Anti-epidermal Growth Factor Receptor Antibody Research Articles

The occurrence of mutant KRAS clones in the blood of RAS wild type colorectal cancer patients: Impact of response or failure under anti-EGFR therapy.

600 Background: Colorectal cancer (CRC) is characterized by a high level of genetic heterogeneity. In addition, changes in the genetic profile induced by chemotherapy affect treatment results. Acquired resistance of tumors is defined as a result of clonal evolution and clonal selection under systemic chemotherapy. Repeated tumor tissue biopsies are difficult to obtain and cannot be easily used for dynamic monitoring of therapy response or failure due to marked tumor heterogeneity. Promising data for circulating cell-free tumor DNA (ctDNA) as a tool for studying tumor evolution were recently published. Methods: In this study we analyzed ctDNA from patients with metastatic CRC during treatment with anti-epidermal growth factor receptor (EGFR) antibodies (cetuximab/panitumumab). By droplet digital PCR we performed genotyping of CRC tissue and tracking of clonal evolution of the most frequent KRAS mutations (G12A, G12C, G12D, G12R, G12S, G12V, G13C, G13D, Q61R, A146T and A59T) in plasma ctDNA. Results: In initial KRAS wild type tumors several mutated KRAS clones occurred in plasma under the course of anti-EGFR-therapy indicating an increasing acquired resistance to the given therapy leading to a disease progression. Some of these mutations declined upon discontinuation of anti-EGFR therapy. Conclusions: Based on these results we hypothesize that the initial state of KRAS wild type situation seems to be restored in some cases. This opens up the possibility to reinduce anti-EGFR therapy in later therapy lines.

Biomarkers of skin toxicity induced by anti-epidermal growth factor receptor antibody treatment in colorectal cancer.

Skin toxicity is a common symptom of anti-epidermal growth factor receptor (EGFR) antibody treatment and is also a predictive marker of its efficacy in colorectal cancer patients. However, severe skin disorders induced by such antibodies negatively impact on the quality of life of patients and decreases drug compliance during treatment. If we can predict the high-risk group susceptible to severe skin toxicity before treatment, we can undertake the early management of any arising skin disorders and formulate a more accurate prognosis for anti-EGFR antibody treatment. Previous studies have identified molecular markers of skin toxicity induced by anti-EGFR antibody, such as EGFR polymorphisms, the expression of inflammatory chemokines and serum levels of EGFR ligands. A clinical trial was undertaken involving the escalation of cetuximab doses, guided by the grade of skin toxicity observed, such as no or low-grade, in metastatic colorectal cancer (the EVEREST study). The dose escalation of cetuximab was confirmed by a safety profile and had the tendency to achieve a higher response rate in KRAS wild-type patients. A large, prospective randomized trial is now ongoing (EVEREST 2) and the results of this trial may contribute to personalized medicine in KRAS wild-type colorectal cancer patients.

L-Tyrosine Confers Residualizing Properties to a d-Amino Acid-Rich Residualizing Peptide for Radioiodination of Internalizing Antibodies.

Purpose:The aims of the study were to develop and evaluate a novel residualizing peptide for labeling internalizing antibodies with 124I to support clinical development using immuno-positron emission tomography (PET).Methods:The anti-epidermal growth factor receptor antibody ch806 was radiolabeled directly or indirectly with isotopes and various residualizing peptides. Azido-derivatized radiolabeled peptides were conjugated to dibenzylcyclooctyne-derivatized ch806 antibody via click chemistry. The radiochemical purities, antigen-expressing U87MG.de2-7 human glioblastoma cell-binding properties, and targeting of xenografts at 72 hours post injection of all radioconjugates were compared. Biodistribution of 124I-PEG4-tptddYddtpt-ch806 and immuno-PET imaging were evaluated in tumor-bearing mice.Results:Biodistribution studies using xenografts at 72 hours post injection showed that 131I-PEG4-tptddYddtpt-ch806 tumor uptake was similar to 111In-CHX-A″-DTPA-ch806. 125I-PEG4-tptddyddtpt-ch806 showed a lower tumor uptake value but higher than directly labeled 125I-ch806. 124I-PEG4-tptddYddtpt-ch806 was produced at 23% labeling efficiency, 98% radiochemical purity, 25.9 MBq/mg specific activity, and 64% cell binding in the presence of antigen excess. Tumor uptake for 124I-PEG4-tptddYddtpt-ch806 was similar to 111In-CHX-A″-DTPA-ch806. High-resolution immuno-PET/magnetic resonance imaging of tumors showed good correlation with biodistribution data.Conclusions:The mixed d/l-enantiomeric peptide, dThr-dPro-dThr-dAsp-dAsp-Tyr-dAsp-dAsp-dThr-dPro-dThr, is suitable for radiolabeling antibodies with radiohalogens such as 124I for high-resolution immuno-PET imaging of tumors and for evaluation in early-phase clinical trials.

