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Kaplan Meier curves for EGFL7 mRNA levels in CLM for RFS (complete response, CR)

Patients' characteristics of JACCRO CC-05/06 and 05/06AR trials

Molecular differences in tumor locations may contribute to the sidedness-specific response to cetuximab in metastatic colorectal cancer (mCRC). We investigated genes associated with the response to cetuximab treatment depending on tumor sidedness. Our study included 77 patients with mCRC (13/63, right/left) with KRAS exon 2 wild-type tumors from phase II trials of first-line therapy with cetuximab. Expression levels of 2,551 genes were measured in tissue samples by HTG EdgeSeq Oncology Biomarker Panel. Univariate Cox regression analysis using log2 values of counts per million (CPM) was conducted in each sidedness to assess associations with clinical outcomes, and to define the optimal cut-off point for clinically significant genes. In addition, a gene set enrichment analysis (GSEA) was performed to identify significant gene pathways in each sidedness. Sixty-nine patients were assessable for gene expression data. Overexpression of BECN1 [log2(CPM) ≥ 6.8] was associated with favorable survival, regardless of tumor sidedness. High expression of NOTCH1 [log2(CPM) ≥ 7.5] predicted significantly longer progression-free survival (PFS; median 14.7 vs. 11.1 months, HR 0.43, P = 0.01) and overall survival (OS; median 42.8 vs. 26.5 months, HR 0.35, P = 0.01) in left side but not in right side. The GSEA showed that regulation of DNA replication gene set correlated with favorable survival in the left, whereas the subcellular component and leukocyte migration gene sets were associated with good survival in the right. In conclusion, genes contributing to the efficacy of cetuximab treatment may differ according to the sidedness in mCRC. NOTCH1 may potentially discriminate favorable responders to cetuximab in patients with left-sided tumors.

Angiogenesis-related gene expression is associated with the efficacy of anti-VEGF therapy. We tested whether intratumoral mRNA expression levels of genes involved in vascular morphogenesis and early vessel maturation predict response, recurrence-free survival (RFS), and overall survival (OS) in a unique cohort of patients with colorectal liver metastases (CLM) treated with bevacizumab-based chemotherapy followed by curative liver resection. Intratumoral mRNA was isolated from resected bevacizumab-pretreated CLM from 125 patients. In 42 patients, a matching primary tumor sample collected before bevacizumab treatment was available. Relative mRNA levels of 9 genes (ACVRL1, EGFL7, EPHB4, HIF1A, VEGFA, VEGFB, VEGFC, FLT1, and KDR) were analyzed by RT-PCR and evaluated for associations with response, RFS, and OS. P values for the associations between the individual dichotomized expression level and RFS were adjusted for choosing the optimal cut-off value. In CLM, high expression of VEGFB, VEGFC, HIF1A, and KDR and low expression of EGFL7 were associated with favorable RFS in multivariable analysis (P < 0.05). High ACVRL1 levels predicted favorable 3-year OS (P = 0.041) and radiologic response (PR = 1.093, SD = 0.539, P = 0.002). In primary tumors, low VEGFA and high EGFL7 were associated with radiologic and histologic response (P < 0.05). High VEGFA expression predicted shorter RFS (10.1 vs. 22.6 months; HR = 2.83, P = 0.038). High VEGFB (46% vs. 85%; HR = 5.75, P = 0.009) and low FLT1 (55% vs. 100%; P = 0.031) predicted lower 3-year OS rates. Our data suggest that intratumoral mRNA expression of genes involved in vascular morphogenesis and early vessel maturation may be promising predictive and/or prognostic biomarkers. Mol Cancer Ther; 15(11); 2814–21. ©2016 AACR.

Consort flow diagram of the study

TPS3149 Background: Human papillomavirus 16 (HPV16) is the most prominent subtype across invasive head and neck cancers, as well as cervical cancer and other anogenital cancers (Saraiya M, et al. J Natl Cancer Inst. 2015). The HPV16 E7 (E7) viral antigen is important for the survival of HPV-positive tumor cells but is absent from normal human tissue, making it an attractive target for anti-cancer therapy. Preclinical efficacy has been observed with MHC class I-restricted T cell receptor (TCR)-engineered T cells targeting E7 on HPV16-positive tumor cells (Jin BY, et al. JCI Insight. 2018). This Phase 1, first-in-human, open-label, multicenter study (NCT03912831) will evaluate the safety and efficacy of KITE-439, an autologous TCR-engineered T cell therapy targeting E7, in HLA-A*02:01–positive patients with relapsed/refractory HPV16-positive cancers. Methods: A single-patient dose-escalation schema will be used in Phase 1A of the study, enrolling up to 30 patients. Phase 1A will evaluate safety and inform the recommended dose of KITE-439 for Phase 1B. Approximately 45 patients with squamous cell cancer of the head and neck, cervical cancer, and other HPV16-positive tumors will be included in Phase 1B. Patients in Phase 1A and Phase 1B may receive optional bridging therapy followed by cyclophosphamide and fludarabine conditioning chemotherapy. Patients will then receive an infusion of KITE-439 at 1 × 106 up to 1 × 108 TCR-transduced T cells/kg along with daily subcutaneous IL-2 therapy for a maximum of 14 doses post-infusion. The primary endpoint for Phase 1A is the incidence of adverse events defined as dose-limiting toxicities. The primary endpoint for Phase 1B is investigator-assessed objective response rate per modified RECIST v1.1 criteria (Eisenhauer EA, et al. Eur J Cancer. 2009). Secondary endpoints for Phase 1B include duration of response, progression-free survival, overall survival, and safety. Patients ≥ 18 years must be HLA-A*02:01–positive and have relapsed/refractory HPV16-positive cancer, an ECOG PS of ≤ 1, and adequate bone marrow and organ function. Key exclusion criteria include a history of stroke, myocardial infarction, or symptomatic deep vein thrombosis/pulmonary embolism, known infection with human immunodeficiency virus, detectable hepatitis C, or detectable hepatitis B. This study is currently open and accruing patients. Clinical trial information: NCT03912831 .

101 Background: Genetically engineered T-cell therapy has shown remarkable clinical activity in hematologic malignancies. It is not known if this type of treatment can be applied effectively to epithelial cancers, which account for 80% to 90% of human malignancies. Methods: We conducted a phase I clinical trial with a 3 + 3 dose escalation in which patients with metastatic HPV-16+ epithelial cancers were treated with a one-time infusion of genetically engineered T cells expressing a T-cell receptor targeting an HLA-A*02:01-restricted epitope of HPV-16 E7 (E7 TCR-T cells). A lymphocyte-depleting conditioning regimen was administered before cell infusion, and high-dose systemic aldesleukin was administered after cell infusion. Results: Twelve patients, previously treated with a median of 4 (range, 3 to 7) anticancer agents, were treated. The cell dose was not limited by toxicity. Six patients demonstrated objective clinical responses, which included regression of bulky tumors and complete elimination of some tumors. Responses occurred in patients with vulvar, anal, head and neck, and cervical cancer. Four patients who previously received PD-1-based therapy responded. Response duration ranged from 3 to 9 months. Sustained, high-level engraftment of E7 TCR-T cells in peripheral blood was observed (median after approximately 6 weeks, 66% of total T cells, range 1% to 88%) and correlated with cell dose but not with clinical response. Infused T cell characteristics did not correlate strongly with response. Of the 4 resistant tumors that were studied, 3 demonstrated genetic defects in HLA-A*02:01 or B2M (necessary components of the target complex) and 1 demonstrated copy loss with decreased expression of antigen presentation and interferon response molecules (i.e. TAP1, TAP2, IFNGR1, IFNGR2). Of the 3 sensitive tumors studied, 0 showed genetic defects in these molecules. Conclusions: E7 TCR-T cells demonstrated safety and clinical activity in the treatment of highly refractory metastatic HPV-16+ cancers. Treatment resistance was linked to definitive genetic defects in the targeted peptide-HLA complex and to manifold defects in antigen processing and interferon response. Clinical trial information: NCT02858310.

