly longer PFS and OS in comparison to the control group (P<0.05). The patients who underwent cetuximab treatment for ≥10 months had a slightly higher rate of K-Ras mutations than those treated with cetuximab for <10 months (9.1% versus 7.3%).ConclusionsOxaliplatin/5-fluorouracil/capecitabine plus cetuximab exhibited better efficacy as initial treatment than the alternative treatment; it was also highly safe. Unfortunately, some patients might develop K-Ras mutations after long duration of cetuximab treatment, suggesting that K-Ras mutations are correlated with tumor progression and depend on the duration or dose of cetuximab treatment.

Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation.

Background and Purpose: RAS mutations limit the effectiveness of anti-epidermal growth factor receptor (EGFR) monoclonal antibodies in combination with chemotherapy for metastatic colorectal cancer (mCRC) patients. Therefore, new cell death forms have focused on identifying indirect targets to inhibit Ras-induced oncogenesis. Recently, emerging evidence has shown the potential of triggering ferroptosis for cancer therapy, particularly for eradicating aggressive malignancies that are resistant to traditional therapies.Methods: KRAS mutant CRC cell HCT116 and Lovo were treated with cetuximab and β-elemene, a bioactive compound isolated from Chinese herb Curcumae Rhizoma. Ferroptosis and epithelial-mesenchymal transformation (EMT) were detected in vitro and in vivo. Orthotopic CRC animal model were established and the tumor growth was monitored by IVIS bioluminescence imaging. Tumor tissues were collected to determine ferroptosis effect and the expression of EMT markers after the treatment.Results: CCK-8 assay showed that synergetic effect was obtained when 125 µg/ml β-elemene was combined with 25 µg/ml cetuximab in KRAS mutant CRC cells. AV/PI staining suggested a non-apoptotic mode of cell death after the treatment with β-elemene and cetuximab. In vitro, β-elemene in combination with cetuximab was shown to induce iron-dependent reactive oxygen species (ROS) accumulation, glutathione (GSH) depletion, lipid peroxidation, upregulation of HO-1 and transferrin, and downregulation of negative regulatory proteins for ferroptosis (GPX4, SLC7A11, FTH1, glutaminase, and SLC40A1) in KRAS mutant CRC cells. Meanwhile, combinative treatment of β-elemene and cetuximab inhibited cell migration and decreased the expression of mesenchymal markers (Vimentin, N-cadherin, Slug, Snail and MMP-9), but promoted the expression of epithelial marker E-cadherin. Moreover, ferroptosis inhibitors but not other cell death suppressors abrogated the effect of β-elemene in combination with cetuximab on KRAS mutant CRC cells. In vivo, co-treatment with β-elemene and cetuximab inhibited KRAS mutant tumor growth and lymph nodes metastases.Conclusions: Our data for the first time suggest that the natural product β-elemene is a new ferroptosis inducer and combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant CRC cells by inducing ferroptosis and inhibiting EMT, which will hopefully provide a prospective strategy for CRC patients with RAS mutations.

Biomarkers of cetuximab resistance in patients with head and neck squamous cell carcinoma.

Objective: In patients with head and neck squamous cell carcinoma (HNSCC), cetuximab [a monoclonal antibody targeting epidermal growth factor receptor (EGFR)] has been shown to improve overall survival when combined with radiotherapy in the locally advanced setting or with chemotherapy in first-line recurrent and/or metastatic (R/M) setting, respectively. While biomarkers of resistance to cetuximab have been identified in metastatic colorectal cancer, no biomarkers of efficacy have been identified in HNSCC. Here, we aimed to identify biomarkers of cetuximab sensitivity/resistance in HNSCC.Methods: HNSCC patients treated with cetuximab at the Curie Institute, for whom complete clinicopathological data and formalin-fixed paraffin-embedded (FFPE) tumor tissue collected before cetuximab treatment were available, were included. Immunohistochemistry analyses of PTEN and EGFR were performed to assess protein expression levels. PIK3CA and H/N/KRAS mutations were analyzed using high-resolution melting (HRM) and Sanger sequencing. We evaluated the predictive value of these alterations in terms of progression-free survival (PFS).Results: Hot spot activating PIK3CA and KRAS/HRAS mutations were associated with poor PFS among HNSCC patients treated with cetuximab in the first-line R/M setting, but not among HNSCC patients treated with cetuximab in combination with radiotherapy. Loss of PTEN protein expression had a negative predictive value among HNSCC patients treated with cetuximab and radiotherapy. High EGFR expression did not predict cetuximab sensitivity in our patient population.Conclusions: Hot spot activating PIK3CA and RAS mutations predicted cetuximab resistance among HNSCC patients in the first-line R/M setting, whereas loss of PTEN protein expression predicted resistance to cetuximab when combined to radiotherapy.