Possibility of TAS-102 as an Early Line Therapy Against Advanced Colorectal Cancer

Introduction: New drugs have improved the outcome of metastatic colorectal cancer treatment. In Japan, more than 10 agents with at least seven different mechanisms such as 5-fluorouracil (5-FU), irinotecan (IRI), oxaliplatin, angiogenesis inhibitors, anti-epidermal growth factor receptor antibodies, regrafenib, and TAS-102, have been approved. With the increase in the number of alternatives, the importance of choosing the agents increases. To suppress the toxicities of agents is also necessary. To increase the quality of life of the patients should be taken into consideration for their longer survival. TAS-102 has proved to be an effective and safe agent for the 3rd or 4th lines of advanced colorectal cancer. Presentation of case: We report a case of a patient who had suffered severe adverse events from standard firstline chemotherapy with 5-FU derivative and IRI. She did not want to undergo any more intensive chemotherapy. Toxicity and anxiety made her evade the standard combination chemotherapy. For this patient, TAS-102 achieved over 30% tumor shrinkage and more than 1-year progression-free survival with acceptable toxicities. Conclusion: TAS-102 can result in a better outcome for a responder when given as an earlier line of therapy. In future, predictive biomarkers of TAS-102 should be identified for better use of this agent.

Pitfalls of KRAS Testing for Treating Patietns with Metastatic Colorectal Cancer

Background: KRAS testing is a critical initial step of the treatment for metastatic colorectal cancer patients. However, recent clinical trials have raised the issue of whether patient selection as determined by KRAS testing is appropriate. Method: Here, we reviewed guidelines, literatures and clinical trials to elucidate whether detection threshold of KRAS testing is appropriate. Results: It has been recommended that a detection threshold within 1–10% should be considered for RAS testing in Japan, but there is insufficient evidence for widespread adoption in the guideline. In literatures, retrospective studies have shown that high-sensitivity KRAS testing is more effective in the secondor third-line setting but no study has been conducted in the first-line setting. High-sensitivity KRAS testing in recent clinical trials has revealed undetectable mutant clones in tumors before chemotherapy. These clones have been shown to influence the outcome of second-line treatment, but there is no evidence of their influence in the first-line setting. On the other hands, there are accumulating data showing an additional effect of anti-epidermal growth factor receptor (EGFR) antibodies in patients harboring minor KRAS mutations determined by a low detection threshold of 5–10% in the first-line setting. These findings indicate that high-sensitivity KRAS testing have enabled to select super-responders, but may lead to exclusion of patients who could benefit from drug therapy. Conclusion: In order not to deprive patients of the chance to benefit from anti- EGFR antibodies, the optimal cut-off value for KRAS testing should be determined for selection of patients likely to benefit from treatment in the first-line setting.

Immunological Effect of Anti-Epidermal Growth Factor Receptor (EGFR) Antibodies in Squamous Cell Carcinoma of the Head and Neck (HNSCC): the Present and the Future

Targeted therapy with anti Epidermal growth factor receptor (EGFR) monoclonal Antibodies (mAbs) offers the potential to improve outcomes in HNSCC. EGFR is over-expressed in 80 to 90% of HNSCC and leads to tumor cell proliferation and invasion. HNSCC is an immunogenic disease, it has a multiplicity of non-mutually exclusive mechanisms of immune suppression (e.g., reduction CD8+ cell influx and altered function of intra-tumoral CD4+ cells). Monoclonal Abs possess the potential advantage of recruiting immune effector mechanisms, such as complement-dependent cytotoxicity (CDC) and Ab-dependent cellular cytotoxicity (ADCC), as additional mechanisms of tumor cell killing. However immunotherapy with the EGFR-specific mAb Cetuximab is clinically effective in 10-20% of patients. There is a need to further understand the immunological mechanism of the mAbs to optimize the design of a target-based immunotherapy.

Abstract B149: Clinical pharmacokinetics (PK) of an anti-epidermal growth factor receptor (EGFR) antibody drug conjugate (ADC), ABT-414, in patients with glioblastoma multiforme (GBM) or advanced solid tumors likely to overexpress EGFR: Results from phase 1 studies