433 Background: MAGE is an attractive target for IO given its expression restricted to carcinomas and the testes. Engineered T-cell targeting MAGE A3/6 has shown promise. However, successful treatment is dependent on circumventing the potentially hostile immune microenvironment in metastatic tumors. In this study, we aim to assess the effects of ICI on the immune microenvironment in patients who progressed on ICI. Methods: We obtained FFPE tissue from 16 patients with mUC across 3 institutions. Samples from the primary or metastatic tumor prior to treatment with PD1/PD-L1 inhibitors (ICI) were paired with samples from metastatic sites post-treatment. Differential mRNA expression was assessed using NanoString PanCancerIO360 panel for the main genes of interest ( MAGEA3/6, CD274, HLADP, HLAA, B2M and PDCD1) in addition to immune profiling of the tumor samples. Also, IHC was done on the paired samples to assess DE for the genes of interest using an H-score analyzed via the paired T-test. p-values

Molecular differences in tumor locations may contribute to the sidedness-specific response to cetuximab in metastatic colorectal cancer (mCRC). We investigated genes associated with the response to cetuximab treatment depending on tumor sidedness. Our study included 77 patients with mCRC (13/63, right/left) with KRAS exon 2 wild-type tumors from phase II trials of first-line therapy with cetuximab. Expression levels of 2,551 genes were measured in tissue samples by HTG EdgeSeq Oncology Biomarker Panel. Univariate Cox regression analysis using log2 values of counts per million (CPM) was conducted in each sidedness to assess associations with clinical outcomes, and to define the optimal cut-off point for clinically significant genes. In addition, a gene set enrichment analysis (GSEA) was performed to identify significant gene pathways in each sidedness. Sixty-nine patients were assessable for gene expression data. Overexpression of BECN1 [log2(CPM) ≥ 6.8] was associated with favorable survival, regardless of tumor sidedness. High expression of NOTCH1 [log2(CPM) ≥ 7.5] predicted significantly longer progression-free survival (PFS; median 14.7 vs. 11.1 months, HR 0.43, P = 0.01) and overall survival (OS; median 42.8 vs. 26.5 months, HR 0.35, P = 0.01) in left side but not in right side. The GSEA showed that regulation of DNA replication gene set correlated with favorable survival in the left, whereas the subcellular component and leukocyte migration gene sets were associated with good survival in the right. In conclusion, genes contributing to the efficacy of cetuximab treatment may differ according to the sidedness in mCRC. NOTCH1 may potentially discriminate favorable responders to cetuximab in patients with left-sided tumors.

Background Few clinical studies have investigated the association between neutrophil-lymphocyte ratio (NLR) and treatment with cetuximab-based chemotherapy in metastatic colorectal cancer (mCRC). The NLR may reflect immune cells modulating specific cytokine signals in the tumor microenvironment; however, which immune-related genes affect the NLR remain unclear. Patients and Methods In 77 patients with KRAS exon2 wild-type mCRC from prospective trials of first-line chemotherapy with cetuximab, expression levels of 354 immune-related genes were measured in tissue samples obtained from all patients by the HTG EdgeSeq Oncology Biomarker Panel. The association between the NLR and clinical outcomes was evaluated using the Spearman rank correlation coefficient. In addition, 2-sample t tests were performed to investigate which genes among the top 100 genes associated with survival had significantly different expression levels between the NLR-low and NLR-high groups among all measured genes. Results NLR data were available for 71 patients. The NLR was associated with progression-free survival and overall survival (r = −0.24; P = .040 and r = −0.29; P = .010, respectively). When stratified by the median value of the NLR, the Kaplan-Meier curve of NLR-low versus NLR-high differed significantly for both progression-free survival (median, 11.8 vs. 9.1 months; P = .036) and overall survival (median, 42.8 vs. 26.7 months; P = .029). The 2-sample t test revealed that the expression levels of the LYZ, TYMP, and CD68 genes differed significantly between the NLR-low and NLR-high groups (t test P-value Conclusion NLR is significantly associated with survival in patients with mCRC treated with first-line chemotherapy with cetuximab. Genes encoding for activities on macrophages may affect the NLR.

Melanoma-associated antigen-A (MAGE-A) and programmed-death ligand 1 (PD-L1) are present in urothelial carcinoma (UC). We assessed survival outcomes in patients with MAGE-A and PD-L1 expression. MAGE-A and PD-L1 expression on neoplastic cells was analyzed using tissue microarrays from patients with UC. We compared differential expression between disease stage and grade. MAGE-A and PD-L1 co-expression was subcategorized. Fisher’s exact test was done for categorical variables followed by univariable and multivariable analysis of recurrence-free survival (RFS) and progression-free survival (PFS). Co-expression of MAGE+/PD-L1+ was higher in advanced disease; however, only MAGE+/PD-L1− was associated with shorter RFS [hazard ratio (HR) 1.89; 95% confidence interval (CI) 1.19–2.99; p = .006]. MAGE+/PD-L1+ was associated with the worst PFS (HR 17.1; 95% CI 5.96–49.4; p ≤ .001). MAGE-A expression was more prevalent with high-grade (p = .015), and higher-stage ≥ pT2 (p = .001) disease. The 5-year RFS was 44% for MAGE+ versus 58% for MAGE− patients. On multivariable analysis, MAGE+ was also associated with shorter RFS (HR 1.55; 95% CI 1.05–2.30; p = .03). Similarly, MAGE+ was associated with shorter PFS (HR 3.12; 95% CI 1.12–8.68; p = .03). MAGE-A and PD-L1 expression is increased in advanced disease and associated with shorter PFS. Furthermore, MAGE-A expression was significantly associated with higher-grade and -stage disease and associated with shorter RFS and PFS. The worse prognosis associated with MAGE-A+/PD-L1+ provides evidence that a combinatorial treatment strategy co-targeting MAGE/PD-L1 might be feasible. Further studies are needed to validate these findings.

BACKGROUND No biomarkers exist to predict benefit from antiangiogenic therapy in metastatic colorectal cancer patients. ACVRL1 (activin receptor like-protein 1) encodes for ALK1, a member of the transforming growth factor-β receptor family, which directs pathologic angiogenesis. We examined the intratumoral expression of ACVRL1 and other angiogenesis pathway-related genes to identify molecular markers in the TRIBE study. MATERIALS AND METHODS Of 503 randomized patients, 228 had sufficient tissue for analysis. Formalin-fixed paraffin-embedded specimens were examined for expression of VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2, ACVRL1, EphB4, and EGFL7 using reverse transcription polymerase chain reaction. A maximal χ approach was used to determine the messenger RNA levels associated with progression-free survival (PFS), overall survival (OS), response rate, early tumor shrinkage, and depth of response. Recursive partitioning trees were constructed to identify composite prognostic biomarker profiles. External validation was conducted in silico using the Oncomine database. RESULTS High ACVRL1 expression was associated with superior OS in both treatment arms (FOLFOXIRI [5-fluorouracil, leucovorin, oxaliplatin, irinotecan]-bevacizumab, 32.7 vs. 13.5 months, hazard ratio [HR], 0.38, P = .023; FOLFIRI [5-fluorouracil, leucovorin, irinotecan]-bevacizumab, 35.1 vs. 22.0 months, HR, 0.36, P = .006) and prolonged PFS (11.7 vs. 5.9 months, multivariate HR, 0.17; P = .001) for patients receiving FOLFOXIRI-bevacizumab on univariate and multivariate analyses. In recursive partitioning analysis, ACVRL1 was the strongest discriminator of the response rate, PFS, and OS in patients receiving FOLFOXIRI-bevacizumab and of OS in patients receiving FOLFIRI-bevacizumab. In silico validation revealed significant associations between ACVRL1 expression, disease recurrence, and 1-year survival (P < .05) among all colorectal cancer stages. CONCLUSION ACVRL1 expression could serve as a prognostic biomarker in metastatic colorectal cancer patients receiving chemotherapy and bevacizumab and warrants further evaluation in prospective studies.

© 2015 Elsevier Inc. New approaches to optimization of cancer drug development in the laboratory and the clinic will be required to fully achieve the goal of individualized, precision cancer therapy. Improved preclinical models that more closely reflect the now recognized genomic complexity of human cancers are needed. Here we describe a collaborative research project that integrates core resources of The Jackson Laboratory Basic Science Cancer Center with genomics and clinical research facilities at the UC Davis Comprehensive Cancer Center to establish a clinically and genomically annotated patient-derived xenograft (PDX) platform designed to enhance new drug development and strategies for targeted therapies. Advanced stage non-small-cell lung cancer (NSCLC) was selected for initial studies because of emergence of a number of "druggable" molecular targets, and recent recognition of substantial inter- and intrapatient tumor heterogeneity. Additionally, clonal evolution after targeted therapy interventions make this tumor type ideal for investigation of this platform. Using the immunodeficient NOD scid gamma mouse, > 200 NSCLC tumor biopsies have been xenotransplanted. During the annotation process, patient tumors and subsequent PDXs are compared at multiple levels, including histomorphology, clinically applicable molecular biomarkers, global gene expression patterns, gene copy number variations, and DNA/chromosomal alterations. NSCLC PDXs are grouped into panels of interest according to oncogene subtype and/or histologic subtype. Multiregimen drug testing, paired with next-generation sequencing before and after therapy and timed tumor pharmacodynamics enables determination of efficacy, signaling pathway alterations, and mechanisms of sensitivity-resistance in individual models. This approach should facilitate derivation of new therapeutic strategies and the transition to individualized therapy.

Colorectal cancer (CRC) is a heterogeneous disease with genetic profiles and clinical outcomes dependent on the anatomic location of the primary tumor. How location has an impact on the molecular makeup of a tumor and how prognostic and predictive biomarkers differ between proximal versus distal colon cancers is not well established. We investigated the associations between tumor location, KRAS and BRAF mutation status, and the messenger RNA (mRNA) expression of proteins involved in major signaling pathways, including tumor growth (epidermal growth factor receptor (EGFR)), angiogenesis (vascular endothelial growth factor receptor 2 (VEGFR2)), DNA repair (excision repair cross complement group 1 (ERCC1)) and fluoropyrimidine metabolism (thymidylate synthase (TS)). Formalin-fixed paraffin-embedded tumor specimens from 431 advanced CRC patients were analyzed. The presence of seven different KRAS base substitutions and the BRAF V600E mutation was determined. ERCC1, TS, EGFR and VEGFR2 mRNA expression levels were detected by reverse transcriptase-PCR. BRAF mutations were significantly more common in the proximal colon (P

Introduction: Chimeric antigen receptor (CAR) T cell therapy is a transformative treatment modality in B cell malignancies. As next generation CARs are developed to improve durable response rates and expand into other indications of unmet need, the proper tools to identify, characterize, and modulate CAR activity will play a pivotal role in the optimization of this therapeutic option. This study developed and evaluated one approach, that of using monoclonal antibodies (mAbs) against common elements utilized in approved and investigational CARs. Methods: The mAbs KIP-1 and KIP-4 were raised in rabbits against the linkers of 2 single-chain variable fragments, the Whitlow linker (Whitlow, et al. Protein Eng. 1993) and the G4S linker (Huston JS, et al. PNAS. 1988), respectively, to generate universal detection reagents without adding an exogenous peptide recognition motif. Specificity and sensitivity were assessed by flow cytometric analysis of CAR T cells vs nontransduced T cells, immunohistochemistry (IHC) staining of embedded cell pellets with or without a CAR carrying the relevant linker, and epitope mapping by ELISA. Activation was assessed in a competitive stimulation assay in which nontransduced T cells and CAR T cells were mixed at defined ratios and stimulated broadly with OKT3 or specifically with KIP-1 or KIP-4. Results: Flow cytometric analysis demonstrated that CAR T cells with the Whitlow linker could be detected by phycoerythrin-conjugated KIP-1 at ≤ 10 ng per million cells with no detectable staining on nontransduced T cells above background levels. To further demonstrate specificity, IHC data revealed that KIP-1 bound to T cell pellets expressing CARs containing the Whitlow linker, but not the G4S linker. Epitope mapping revealed that the minimal KIP-1 epitope was contained within the amino acid sequence SGKPGSGE. Furthermore, KIP-1 was able to effectively identify CAR T cells in patient samples by both flow cytometry and IHC. Having established that KIP-1 was specific and sensitive, activation assays were performed to determine its CAR T cell-specific activating capacity. KIP-1 specifically activated CAR T cells, but not nontransduced T cells, as demonstrated by CAR T cell expansion and upregulation of cell surface activation markers, including CD69 and 4-1BB. KIP-4 was also assessed for specificity, sensitivity, and activation capacity with similar results. Conclusions: Taking advantage of the linear epitopes within the commonly-used Whitlow and G4S linkers, KIP-1 and KIP-4, respectively, can be used in a wide variety of phenotypic and functional assays with CARs of various specificities that share these linkers. These tools provide the means to realize the full potential of investigational CAR T cell products by supporting research, clinical, and manufacturing efforts. Citation Format: Stuart A. Sievers, Keith A. Kelley, Stephanie H. Astrow, Adrian Bot, Jed J. Wiltzius. Design and development of anti-linker antibodies for the detection and characterization of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1204.

On the basis of the results of recent clinical trials, histology-based decision-making for therapy of non-small-cell lung cancer has been advocated. We hypothesized associations of the biomarkers excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase M1 (RRM1), and thymidylate synthase (TS) with histology as a contributing factor to reported differences in chemotherapy outcomes between squamous cell carcinoma (SCCA) and adenocarcinoma (AC) subtypes. Here, we report analysis of the Response Genetics Inc., database and implications for histology-based therapy.RNA from microdissected formalin-fixed paraffin-embedded tumors was extracted and analyzed as previously described. Specimens from 2540 individual non-small-cell lung cancer patients were analyzed for one or more biomarkers, of which 1457 were categorized as AC or SCCA.For each biomarker, gene expression was lower in AC compared with SCCA (0.001), although there was a wide range between individual patients. Gene expression was higher in men versus women: ERCC1: 2.51 versus 2.22 (p = 0.005); RRM1: 1.41 versus 1.24 (p = 0.004); TS: 3.23 versus 2.83 (p0.001). However, SCCA was more frequent in men versus women (30%/19%; p0.001). When AC and SCCA were assessed separately, the statistical significance between gene expression and sex was lost (in SCCA: ERCC1, p = 0.14; RRM1, p = 0.26; TS, p = 0.11).This analysis represents the largest data set for gene expression of these biomarkers reported so far. Significant histology-related associations for ERCC1, RRM1, and TS are seen. However, marked heterogeneity exists in individual patient tumor expression levels. Randomized phase III trials assessing the predictive value of these chemotherapy-related biomarkers are warranted.

Angiogenesis-related gene expression is associated with the efficacy of anti-VEGF therapy. We tested whether intratumoral mRNA expression levels of genes involved in vascular morphogenesis and early vessel maturation predict response, recurrence-free survival (RFS), and overall survival (OS) in a unique cohort of patients with colorectal liver metastases (CLM) treated with bevacizumab-based chemotherapy followed by curative liver resection. Intratumoral mRNA was isolated from resected bevacizumab-pretreated CLM from 125 patients. In 42 patients, a matching primary tumor sample collected before bevacizumab treatment was available. Relative mRNA levels of 9 genes (ACVRL1, EGFL7, EPHB4, HIF1A, VEGFA, VEGFB, VEGFC, FLT1, and KDR) were analyzed by RT-PCR and evaluated for associations with response, RFS, and OS. P values for the associations between the individual dichotomized expression level and RFS were adjusted for choosing the optimal cut-off value. In CLM, high expression of VEGFB, VEGFC, HIF1A, and KDR and low expression of EGFL7 were associated with favorable RFS in multivariable analysis (P < 0.05). High ACVRL1 levels predicted favorable 3-year OS (P = 0.041) and radiologic response (PR = 1.093, SD = 0.539, P = 0.002). In primary tumors, low VEGFA and high EGFL7 were associated with radiologic and histologic response (P < 0.05). High VEGFA expression predicted shorter RFS (10.1 vs. 22.6 months; HR = 2.83, P = 0.038). High VEGFB (46% vs. 85%; HR = 5.75, P = 0.009) and low FLT1 (55% vs. 100%; P = 0.031) predicted lower 3-year OS rates. Our data suggest that intratumoral mRNA expression of genes involved in vascular morphogenesis and early vessel maturation may be promising predictive and/or prognostic biomarkers. Mol Cancer Ther; 15(11); 2814–21. ©2016 AACR.