Abstract Introduction: ABT-414 is an ADC consisting of an anti-EGFR monoclonal antibody ABT-806 conjugated to a potent microtubule toxin monomethylauristatin F (MMAF) via a non-cleavable maleimidocaproyl (mc) linker. The antibody component of ABT-414 delivers MMAF to antigen-positive cells, where it disrupts microtubule network and induces cell cycle arrest and apoptosis. ABT-414 is currently in phase 2 clinical trials evaluating its efficacy in GBM subjects with EGFR amplification. Here we present the PK results from phase 1 studies. Methods: Two ongoing phase 1 studies are examining the safety, PK, and efficacy of ABT-414 in patients with GBM and solid tumors. Three analytes, including, ABT-414, total ABT-806, cys-mcMMAF, were measured to characterize ABT-414 PK. In GBM subjects, ABT-414 was administered Q2W in 3 arms: 1. in combination with radiation plus temozolomide (TMZ); 2. in combination with TMZ; 3. As monotherapy. In subjects with advanced solid tumors, ABT-414 monotherapy was administered as either a Q3W or as a weekly (2 week on/1 week off) IV dosing. An electrochemiluminescence immunoassay was used to determine ABT-414 and total ABT-806 concentrations and an LCMS/MS assay was used to measure cys-mcMMAF concentrations. PK parameters were computed using non compartmental analysis. Immunogenicity was assessed by measuring anti-drug antibody (ADA) against ABT-414. Results: The maximum concentrations (Cmax) of ABT-414 and total ABT-806 are typically achieved following the end of infusion and the exposures are approximately dose proportional in both studies. The harmonic mean t1/2 of ABT-414, total ABT-806, and cys-mcMMAF in days (%CV) were 7.2 (32.6), 9.4 (40.8), 4.3 (33.2), respectively. The mean clearance (%CV) of ABT-414 is 0.176 (23.3) and 0.267 (49.6) mL/hr/kg in GBM and solid tumor subjects, respectively. The systemic cys-mcMMAF exposures are ∼500-fold lower than ABT-414 on molar basis. The PK of ABT-414 at 1.25 mg/kg is not affected by temozolomide in recurrent GBM subjects [ABT-414 AUC (mg*hr/mL): with TMZ: 9.14 ± 2.75 without TMZ: 10.4 ± 1.98]. The exposures of cys-mcMMAF are comparable between subjects with normal renal function and subjects with mild or moderate renal impairment [cys-mcMMAF Cmax (ng/ml/mg/kg): Normal: 0.217 (0.189 - 0.249); mild: 0.199 (0.166 - 0.240); moderate: 0.168 (0.09 - 0.313)]. No correlation between ABT-414 exposures and creatinine clearance was observed. The exposures of ABT-414 and cys-mcMMAF are comparable between subjects with or without EGFR amplification [ABT-414 AUC (mg*hr/mL/mg/kg): EGFR (+): 5.3 ± 1.8; EGFR (-): 4.2 ± 1.0; Cys-mcMMAF AUC (ng*hr/mL/mg/kg): EGFR (+): 32.4 ± 20.8; EGFR (-): 28.8 ± 12.8]. Of 152 patients treated with ABT-414 and evaluable for ADA, 5.3% and 2.6% tested ADA positive at baseline and post-treatment, respectively. Conclusions: ABT-414 showed linear PK in the ongoing phase 1 studies in the dose ranges studied. Systemic exposures of cys-mcMMAF are significantly lower than ABT-414. Temozolomide appears to have no effect on ABT-414 PK or vice versa. EGFR amplification, mild or moderate renal impairment appear to have little effect on the PK of ABT-414 or cys-mcMMAF. Available data show lack of immunogenicity to ABT-414 treatment. Citation Format: Rajendar K. Mittapalli, Wijith P. Munasinghe, Zaw-Myo-Htet F. Myasein, David M. Hoffmann, Rose K. Lai, Kyle D. Holen, Hao Xiong. Clinical pharmacokinetics (PK) of an anti-epidermal growth factor receptor (EGFR) antibody drug conjugate (ADC), ABT-414, in patients with glioblastoma multiforme (GBM) or advanced solid tumors likely to overexpress EGFR: Results from phase 1 studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B149.

Abstract B130: Correlation of in vitro cytotoxicity and catabolism of antibody-drug conjugates (ADCs) in target cancer cells

Abstract A sensitive and convenient ELISA method has been developed to quantify catabolites generated upon treatment of target cancer cells with an antibody-drug conjugate (ADC). The levels of catabolites generated by different ADCs in target cells were correlated with their in vitro cytotoxic potencies. The ELISA method, exemplified for maytansinoid ADCs, measures catabolite levels as low as 0.03 pmol, requiring only 1 million cells, and is suitable as a high-throughput assay to compare processing efficiency of multiple conjugates with different antibodies or linkers. An anti-epidermal growth factor receptor antibody conjugate, anti-EGFR-CX-DM1, using our maytansinoid payload and a new, triglycyl (CX) peptide linker was highly active in vitro and in vivo against xenografts of the HSC-2 cell line. In contrast, an anti-EGFR-SMCC-DM1 conjugate using the non-cleavable SMCC linker had little activity. The CX-DM1 conjugate generated 1.7-fold greater amount of total maytansinoid catabolite compared to SMCC-DM1 (3.5 and 2.0 pmol per million cells in one day, respectively), with an 11-fold higher level of catabolite in the media for CX-DM1 (2.8 and 0.25 pmol per million cells, respectively), suggesting trapping of SMCC-DM1 catabolite in the lysosomes of this cell line. The carboxylic acid catabolite of CX-DM1 is presumably effluxed as an uncharged molecule out of acidic lysosomes into the cytoplasm, where it binds to and inhibits microtubules, and is also effluxed out of cells into the media. In contrast, the SMCC-DM1 conjugate generates a charged Lys-SMCC-DM1 catabolite that is not effectively transported out of the lysosome. In another cell line, HCC827, where both anti-EGFR CX-DM1 and SMCC-DM1 conjugates were equally cytotoxic, high levels of total catabolites were generated (2.7 and 2.0 pmol/million cells, respectively) and relatively high levels detected in the media (1.7 and 0.9 pmol/million cells, respectively), suggesting efficient processing and catabolite transport for both CX-DM1 and SMCC-DM1 conjugates in this cell line. The activity and processing of maytansinoid conjugates of an anti-epithelial cell adhesion molecule antibody (anti-EpCAM) with a hindered-disulfide linker (SPDB-DM4) and a charged sulfonate-bearing hindered-disulfide linker (sulfo-SPDB-DM4) were compared in a multi-drug resistant (MDR) and a non-MDR cell line. The sulfo-SPDB-DM4 conjugate was cytotoxic against the MDR cell line, HCT-15, while the SPDB-DM4 conjugate was inactive. A greater amount of total catabolite was generated for sulfo-SPDB-DM4 than for SPDB-DM4 (0.7 and 0.4 pmol/million cells, respectively), with lower MDR-mediated efflux for sulfo-SPDB-DM4 compared to SPDB-DM4 (intracellular amounts of 0.5 vs 0.1 pmol/million cells, respectively). In a non-MDR cell line, COLO 205, both sulfo-SPDB-DM4 and SPDB-DM4 conjugates were cytotoxic and generated high levels of total catabolites (2.6 and 2.0 pmol/million cells, respectively) and similar levels in the media (0.6 and 0.8 pmol/million cells, respectively), suggesting efficient catabolism and catabolite transport for both conjugates in this cell line. The ELISA method is a convenient and sensitive tool to measure the intra- and extracellular levels of maytansinoid catabolites generated by processing of ADCs. In several cell lines and ADCs, the method gave good correlation with in vitro cytotoxicity. Citation Format: Rajeeva Singh, Paulin Salomon, Yulius Setiady, Jose Ponte. Correlation of in vitro cytotoxicity and catabolism of antibody-drug conjugates (ADCs) in target cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B130.

Abstract A27: Discovery of predictive biomarker of adjuvant chemotherapy in stage III colorectal cancer related to KRAS/NRAS mutation status using proteomic approach: results from the two randomized phase 3 trials (NSAS-CC/RC)

Abstract Ras is a well-known oncogene, and patients with metastatic colorectal cancer that harbored activating mutations in KRAS and NRAS did not benefit from anti-epidermal growth factor receptor antibodies in previous clinical trials. However, the value of RAS mutations in stage III colorectal cancer has been controversial. NSAS-CC/RC studies consisting of two multicenter phase 3 randomized trials showed that postoperative adjuvant chemotherapy with uracil-tegafur (UFT) significantly improved relapse-free survival (RFS) and overall survival (OS) in patients with stage III colorectal cancer. Here, we performed an integrated analysis of two trials in order to evaluate the association of RAS (KRAS exons 2, 3, 4 and NRAS exons 2, 3) mutations with effectiveness of adjuvant UFT therapy. The analysis included 178 patients with colon cancer and 146 with rectal cancer. RAS mutations were detected by direct-sequencing with a polymerase chain reaction method and observed in 134/304 (44.0%) patients. In patients with RAS mutations, a significant survival benefit was associated with adjuvant UFT chemotherapy in terms of both RFS (HR = 0.49; p = 0.02) and OS (HR = 0.51; p = 0.03). In contrast, among patients without RAS mutations, there was no difference in RFS (HR = 0.89; p = 0.67) and OS (HR = 0.94; p = 0.83) between the surgery-alone group and surgery plus adjuvant UFT group. Therefore, we propose that KRAS/NRAS mutations are predictive indicators of the efficacy of adjuvant UFT chemotherapy. Thereafter, we performed a proteomic analysis of 73 tumors of the patients participating in this study by the system of 2-dimensional image-converted analysis of liquid chromatography and mass spectrometry (2DICAL). The quantity of detected 2652 proteins was compared between RAS mutation group and RAS wild-type groups. Significant differences were observed in the expression of 36/2652 proteins. Moreover, we divided the patients treated with adjuvant UFT chemotherapy into an early-recurrence group (recurrence within 12 months after surgery) and a late recurrence/ no recurrence group. Among the 36 proteins, the quantities of 4 proteins (KRT3, CLU, C6orf70 and FBXW8) were significantly different between these groups. In conclusion, our study suggested that the KRAS/NRAS mutation is a biomarker to predict the benefit of adjuvant chemotherapy in stage III colorectal cancer. The benefit may be caused by differences in the expression of several proteins. Citation Format: Yusuke Sasaki, Yasuhide Yamada, Masahiro Kamita, Masaya Ono. Discovery of predictive biomarker of adjuvant chemotherapy in stage III colorectal cancer related to KRAS/NRAS mutation status using proteomic approach: results from the two randomized phase 3 trials (NSAS-CC/RC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A27.

Therapeutic potential of TAS-102 in the treatment of gastrointestinal malignancies.