Several studies have reported that epidermal growth factor receptor (EGFR)-related molecules may serve as predictors of cetuximab treatment for metastatic colorectal cancer (mCRC), such as EGFR gene copy number (GCN), expression of 2 ligands of EGFR, amphiregulin (AREG) and epiregulin (EREG), and EGFR CA simple sequence repeat 1 (CA-SSR1) polymorphism; however, these biomarkers still remain not useful in clinical practice since they have been evaluated using cohorts with patients treated in various settings of chemotherapy. We therefore analyzed associations of mRNA expression of AREG and EREG, EGFR GCN, and CA-SSR1 polymorphism [short (S;≤ 19) / long (L; ≥ 20)] with clinical outcomes in 77 Japanese patients with KRAS exon 2 wild-type mCRC enrolled in phase II trials of FOLFOX (n = 28/57, UMIN000004197) or SOX (n = 49/67, UMIN000007022) plus cetuximab as first-line therapy. High AREG expression correlated with significantly better progression-free survival (median 11.6 vs. 66 months, HR 0.52, P = 0.037); moreover, it remained statistically significant in multivariate analysis (HR: 0.48, P = 0.027). S/S genotype of CA-SSR1 predicted severe skin toxicity (P = 0.040). Patients with both AREG-low and EGFR low-GCN had significantly shorter overall survival than the others (median 22.2 vs. 42.8 months, HR 2.34, P = 0.042). The multivariate analysis showed that molecular status with both AREG-low and EGFR low-GCN was a predictor of worse survival (P = 0.006). In conclusion, AREG mRNA expression and EGFR CA-SSR1 polymorphism predict survival and skin toxicity, respectively, of initial chemotherapy with cetuximab. Our results also suggest potential prognostic value of the combined assessment of AREG and EGFR GCN for first-line cetuximab treatment.

Reduced phosphatase and tensin homolog (PTEN) expression in breast cancer confers susceptibility to phosphatidylinositol 3 kinase/Akt pathway inhibitors. As inhibitors of this pathway are in development for treatment of breast and other cancers, it is of interest to minimize the frequency at which PTEN signal is reduced because of poor antigen preservation, rather than to decreased protein expression. Methods for preserving antigenicity include coating sections in paraffin, refrigerated storage, desiccation, and storage in nitrogen. This study examined various combinations of the first 2 methods with respect to PTEN immunoreactivity. This was assessed semiquantitatively in freshly cut sections of paraffin blocks from 26 breast cancer specimens. The histoscore was compared with that observed in cut sections stored for 2 to 6 months with or without paraffin coating at room temperature (RT; 20 to 25°C) or at 2 to 8°C. At 2 months, the histoscore was strongest in freshly cut sections. Among the other treatments, refrigerated paraffin-coated sections had the highest histoscore followed by refrigerated uncoated sections. It was lower in RT paraffin-coated sections. Uncoated sections stored at RT showed the greatest signal loss. The greatest magnitude of changes between the various storage conditions and freshly cut slides ranged from 15% for refrigerated paraffin-coated slides to 75% for RT uncoated slides at 4 months. Although freshly cut paraffin-embedded sections were optimal, sections can be stored with adequate antigen preservation for 2 months using refrigeration with or without paraffin coating, and for 4 months using refrigeration with paraffin coating.

Background: Adoptive T cell therapy with gene-engineered T cells is an emerging cancer treatment strategy that has been applied successfully to the treatment of hematological cancers. We conducted a clinical trial to test proof of principle for this type of treatment in an epithelial cancer. Patients with human papillomavirus (HPV) 16-associated cancers were treated with gene-engineered T cells targeting HPV16 E7. Methods: The clinical trial was a phase I study with a 3 + 3 design and three dose levels (DL) of gene-engineered T cells (DL1: 1 x 109, DL2: 1 x 1010, DL3: 1 x 1011). Patients had metastatic HPV-16+ cancers from any primary tumor site. Treatment consisted of a one-time infusion of autologous T cells that were gene-engineered to express an HLA-A*02:01-restricted T-cell receptor (TCR) that targets HPV-16 E7 (E7 T cells). A lymphocyte-depleting conditioning regimen was administered before treatment. E7 T cell infusion was followed by high-dose systemic aldesleukin. Results: Twelve patients were treated (DL1, n=3; DL2, n=3; DL3, n=6). The age range was 31 to 59 years. The site of the primary cancer was vulva (n=1), head and neck (n=4), uterine cervix (n=5), and anus (n=2). Each patient had multiple metastases and had previously received 3 to 7 anti-cancer agents. The conditioning regimen consisted of cyclophosphamide 30 mg/Kg (n=6) or 60 mg/Kg (n=6) iv daily for 2 days overlapping with fludarabine 25 mg/m2 iv daily for 5 days. The E7 TCR was expressed by 90-99% of the infused T cells for each patient. E7 T cell cross-reactivity against healthy tissues was not identified. Cytokine-release syndrome was not observed. A single patient, at DL3, experience dose-limiting toxicity. Four patients experienced confirmed responses, and two patients experienced unconfirmed responses (i.e. met criteria for response at only one assessment) (Figure 1). Confirmed responses occurred in patients with cervical cancer, oropharyngeal cancer, vulvar cancer, and anal cancer. The duration of responses was 3 months (ongoing), 4 months, 8 months, and 9 months, respectively. These patients had previously received 7, 4, 7 and 3 prior anti-cancer agents, respectively. Three patients with confirmed responses had previously received PD-1 or PD-L1 checkpoint blockade. Four patients whose cancer progressed after E7 T cells received PD-1 or PD-L1 checkpoint blockade; none responded. Conclusions: Tumor regression can occur following treatment of an epithelial cancer with gene-engineered T cells. These findings support continued study of E7 T cells and possibly other types of gene-engineered T cells in epithelial cancers. Disclosures Adhikary: Kite Pharma: Employment. Schweitzer:Kite Pharma: Employment. Astrow:Kite Pharma: Employment. Hinrichs:Kite Pharma: Research Funding; NIH: Patents & Royalties: NIH patents related to cell therapy.

3043Background: Adoptive T-cell therapy with gene-engineered T cells is an emerging cancer treatment strategy. Study of this approach in epithelial cancers, the most common types of malignancies, h...