Fluoropyrimidines form the mainstay in treatment of gastrointestinal malignancies. For decades 5-fluorouracil (5FU), was the major fluoropyrimidine. Currently it is usually given in a combination with leucovorin and oxaliplatin, i.e. FOLFOX, or irinotecan, i.e. FOLFIRI, or all three, i.e. FOLFIRINOX, but gradually it has been replaced by oral fluoropyrimidine prodrug formulations, such as tegafur-uracil and S-1 (both contain ftorafur), and capecitabine (Xeloda®). Novel drugs such as the antivascular endothelial growth factor antibody, bevacizumab, and the anti-epidermal growth factor receptor antibody, cetuximab, are often combined with one of these treatment options. However, when resistance emerged, no alternatives were available. TAS-102, a combination of trifluorothymidine and the thymidine phosphorylase inhibitor TPI in a 1:0.5 ratio, is a novel oral formulation, which is active in 5FU-resistant models, both in vitro and in xenograft models. In addition to inhibition of thymidylate synthase, the major mechanism of action of classical fluoropyrimidines, TAS-102's major mechanism of action is incorporation into DNA, thereby causing DNA damage. TAS-102 also follows an alternative activation pathway via thymidine kinase, and is not a substrate for dihydropyrimidine dehydrogenase. All together this explains the efficacy in 5FU-resistant models. In early clinical studies, the twice-daily schedule (5 days on, 2 days rest) for 2 weeks every 4 weeks, led to a significant disease control rate in various malignancies. This schedule showed consistent activity in two randomized trials on fluoropyrimidine refractory colorectal cancer patients, reflected by an increase of 2-3 months in overall survival in the TAS-102 group compared with placebo. Considering the impressive preclinical potential of various combinations TAS-102 has the promise to become an alternative for 5FU-resistant cancer.

Routine multiplexed mutational analysis of colorectal cancers (CRCs): A single-institution experience.

599 Background: Colorectal tumors harbor mutations in genes that play key roles in CRC carcinogenesis and that may enable prioritization of appropriate anti-cancer therapy. KRAS testing is part of standard of care testing prior to initiation of anti-epidermal growth factor receptor antibodies. Other mutations are emerging as significant drivers, but they are not yet part of routine screening. Methods: As part of Vanderbilt-Ingram Cancer Center’s Personalized Cancer Medicine Initiative, we developed a clinical algorithm to genotype patient’s colorectal tumors using a SNaPshot-based assay (multiplexed PCR, primer extension, and capillary electrophoresis) designed to detect 65 somatic mutations in 7 genes (KRAS, BRAF, AKT1, PIK3CA, SMAD4, PTEN, and NRAS) important in CRC carcinogenesis. Adult patients with metastatic CRC were consented for genotyping of tumor tissue. Mutational profiling was performed in Vanderbilt’s CLIA-certified Molecular Diagnostics Laboratory. Results were reported in the electronic medical record for clinical use. Results: Study enrolled from May 2012 to February 2014. SNaPshot results were available from 126 patients. 49 tumors (38.9%) were wildtype for all mutations tested. 58 (46.0%) tumors had a KRAS mutation, including one with two KRAS mutations; the most common was G12D (n=23), but two had a Q61H mutation and two had a K117N mutation, which are not included in some commercial KRAS tests. Ten (8.9%) tumors carried the V600E BRAF mutation. Nine (7.1%) tumors harbored a PIK3CA mutation. Three (2.4%) tumors had mutations in SMAD4. Four (3.2%) tumors had a NRAS mutation. One (0.8%) tumor had a PTEN mutation. Nine (7.1%) tumors harbored double mutations, the most common being KRAS and PIK3CA double mutations (N=6). 24 (19%) subjects subsequently were treated on clinical trial therapy, including 6 subjects on therapy based on the SNaPshot results. Not surprisingly, RAS results impacted use of anti-EGFR therapy. Conclusions: 61% of the tumors carried at least one mutation in the seven genes tested, suggesting that trials of tumor genotyping-based treatment assignment are feasible. Study was supported by Vanderbilt University Medical Center.

The cancer pain related factors affected by celecoxib together with cetuximab in head and neck squamous cell carcinoma

Pain is the most disruptive influence on the quality of prognosis among head and neck squamous cell carcinoma (HNSCC) patients. The development of pain is closely associated with tumor growth and inflammation in the cancer patients. Notably, cyclooxygenase-2 (COX-2) is an important mediator during inflammation. Celecoxib, a selective inhibitor of COX-2, was hailed as a promising chemopreventive agent for HNSCC. Dose-dependent cardiac toxicity limits long-term use of celecoxib. However, the toxicity can be diminished by lowering the dosage. In this study, we hypothesized that a combinatory strategy to reduce cancer pain via two distinct pathways, tumor grown inhibition and inflammation blockade, which would enhance analgesia effect induced by HNSCC. We found that treatment of cetuximab (C225), a monoclonal anti-epidermal growth factor receptor (EGFR) antibody, with low-dose celecoxib results in a more pronounced anticancer effect in HNSCC than either agent alone. More noticeably, the combination could downregulate the phosphorylation of constitutively active extracellular signal regulated kinase (ERK) in CAL-27 and Fadu cells. Furthermore, combination therapy enhancing S phase arrest and downregulating cyclin D1 was observed in Fadu cells. The COX-2 expression was significantly blocked by celecoxib combined with C225, and other cancer pain related factors, such as ET-1 and NGF, was also downregulated by combination treatment. Taken together, these results strongly suggest that combination of celecoxib with C225 holds potential as a new therapy strategy in developing cancer pain treatment in HNSCC.