Purpose: Therapeutic options are limited for patients with advanced solid tumors failing conventional chemotherapy. Clinical responses have been observed with adoptively transferred tumor-specific T cells, including a T-cell receptor (TCR)-based therapy targeting the Cancer Testis Antigens MAGE-A3 and MAGE-A6 (Lu et al., J Immunother Cancer 2015;3(Suppl 2) P158). A T-cell product comprising this TCR is currently being tested in a novel phase 1 multicenter trial (NCT03139370). The programmed cell death-1/programmed cell death ligand 1 (PD-1/PD-L1) pathway may interfere with antitumor immune mechanisms. Several inhibitors of PD-1/PD-L1 signaling have shown activity and acceptable safety in multiple advanced cancer histologies. The expression of MAGE A antigens is prevalent in diverse solid tumors (Kerkar et al., J Immunother 2016;39(4):181-7). The prevalence of MAGE A3/A6 expression among MAGE A-positive tumors, and the rate and pattern of PD-L1 expression in relation to MAGE expression, are of interest as they may create a rationale for MAGE A3/A6 directed TCR therapy, alone or in conjunction with PD1/PD-L1 inhibition. Here, we describe the development and analytical validation of a two-part assay system combining immunohistochemistry (IHC) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for the detection of MAGE A3/A6 expression, and examine its performance in NSCLC, UC, SCCHN and CC. We also assess the coexpression of PD-L1 and other immune-related genes (e.g., HLAs) in tumor cells and/or immune cells to better understand the tumor microenvironment. Methods: Sections from approximately 200 formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens were screened by IHC for MAGE-A (mAb 6C1; recognizes MAGE A1, A2, A3, A4, A6, A10, A12) and PD-L1 (mAb E1L3N) expression. Gene expression analyses (RT-qPCR and RNA sequencing) were carried out on RNA extracted from adjacent tissue sections. Results: Analytical validation of the MAGE A3/A6 assay system successfully met predefined acceptance criteria appropriate for clinical sample testing. 75-80% of MAGE A IHC+ tumors were positive for MAGE A3/A6 mRNA. The rate of PD-L1 expression in tumor cells and/or immune cells in MAGE+ tumors was 89% for NSCLC (n=37), 41% for UC (n=32), 95% for SCCHN (n=19), and 100% for CC (n=7). Distinct tumor cell staining patterns were identified including samples in which PD-L1 and MAGE-A staining overlapped extensively, partially, or were nearly mutually exclusive. In some tumors, diffuse PD-L1+ immune cells were observed in the tumor bed; in other samples, PD-L1+ immune cells appeared to surround the tumor bed, while others showed lack of PD-L1+ immune cells. Conclusions: The results support clinical evaluation of a MAGE A3/A6 TCR T-cell product across these major histologies. This two-part MAGE A3/A6 screening assay may reduce the potential for false-positive results obtained using the 6c1 IHC assay alone , and be useful in screening subjects for anti-MAGE A3/A6 T-cell therapy. While the significance of staining patterns remains to be determined, the presence of PD-L1 positive TC and IC in MAGE A3/A6+ tumors supports the concept of checkpoint blockade in combination with T-cell therapy to treat advanced solid tumors. Citation Format: Stephanie H. Astrow, Izak Faiena, Rajul Jain, Alexandra Drakaki, Wesley S. Chang, Clark C. Fjeld, Jin Li, Adrian Bot. Gene and protein expression of MAGE, PD-L1, and associated immune landscape elements in non-small cell lung carcinoma (NSCLC), urothelial carcinoma (UC), squamous cell cancer of the head and neck (SCCHN), and cervical carcinoma (CC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A040.

Increased ornithine decarboxylase (ODC) activity, measured biochemically in breast cancers, has been associated with increased risk for recurrence of disease and death. Recently an immunohistochemical (IHC) method for ODC determinations in formalin-fixed paraffin-embedded tissues has been developed. We used this IHC ODC assay to evaluate primary breast cancers from 433 Vietnamese premenopausal women participating in a clinical trial of adjuvant combined hormonal therapy. Using an H SCORE system (intensity of staining 0-3 x percentage of all cells; possible range 0-300), 52% of tumors had an ODC score ofor = 35; 12% had a score ofor = 100. No statistically significant correlations of ODC H SCORES and usual prognostic factors were found; a negative weak correlation with weight was demonstrated (Spearman -0.12; p = 0.01). Using two cutoff scores, high and low ODC groups were similar in prognostic factors, except for high histologic grade which was more common with higher ODC H SCORES. Univariate, Kaplan-Meier and multivariate Cox analyses showed no evidence of relationships of ODC by H SCORE to disease-free or overall survival.

11613 Background: The antitumor activity of cetuximab (cet) may be affected by extracellular immune mechanisms. We have reported that immune-related genes are associated with survival in mCRC patients (pts) treated with cet (ASCO 2016 abstract#11591). NLR reflects cancer-related inflammation and is a validated prognostic maker in many types of cancers; however, it is not currently used for treatment decision-making. The association between NLR and clinical outcome of cet treatment for mCRC is unknown, and which genes are affecting the NLR remain to be identified. Methods: We enrolled 77 pts (57% males and 15% right-colon cancer) with KRAS exon 2 wild-type from 2 phase II trials (JACCRO CC-05 or CC-06) of 1st-line therapy with FOLFOX or SOX plus cet. All patients’ tissues were measured for expression levels of 354 immune-related genes by HTG EdgeSeq Oncology Biomarker Panel using next generation sequencing for quantitative analysis of targeted RNAs. The association between the NLR and clinical outcome was evaluated using Spearman’s rank correlation coefficient. In addition, the two-sample t-test was performed to investigate which genes had significantly different expression level between NLR-low and high groups in top 100 genes associated with survival among all measured genes. Results: Seventy-one of 77 pts were available for NLR data. The NLR was associated with progression-free survival (PFS) and overall survival (OS) (r = 0.24; p= 0.04, r = 0.29; p= 0.01, respectively). When stratified by median value of NLR, the Kaplan-Meier curve of NLR-low (n = 36) vs. high (n = 35) had a significant difference in both PFS (median 11.8 vs. 9.1 m, p= 0.036) and OS (median 42.8 vs. 26.7 m, p= 0.029). The two-sample t-test revealed that LYZ, TYMP, and CD68 genes expressed significantly differently between NLR-low and high groups (t-test p-value < 0.005, FDR p-value < 0.150). Conclusions: NLR is significantly associated with survival of 1st-line cet treatment for mCRC. Genes encoding for activities on tissue macrophages and endothelial cells may affect the level of NLR associated with outcome of cet combination chemotherapy. Clinical trial information: UMIN000010635.

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or “fingers” that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 µ at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.

Molecules localized to the synapse are potential contributors to processes unique to this specialized region, such as synapse formation and maintenance and synaptic transmission. We used an immunohistochemical strategy to uncover such molecules by generating antibodies that selectively stain synaptic regions and then using the antibodies to analyse their antigens. In this study, we utilized a monoclonal antibody, mAb 6D7, to identify and characterize an antigen concentrated at frog neuromuscular junctions and in peripheral nerves. In adult muscle, immunoelectron microscopy indicates that the antigen is located in the extracellular matrix around perisynaptic Schwann cells at the neuromuscular junction and in association with myelinated and nonmyelinated axons in peripheral nerves. The maintenance of the mAb 6D7 epitope is innervation-dependent but is muscle-independent; it disappears from the synaptic region within 2 weeks after denervation, but persists after muscle damage when the nerve is left intact. mAb 6D7 immunolabelling is also detected at the neuromuscular junction in developing tadpoles. Biochemical analyses of nerve extracts indicate that mAb 6D7 recognizes a glycoprotein of 127 kDa with both N- and O-linked carbohydrate moieties. Taken together, the results suggest that the antigen recognized by mAb 6D7 may be a novel component of the synaptic extracellular matrix overlying the terminal Schwann cell. The innervation-sensitivity of the epitope at the neuromuscular junction suggests a function in the interactions between nerves and Schwann cells.

KRAS mutations are associated with diverse biologic functions as well as prognostic and predictive impact in non-small cell-lung cancer (NSCLC) and colorectal cancer (CRC). In CRC, benefit from monoclonal antibody therapies targeting EGFR is generally limited to patients whose tumors have wild-type (WT) KRAS, whereas data suggest that this association is not present for NSCLC. We hypothesized that the unique tobacco-related carcinogenesis of NSCLC results in a divergence of KRAS MT genotype compared with CRC, contributing to differences in outcomes from EGFR-targeted therapies.Tumor from 2603 patients (838 CRC and 1765 NSCLC) was analyzed for KRAS mutations. DNA was extracted from microdissected formalin-fixed-paraffin-embedded specimens (FFPE) and 7 different base substitutions in codons 12 and 13 of KRAS were determined.KRAS mutation genotype differed significantly between NSCLC and CRC in frequency (25% vs. 39%; p0.001), smoking-associated GT transversions (73% versus 27%; p0.001), and ratio of transversions to transitions (3.5 vs. 0.79; p0.001). In NSCLC GLY12Cys mutations, resulting from a codon 12 GGTTGT substitution, were observed in 44% compared to 10% for CRC. In contrast, codon 12 or 13 GLYASP substitutions (resulting in a GA transition) were more frequent in CRC (42%) compared with NSCLC (21%).In this large dataset, KRAS mutation patterns are quantitatively and qualitatively distinct between NSCLC and CRC, reflecting in part differences in tobacco-related carcinogenesis. In light of differences in predictive value for EGFR-directed monoclonal antibody therapy and prognosis for specific KRAS mutations between NSCLC and CRC, these data provide an underlying biologic rationale.

e20056Background: The Vysis ALK Break Apart FISH Probe assay (ALK FISH) detects ALK gene chromosome 2p23 rearrangements in NSCLC FFPE tissue by fluorescence in situ hybridization (FISH) and involve...

11591Background: CRC Subtyping Consortium identified 4 molecular subtypes, one of which was classified by factors including activation or expression of immune-related pathways (Guinney J, et al. Na...

589 Background: Cet exerts its therapeutic effects not only through EGFR-signal blockade but also through antibody-dependent cell-mediated cytotoxicity (ADCC), mainly driven by natural killer (NK) cells. NK cells play a key role by releasing granzyme B (GZMB) via perforin (PRF1)-formed pores, leading to tumor cell apoptosis. CD137 (TNFRSF9) is expressed on NK cells, which regulates activation and proliferation of T cells. We hypothesized that genetic alteration in NK cell-mediated ADCC may serve as a predictor of cet in mCRC pts. Methods: Genomic DNA and RNA were isolated from tumor tissues of 77 KRAS exon2 wild-type (wt) pts enrolled in 2 Japanese phase II trials of cet plus oxaliplatin-based chemotherapy as 1st-line therapy, FOLFOX (n= 28/57, UMIN000004197) and SOX (n= 49/67, UMIN000007022). We evaluated associations between functional polymorphisms and gene expression of TNFRSF9, GZMB, and PRF1, and clinical outcome using PCR-based direct sequencing and molecular profiling panel (HTG EdgeSeq Oncology Biomarker Panel) based on next generation sequencing. Recursive partitioning (RP) method was also used to explore the associations. Results: In the population with median age of 63 years and follow-up time of 31.4 months (m), response rate (RR), median progression-free survival (PFS), and overall survival (OS) were 73 %, 10.0 m, and 33.9 m, respectively. TNFRSF9 rs161826 (A > G) was associated with PFS but not RR or OS in both univariate (any G vs A/A: 9.2 m vs 13.8 m, HR 1.75, P= 0.030) and multivariate analyses (P= 0.050). No association of rs161826 with gene expression was observed. PRF1 polymorphisms were not associated with outcome, while high PRF1 mRNA expression had association with shorter PFS (9.7 m vs 18.0 m, HR 2.60, P= 0.028), and it remained significant in multivariate analysis (P= 0.002). In RP analysis, PRF1 mRNA expression also showed significant association with PFS. Conclusions: TNFRSF9 rs161826 and PRF1 gene expression are significantly associated with PFS in pts with KRAS wt mCRC treated with cet-based chemotherapy. Gene expression and variation in NK cell-mediated ADCC may predict efficacy of cet (UMIN000010635). Clinical trial information: UMIN000010635.

616 Background: Prior work identified the fibroblast growth factor (FGF) pathway as a mediator of resistance to anti-VEGF therapy. We tested dovitinib, an oral inhibitor of both FGFRs and VEGFRs in pts progressing on anti-VEGF treatment. Methods: Pts with measurable advanced colorectal cancer (CRC) or non-small cell lung cancer (NSCLC) with progression despite treatment with an anti-VEGF agent within 56 days, good performance status (PS) and adequate organ function were eligible. A pre-treatment tumor biopsy was followed by treatment with dovitinib 500 mg on a 5 day on / 2 day off schedule for 28-day cycles. The primary endpoint of response (RECIST 1.1) was evaluated every 2 cycles. Secondary endpoints included toxicity and the disease control rate at 8 weeks. Intratumor mRNA expression was analyzed using a Next Generation Sequencing based expression array and circulating angiogenic factors analyzed by a multiplex bead-based assays and ELISAs. Results: Ten pts (9 CRC, 1 NSCLC) treated previously with bevacizumab (8) or ziv-aflibercept (2) enrolled. The study closed with termination of dovitinib development. No responses were observed in 7 evaluable pts; 3 withdrew consent during cycle 1. The best response was stable disease in 1 patient. Common toxicities included gastrointestinal, metabolic, and biochemical derangements. All pts experienced at least 1 grade 3 or higher treatment-related adverse event, most commonly fatigue, elevated GGT, and lymphopenia. Expression of multiple angiogenic mediators was common in tumors progressing on anti-VEGF therapy including high levels of FGFR1 and VEGFA. Dovitinib modulated circulating VEGF pathway components; however, no hyperphosphatemia was observed and plasma FGF2 increased continuously. Conclusions: We found no evidence for the activity of dovitinib in pts with CRC progressing on anti-VEGF therapy and could not confirm potent FGFR inhibition. Toxicities were significant. In tumors progressing despite anti-VEGF therapy, a multitude of pro-angiogenic mediators are expressed, including members of the FGF pathway. Clinical trial information: NCT01676714.

As in arthropods, several major organ systems in leeches, including body musculature, nervous system and nephridia, are organized into a fixed number of longitudinally iterated units called segments. Many cells, especially neurons, can be uniquely identified from segment to segment. Leech embryos comprise identified cells, which facilitates developmental analysis. So far as it is known, cell lineages in leech are largely determinate. Prior to first cleavage, cytoplasmic reorganization generates domains of yolk-deficient cytoplasm called teloplasm. In situ hybridization experiments suggest that teloplasm is enriched for polyadenylated RNAs. During the first three, unequal cell divisions, teloplasm is segregated to macromere D'; normally, this cell alone cleaves further to generate five bilateral pairs of embryonic stem cells, M, N, O/P and Q teloblasts. Centrifugation experiments have shown a causal link between inheritance of teloplasm and the cleavage pattern that generates teloblasts. Teloblasts undergo highly unequal divisions, generating a longitudinal array of segmental founder cells called m, n, o, p and q blast cells, from which the definitive segmental tissues arise via further stereotyped cell divisions. Microinjecting new-born teloblasts or their precursors with polyadenylic acid induces the formation of supernumerary teloblasts. This discovery permits further analyses of factors specifying the five cell lines generating segmental tissues of the leech.

8036 Background: E1 had been suggested as a predictive marker for platinum drugs. However, it has 4 different isoforms with differential DNA repair functions, antibodies used for quantitation lack ...