SPIRITT: A Randomized, Multicenter, Phase II Study of Panitumumab with FOLFIRI and Bevacizumab with FOLFIRI as Second-Line Treatment in Patients with Unresectable Wild Type KRAS Metastatic Colorectal Cancer

BackgroundSecond-line treatment with chemotherapy and anti-epidermal growth factor receptor or anti-vascular endothelial growth factor antibodies improves outcomes in patients with wild type Kirsten rat sarcoma viral oncogene homolog (KRAS) metastatic colorectal cancer (mCRC). The choice of biological agent in second-line mCRC remains unclear. In this randomized, phase II estimation trial, we compared FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin) in combination with panitumumab or bevacizumab in patients with disease progression during oxaliplatin-based chemotherapy and bevacizumab. Patients and MethodsOne hundred eighty-two patients were randomized to FOLFIRI with panitumumab or bevacizumab. The primary end point was progression-free survival (PFS). Secondary end points included overall survival (OS), objective response rate (ORR), and safety. ResultsPFS was similar between arms, with a hazard ratio (HR) of 1.01 (95% confidence interval [CI], 0.68-1.50; P = .97). Median PFS was 7.7 months (95% CI, 5.7-11.8) in the panitumumab arm and 9.2 months (95% CI, 7.8-10.6) in the bevacizumab arm. OS was also similar between arms, with an HR of 1.06 (95% CI, 0.75-1.49; P = .75). Median OS was 18.0 months (95% CI, 13.5-21.7) in the panitumumab arm and 21.4 months (95% CI, 16.5-24.6) in the bevacizumab arm. ORR was 32% (95% CI, 23%-43%) in the panitumumab arm and 19% (95% CI, 11%-29%) in the bevacizumab arm. Skin disorders, diarrhea, hypomagnesemia, hypokalemia, dehydration, and hypotension were more frequent in the panitumumab arm. Neutropenia was more frequent in the bevacizumab-containing arm. ConclusionPanitumumab or bevacizumab with FOLFIRI as second-line treatment had efficacy similar in patients whose disease progressed during oxaliplatin-based chemotherapy with bevacizumab, with expected toxicities. The development of more accurate biomarkers might help caregivers and patients to better choose between therapies for individual patients.

Regulation of 18F-FDG accumulation in colorectal cancer cells with mutated KRAS.

KRAS gene mutations occur in approximately 40% of colorectal cancers (CRCs) and are associated with resistance to anti-epidermal growth factor receptor antibody therapy. We previously demonstrated that (18)F-FDG accumulation in PET was significantly higher in CRCs with mutated KRAS than in those with wild-type KRAS in a clinical setting. Here, we investigated the mechanisms by which mutated KRAS increased (18)F-FDG accumulation. Using paired isogenic human CRC cell lines that differ only in the mutational status of the KRAS gene, we measured (18)F-FDG accumulation in these cells in vitro and in vivo. We also investigated the roles of proteins that have a function in (18)F-FDG accumulation. Finally, we examined the relationship among mutated KRAS, hypoxia-inducible factor 1α (HIF-1α), and maximum standardized uptake value with 51 clinical CRC samples. In the in vitro experiments, (18)F-FDG accumulation was significantly higher in KRAS-mutant cells than in wild-type controls under normoxic conditions. The expression levels of glucose transporter 1 (GLUT1) and hexokinase type 2 (HK2) were higher in KRAS-mutant cells, and (18)F-FDG accumulation was decreased by knockdown of GLUT1. Hypoxic induction of HIF-1α was higher in KRAS-mutant cells than in wild-type controls; in turn, elevated HIF-1α resulted in higher GLUT1 expression and (18)F-FDG accumulation. In addition, HIF-1α knockdown decreased (18)F-FDG accumulation under hypoxic conditions only in the KRAS-mutant cells. Small-animal PET scans showed in vivo (18)F-FDG accumulation to be significantly higher in xenografts with mutated KRAS than in those with wild-type KRAS. The immunohistochemistry of these xenograft tumors showed that staining of GLUT1 was consistent with that of HIF-1α and pimonidazole. In a retrospective analysis of clinical samples, KRAS mutation exhibited a significantly positive correlation with expressions of GLUT1 and HIF-1α and with maximum standardized uptake value. Mutated KRAS caused higher (18)F-FDG accumulation possibly by upregulation of GLUT1; moreover, HIF-1α additively increased (18)F-FDG accumulation in hypoxic lesions. (18)F-FDG PET might be useful for predicting the KRAS status noninvasively.

Management of colorectal cancer.

Colorectal cancer is one of the most frequent solid tumors in the Western world. Treatment options are dependent on the stage of the disease, the performance status of the patient, and increasingly the molecular makeup of the tumor. In countries with surveillance programs, the incidence rate as well as the mortality rate has gone down because of the earlier stages at which the tumors are detected. For rectal cancer, standard of care differs from that of colon cancer with regard to perioperative treatment. In the metastatic setting, treatment options are uniform for colorectal cancer. Over the years, treatment options have emerged from single-agent 5-fluorouracil (5-FU) treatment to combination regimens using 5-FU and oxaliplatin or irinotecan or both. Treatment efficacy in the metastatic setting has been increased with the introduction of targeted substances. These include (a) the anti-vascular endothelial growth factor-A (anti-VEGF-A) antibody bevacizumab, (b) the anti-epidermal growth factor receptor (anti-EGFR) antibodies cetuximab and panitumumab, (c) the anti-angiogenic multi-kinase inhibitor regorafenib, and (d) the anti-angiogenic compound aflibercept. Anti-EGFR antibodies have shown efficacy only in the subpopulations of tumors that do not have any mutation in KRAS and NRAS exon 2, 3, 4. Physicians have the choice in the first line to use anti-EGFR or anti-VEGF inhibitors in combination with chemotherapy based on treatment goals and patient performance. In recent years, tumor location has been shown to be prognostic and predictive for clinical outcome. Right-sided sporadic colon cancers differ significantly in molecular characteristics and, with the exception of microsatellite instability (MSI-H) tumors, are associated with poor prognosis. Tumors based on hereditary non-polyposis colorectal cancer, on the other hand, have excellent prognosis in stage II and III disease. Recent efforts have focused on the molecular classification of colorectal cancer with the purpose of establishing molecularly defined subgroups.

EL4 - Current Evidence of Chemotherapy for Esophageal Cancer

Chemotherapy is used in many ways for the treatment of esophageal cancer. It has been used not only in the form of systemic chemotherapy but also in the form of definitive chemoradiotherapy and preoperative chemo(radio)therapy. In the JCOG9906 clinical trial, 5-fluorouracil plus cisplatin (FP) and concurrent radiotherapy (RT, 60 Gy) was used as definitive chemoradiotherapy, but this was changed to FP plus RT (50.4 Gy), a regimen that has the advantages of less late toxicities and the possibility of salvage therapy. Currently, this regimen is being evaluated in the JCOG0909 study. As per the JCOG9907 results, 2 courses of FP followed by surgery can be used as standard treatment. In addition, the superiority of docetaxel in combination with FP (DCF), and superiority of RT (41.4 Gy) with FP compared with preoperative FP is being verified in a randomized control trial (JCOG1109, NExT trial). Over the last 2 decades, FP therapy has remained the standard treatment for metastatic or recurrent esophageal cancer, but recently, taxanes have been used as second-line treatment. Evaluation of DCF (biweekly docetaxel with FP) in JCOG0807 indicated good results. Phase III trial, JCOG1314, compared FP to biweekly DCF regimen is planning now. Cetuximab, an anti-epidermal growth factor receptor antibody, was evaluated in a chemoradiotherapy setting, but there was no additive effect of cetuximab to standard chemoradiothearpy. Research for the exploration of new molecular targets is expected to help in the development of novel targeted therapy.

O3-11-6 - A Phase II Study of Pmab + Iri for Mcrc Pts with Wildkras, Resistant to Fluoropyrimidine, Oxaliplatin and Iri(Ogsg1001)

Background: Anti-Epidermal growth factor receptor (EGFR) antibody therapy showed to be effective in treatment for metastatic colorectal cancer (mCRC) with wild KRAS. Especially in BOND-1 trial, combination chemotherapy with anti-EGFR antibody plus irinotecan(IRI) was expected more effective than anti-EGFR antibody alone, resistant to IRI.There were no report of randomized trial to clarify the difference of efficacy between panitumumab(Pmab) plus IRI and Pmab alone, and no report for Pmab plus IRI in Japanese. We conducted a phase II study to evaluate the efficacy and safetyfor Pmab plus IRI.Methods: Subjects were mCRC patients with wild KRAS, who showed resistance to fluoropyrimidine, oxaliplatin(L-OHP) and IRI, and had measurable disease, ECOG PS 0-2. Pmab(6 mg/kg) plus IRI was administered every two weeks. The primary endpoint was response rate(RR). Secondary endpoints were disease control rate (DCR), progression free survival (PFS), overall survival (OS), response duration, and adverse event (AE).Results: A total of 31 subjects were enrolled between July 2010 and July 2012. Median age was 64 years old (range 42-74). Nineteen patients had liver metastasis, 11 had lungmetastasis and 3 had lymph nodemetastasis. An independent review committee evaluated for efficacy in eligible 31 subjects in accordance with the RECIST ver. 1.1. The RR was29.0% (95%CI,14.2-48.0 %). DCR was 74.2% (95%CI,55.4-88.1%), median PFS was 5.6 months(95%CI: 3.4-8.1) and median OS was 10.6 months(95%CI: 6.9-15.8). In 31 subjects, the incidence of any Grade 3 or greater adverse events was 58.1%. Major adverse events of Grade 3 were diarrhea (19.4%), rash acneiform (12.9%), fatigue (9.7%), anorexia (9.7%). A sudden death and an infusion related reaction were occurred.Conclusion: Combination chemotherapy with Pmab plus IRI was demonstaratedto be safe and more effective than Japanese single arm phase II of Pmab alone. This resultis equal to efficacy and safety of other reports for Pmab plus IRI.