519 Background: The selection of targeted therapies is guided by the analysis of somatic mutations. The identification of RAS activating mutations can be used to examine tyrosine kinase inhibitor therapeutic eligibility and prognosis. Beyond known mutations in RAS exon 2 (codons 12 and 13), the identification of additional mutations in RAS exons 3 and 4 (codons 61, 117 and 146) also predict for resistance to EGFR therapy in colorectal cancer (CRC). Meta-analysis supports screening for these additional mutations in any screening strategy prior to administration of EGFR mAb therapy in metastatic CRC patients (Sorich, MJ, et al., Ann Oncol, Aug 12, 2014). Methods: We expanded our existing allele-specific KRAS and sequencing-based NRAS assays to include codons 61, 117, and 146 and analytically validated these assays to CAP/CLIA standards. DNA, from microdissected colon tumor tissue that was wild-type for RAS exon 2, was tested for exon 3 and 4 mutations and included over 15 additional mutations. Results: Forty-two (9.1%) samples were identified as bearing either an exon 3 or 4 KRAS mutation amongst 461 colon cancer specimens. Exon 4 codon 146 mutations were more prevalent than three of the commonly screened exon 2 mutations: G12A, G12R, and G12S. Five (1.8%) of samples were identified as carrying exon 3 or 4 NRAS mutations amongst 272 colon cancer specimens. This included a single codon 146 mutation in exon 4. As is seen with exon 2, RAS mutations at exons 3 and 4 were mutually exclusive of activating BRAF mutations with ~10% of patients harboring V600E. The collection of additional data studying KRAS and NRAS mutation status is currently ongoing. Conclusions: The KRAS expanded coverage contributed an additional 5.5% to overall burden of specimens bearing mutations. The NRAS expanded coverage contributed an additional 1.8% to the mutational burden. These analyses in clinical cohorts support the observations made in a trial population (Douillard JV, et al. NEJM 369:1023-34, 2013). The expanded RAS coverage identifies additional patients unlikely to respond to EGFR-targeted therapies that would otherwise have been assessed as “no mutation detected” in using assays restricted to RAS exon 2.

618 Background: Previous studies have reported that EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), are potential predictive markers for cet and high ERCC1 expression is associated with resistance to platinum-containing chemotherapy. However, there are still few studies to assess predictive values of these expression levels in JPN mCRC pts. The aim of this study was to evaluate the values in JPN mCRC pts treated with cet. Methods: This study enrolled 77 pts with tissue available from 2 prospective clinical trials evaluating combination of cet with oxaliplatin-based chemotherapy as first-line treatment in pts with KRAS wt and EGFR-expressing tumors, modified FOLFOX6 (n=28/57, UMIN000004197) and SOX (n=49/67, UMIN000007022). Total RNA isolated by macro-dissection from tissue was screened by RT-PCR for mRNA expression levels of AREG, EREG, and ERCC1, the cut-off values of which were based on the optimal cut-off value using the maximal chi-square approach on tumor response with adjusted p values. The same cut-off values were used to evaluate associations with progression-free survival (PFS) and overall survival (OS). Results: Median age and follow-up time were 63 (range: 39-79) years old and 24.7 (range: 5.5-39.3) months, respectively. In 73 evaluable pts, response rate (RR) was 77 % (95% CI, 67 to 86), but no significant association was seen in RR. Pts with high AREG expression (>1.59) had significantly longer PFS (11.6 vs. 6.6 months, HR: 0.53, logrank p=0.047) but not OS in 67 assessable pts. Pts with low ERCC1 expression (≤1.35) had significantly longer OS (42.8 vs. 22.0 months, HR: 2.46, logrank p=0.014) but a trend toward longer PFS (11.6 vs. 8.9 months, HR: 1.63, logrank p=0.092) in 68 assessable pts. On the other hand, high EREG expression (>3.21) was not associated with clinical outcome in 64 assessable pts. Conclusions: Our study identified predictive roles for AREG and ERCC1 mRNA expression in JPN mCRC pts treated with cet plus oxaliplatin. However, extended RAS mutations analysis is warranted (UMIN000010635).

3640 Background: Phase III TRIBE trial demonstrated that first-line FOLFOXIRI plus bev improved PFS and RECIST response and, at adjusted analyses, OS as compared to FOLFIRI plus bev. Previous data ...

e14569 Background: The selection of chemotherapies is guided by the analysis of molecular targets in solid tumors for well-known somatic mutations identified in microdissected formalin-fixed and pa...

e15185 Background: Pancreatic adenocarcinoma is one of the most aggressive malignancies with poor overall survival rates. C-Kit is a transmembrane receptor tyrosine kinase known to affect various c...

e15199 Background: Pancreatic ductal adenocarcinoma is one of the most aggressive solid malignancies. The c-MET oncogene plays a crucial role in mediating local invasion, systemic dissemination and...

Increased ornithine decarboxylase (ODC) activity in human breast cancer specimens has recently been shown to be an independent adverse prognostic factor for recurrence and death. Biochemical measurement of ODC, however, is not practical for routine clinical use. Furthermore, it does not take into account the heterogeneous composition of human breast cancers which contain variable proportions of epithelial and stromal elements. Therefore, we developed an immunohistochemical method for ODC determination which can be applied to formalin-fixed, paraffin-embedded tissue sections. We report here our results in a series of 30 human breast cancer samples. ODC expression was detected most consistently in the malignant epithelial component of the tumors. Twenty-seven of 30 samples stained positive with intensities ranging from 1+ to 3+. The fraction of malignant epithelial cells expressing ODC varied among specimens between 10% and > 90%. When quantitated by H-SCORE, ODC expression was significantly higher in the malignant epithelial component than in normal appearing epithelial cells and stroma admixed within the tumor. Normal mammary tissue adjacent to the cancer was available for analysis in six cases. ODC expression was absent in two (while both cancers were positive) but present in four to a degree which was overall comparable to that observed in the corresponding tumors. We believe that this technique will be useful for future studies aimed at expanding our knowledge of the role of ODC and polyamines (PA) in breast cancer biology.

The selection of chemotherapies is guided by the analysis of molecular targets in solid tumors for well-known somatic mutations identified in microdissected FFPE tissue. Combining a molecular targeting panel with select pharmacogenomic targets provides additional clinical information useful in customized patient management. We have developed a combined pharmacogenomic/molecular target approach using FFPE tissue from colon cancer patients. Our test, based on FDA guidance for Irinotecan administration to metastatic colorectal cancer (CRC) patients, is a recommended prescreening assay for UGT1A1 [TA] promoter variations to mimimize neutropenia. Meta-analysis of Irinotecan treated CRC patients has demonstrated an association between the [TA]7 allele (UGT1A1*28) and elevated levels of neutropenia in both homozygous and heterozygous carriers suggesting lowered dosing regimens (Liu, et al Pharmacogenomics J, 2013). Additional alleles, while uncommon in Caucasians, contribute to decreased levels of UGT1A1 enzymatic activity, and are restricted to genetic subgroups. Collectively these alleles influence the pharmacodynamic t1/2 of Irinotecan but are often not widely screened. Thus, a screening palate encompassing multiple ethnic-specific variants provides improved predictive information in a cosmopolitan population of cancer patients. As these polymorphisms are germline changes, UGT1A1 genotypes can be derived from either traditional whole blood assessment or more conveniently from FFPE surgical tissue at the time of diagnosis. We have screened paired blood/tumor samples from CRC patients to examine potential loss of heterozygosity in tumor specimens at this locus. In parallel, we screened microdissected tumor and adjacent normal tissue from FFPE samples to verify the potential of using tumor specimens as a routine alternative to blood. The use of FFPE tissue from CRC patients as part of a molecular work-up can provide genotypes for multiple UGT1A1 alleles of reduced activity (including *28), and may be better at predicting neutropenia in distinct Caucasian, Asian or other genetically distinct patient populations. The development of FFPE-based tests for pharmacogenomic tests such as UGT1A1 premised on genetic ancestry provides actionable information that can be used to maximize patient responses. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A211. Citation Format: Jack Hsiang, Rita El-Khoueiry, Heinz-Josef Lenz, Stephanie H. Astrow, Garrett P. Larson. Multiethnic screening for irinotecan sensitivity in FFPE tissue from colorectal cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A211.

3527 Background: Recent data suggests that CRC from different locations show distinct genetic profiles. Right-sided tumors have a worse prognosis and may have less benefit from targeted therapies. We investigated the tumor locations and genetic profiles (KRAS and BRAF mutation status and ERCC1, TS, EGFR and VEGFR2 mRNA expression) in 580 CRC tumors. Methods: FFPE tumor specimen from 580 patients with advanced CRC adenocarcinoma were microdissected and DNA and RNA were extracted. Specifically designed primers and probes were used to detect 7 different base substitutions in codon 12 and 13 of KRAS, V600E mutations in BRAF and the mRNA expression levels of ERCC1, TS, EGFR and VEGFR2 by RT-PCR. These values were analyzed according to tumor location (left vs. right vs. rectum). Results: BRAF mutations were significantly more common in the right colon (15%), followed by rectum (3.8%) and left colon (2.5%). KRAS mutations occurred at similar frequencies throughout the colon. Gene expression of ERCC1 was significantly higher in right-sided than left-sided colon tumors in KRAS wild-type colon cancers. The highest expression levels for all genes were seen in rectum. These differences reached significant levels for ERCC1 (rectum vs. right and rectum vs. left, p

11062 Background: KRAS is mutated in about 40% of colorectal cancers; it is the only validated predictive marker used in patients with metastatic disease. ERCC1 is a critical enzyme in nucleotide excision repair and is associated with response, progression free and overall survival in patients with NSCLC, colorectal, and gastric cancer treated with platinum based chemotherapy. We tested whether ERCC1 mRNA expression correlates with KRAS and BRAF mutation status in patients with colorectal, pancreatic, and lung cancer. Methods: Formalin fixed paraffin embedded tumor specimens from 1,514 patients (573 colorectal; 91 pancreatic; 850 lung) were microdissected; DNA and RNA were extracted. Specifically designed primers and probes were used to detect 7 different base substitutions in codons 12 and 13 of KRAS and the V600E BRAF mutation. ERCC1 mRNA expression levels were measured by quantitative RT-PCR in a CLIA approved laboratory. Results: Mt KRAS tumors had significantly lower ERCC1 mRNA levels relative to wt KRAS tumors in both colorectal (0.89 vs. 1.06; p

Background: Recent studies have characterized non-small cell lung cancer (NSCLC) as one of the most genomically deranged of all cancers, necessitating that both new drug development and patient therapy account for intra- and inter-patient tumor heterogeneity. Clinically annotated NSCLC patient-derived xenograft (PDX) models represent a novel approach to integrate this genomic complexity into a clinically relevant pre-clinical platform. We here describe molecular characterization to profile all currently “druggable” oncogenes for NSCLC in paired PDXs and original patient NSCLC tumor (PT). Method: Genomic DNA from archival formalin-fixed, paraffin-embedded (FFPE) PT and fresh first human-to-mouse (P0) NSCLC PDX tumors were isolated and subjected to oncogene mutational profiling using Sequenom's OncoCarta Panel v1. This panel detects 238 mutations in 19 genes commonly altered in cancer. RT-PCR-based molecular analyses of EGFR and KRAS mutations, EML4-ALK fusion transcripts, and RNA expression levels of ERCC1, RRM1 and TS genes were performed by Response Genetics, Inc. Genomic DNA from 3 serially passaged NSCLC PDX tumors (2 KRAS and 1 EGFR mutation models) up to 5 passages were also analyzed. Results: In the first 7 of 9 patient-PDX NSCLC models tested, oncogene mutational fidelity was preserved between PDX and PT with a good correlation of molecular biomarker expression (p Conclusion: Our results validate the overall genomic fidelity of PDX tumors compared to original PT. Molecular characterization of individual tumor results in a clinically and genomically annotated PDX model with potential utility for selecting and validating clinically relevant drug target(s) for personalized cancer therapy. Acknowledgement: Supported by UC Davis Comprehensive Cancer Center Developmental Award (NIH/NCI P30CA093373), UL1 RR024146 from the National Center for Research Resources, the Jackson Laboratory, Response Genetics Inc., and the Addario Foundation. Citation Format: Sonal J. Desai, Neal Goodwin, Regina Gandour-Edwards, Royce F. Calhoun, David T. Cooke, Laurel A. Beckett, Martin K.H. Maus, Stephanie H. Astrow, Philip C. Mack, Ralph deVere White, David R. Gandara, Tianhong Li. Genomic annotation of non-small cell lung cancer patient-derived xenograft models for personalized cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2013-336

383 Background: Gene expression levels of ERCC1, TS, EGFR and VEGFR2 may have predictive value for the personalized use of standard chemotherapeutics as well as agents targeting the EGFR and VEGF pathways and the efficacy of EGFR directed monoclonal antibodies like panitumumab and cetuximab has been confirmed to be dependent on wt KRAS and wt BRAF in patients with advanced colorectal cancer. We investigated the correlations between KRAS/BRAF mutational status and the mRNA expression levels of these genes. Methods: Formalin-fixed paraffin-embedded tumor specimens from 600 patients with advanced colorectal adenocarcinoma were microdissected and DNA and RNA was extracted. Specifically designed primers and probes were used to detect 7 different base substitutions in codon 12 and 13 of KRAS, V600E mutations in BRAF and the expression levels of ERCC1, TS, EGFR and VEGFR2 by RT-PCR. Results: Mt KRAS tumors had significantly lower TS and EGFR gene expression levels compared with wt KRAS (p

Repeatedin vivoobservations were used to study regenerated nerve terminals in neuromuscular junctions of the adult frogRana pipiens. Sartorius junctions in living animals were stained with the fluorescent vital dye RH414 and viewed with video fluorescence microscopy. Each junction was observed in the intact muscle and then again 7, 10, and 13 weeks after nerve crush. At 13 weeks, junctions were determined to be mono- or polyneuronally innervated using intracellular recording. Between 7 and 13 weeks, most identified junctions were reinnervated less precisely and completely than described previously. Although some of the original synaptic gutters were reoccupied by regenerated terminal branches, other gutters were only partially occupied, and many appeared abandoned. Junctions showing precise recapitulation of original terminal arborizations comprised a small number of the total examined, as did those where reinnervation was very imprecise. Striking differences in the precision of reinnervation were found within the muscle such that distal terminals regenerated more precisely and completely than did proximal terminals. Terminals in reinnervated muscles were more dynamic than terminals in unoperated muscles over equivalent times. In singly innervated junctions, terminal growth was favored over regression. In doubly innervated junctions, regressive events were more common. Imprecise reinnervation is explained in terms of multisite innervation of muscle fibers and the activity dependence of synaptic stability. We hypothesize that when axons reinnervate the second or third junctions on a fiber, they do so less precisely, because the activity restored by reinnervation of the first junction renders later sites less attractive or less stable.

Monoclonal antibodies 3G2 and 4E2 recognize a postsynaptic component of rat neuromuscular junctions. In contrast to many other postsynaptic junctional antigens, expression of this antigen is nerve-dependent: immunoreactivity disappears form junctions following denervation and returns upon reinnervation (Astrow et al., 1992 J. Neurosci. 12:1602–1615). Here we show that the epitope is also expressed by Schwann cells and that this expression is also neurally regulated. Weak mAb 3G2/4E2 immunoreactivity was found in myelinating Schwann cells but was not detected in either nonmyelinating Schwann cells or in terminal Schwann cells at the neuromuscular junction. Following axotomy, immunoreactivity increased in myelinating Schwann cells, and nonmyelinating and terminal Schwann cells became immmunopositive. Moreover, the immunoreactivity in terminal Schwann cells revealed their extensive sprouting in response to denervation (Reynolds and Woolf, 1992, J. Neurocytol. 21:50–66). After nerve regeneration, mAb 3G2/4E2 immunoreactivity in all Schwann cells returned towards normal: it disappeared from temrinal Schwann cells, returned to low levels in myelinating Schwann cells, and decreased in nonmyelinating Schwann cells. Immunoblots of axotomized nerve and cultured muscle fibers revealed the same set of immunoreactive bands. Therefore, Schwann cells and muscle fibers share the expression of an epitope that is under neural control, but is regulated differently at each site. In Schwann cells, the presence of the nerve suppresses expression of the epitope, whereas in muscle fibers, the nerve terminal promotes this expression. The differential regulation of mAb 3G2/4E2 immunoreactivity in terminal Schwann cells and muscle fibers suggests that the epitope may be involved in interactions between nerve terminals and these cells. 1994 John Wiley & Sons, Inc.