Abstract LB-115: Ultrasensitive detection of cancer mutations in metastatic colorectal cancer FFPE and cell-free DNA samples using multiplexed droplet digital PCR

Abstract Targeted therapies in many forms of cancer today have allowed unprecedented progress in the treatment of disease. Despite these advances, routine implementation of genomic testing is still limited by: 1) methods to detect, with confidence, mutational loads below 10%, 2) limited amounts of sample (pg-ng range) per biological specimen, 3) diagnostic turnaround time, and 4) cost. In metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor antibodies (αEGFR) are used to target the wild-type EGFR receptor. However, KRAS is mutated in approximately 40% of colorectal cancers and is indicative of a negative response to αEGFR therapy. BRAF and PIK3CA mutations are also associated with poor response in the remaining patients with KRAS wild-type genotype. To optimize therapy strategies for personalized care, it is therefore critical to rapidly screen patient samples during the course of disease for the presence of multiple mutations. The low abundance of mutants and limited amount and quality of available clinical samples (FFPE and cfDNA) render it difficult to reliably detect multiple mutations with current platforms and methods. We have developed a multiplexing strategy for screening clinically-actionable KRAS, BRAF, and PIK3CA mutations in mCRC clinical samples using digital PCR. No pre-amplification step was required. This sensitive and inexpensive method reduces the risk of contamination and can be easily implemented in molecular diagnostic laboratories for rapid, routine screening and monitoring of residual disease in cancer patients. Citation Format: Wei Yang, Dawne N. Shelton, Samantha Cooper, Jennifer Berman, Svilen Tzonev, Eli Hefner, John F. Regan. Ultrasensitive detection of cancer mutations in metastatic colorectal cancer FFPE and cell-free DNA samples using multiplexed droplet digital PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-115. doi:10.1158/1538-7445.AM2014-LB-115

Immunohistochemical Evaluation of Epidermal Growth Factor Receptor (EGFR), MMP-2 and Heparanase in Pleomorphic Adenoma of Salivary Glands

Background: Salivary gland tumors are morphologically and clinically diverse group of neoplasms .The most common benign tumor is the pleomorphic adenoma, it contains the epithelial, myoepithelial and mesenchymal component, with variable morphological patterns. Epidermal growth factor receptor (EGFR) is a 170 KD glycoprotein which has a tyrosine kinase activity. It is found at abnormally high levels on the surface of many types of cells, so these cells may divide excessively in the presence of epidermal growth factor.MMP-2 is a widely studied matrix metalloproteinase which participates in extracellular matrix (ECM) degradation, having a wide range of substrates and able to degrade type I, IV, V, VII and X collagens, laminin, elastin, fibronectin and proteoglycans. Heparanase (HPA) is an endo-β-D-glucuronidase that has the activity of cleaving heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPGs), the major proteoglycans of the extracellular matrix (ECM) and basement membrane (BM) which play a key role in preventing tumor cells invasion and metastasis. Aims of the study: This study aimed to evaluate the expression of EGFR, MMP-2 and heparanase in pleomorphic adenoma of salivary glands and to correlate the expression of the aforementioned biomarkers with the clinical parameters, histological subtypes and components, and with each other as well. Materials and methods: Twenty five paraffin blocks of pleomorphic adenoma were included in this study. Sections immunohistochemically stained with anti EGFR, anti MMP-2 and anti heparanase monoclonal antibodies (Mabs). Results: Positive EGFR, MMP-2 and hepranase immunohistochemical expression was found in 22 (88%), 14(56%) and 21 (84%) of the studied cases respectively. Statistically non- significant correlation was found among the aforementioned markers with each other, and between the markers and any of the clinical parameters and histological classifications, except a statistically highly significant negative correlation revealed regarding EGFR expression with the squamous cells of epithelial component (p =0.001) and a highly significant positive correlation found regarding MMP-2 expression with the chondroid stromal component (0.003). Conclusions: Histological variation of pleomorphic adenoma has no influence on its biological behavior concerning EGFR, MMP-2 and heparanase expression. The immunoscores reorded in this study are expected to contribute to a better understanding of the biological behavior of PA.

Patterns of Use and Tolerance of Anti–Epidermal Growth Factor Receptor Antibodies in Older Adults With Metastatic Colorectal Cancer

BackgroundLimited data are available regarding the tolerance of anti–epidermal growth factor receptor (EGFR) antibodies among elderly patients with metastatic colorectal cancer (mCRC). We retrospectively reviewed our experience of treating elderly patients with mCRC with these agents between 2004 and 2011. MethodsPatients with mCRC ≥ 65 years treated with anti-EGFR agents were included in this analysis. We recorded demographic and disease characteristics, treatment regimen and duration, KRAS status, and overall survival (OS). Toxicity evaluation included common hematologic and nonhematologic toxicities seen with these agents. ResultsOne hundred seventeen patients were included, with a median age at treatment initiation of 73 years (range, 65-86 years), 59% of male sex, 82% with colon primary tumors, and 51% with metastatic disease at presentation. Median time on anti-EGFR treatment was 2.4 months. Older age at treatment initiation was associated with use of anti-EGFR antibody as monotherapy versus combination therapy (P = .0009). Worse performance status (PS) at treatment initiation was associated with a shorter overall survival (OS) (P = .013) and shorter treatment duration (P = .01). The incidence of hematologic/nonhematologic grade ≥ 3 was 36% and 15%, respectively. No association was found between age and presence of grade ≥ 3 toxicity. Longer treatment duration and better PS at treatment initiation were the only factors associated with higher incidence of grade 3 toxicity. ConclusionOur data demonstrate that anti-EGFR antibodies can be used in older patients with mCRC, with toxicity profiles similar to those reported in large phase III studies of younger patients. Advanced age was associated with receipt of anti-EGFR agents as monotherapy but did not impact treatment outcomes in this population.