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Purpose: The Src family of kinases (SFKs) regulate multiple signal transduction cascades and influence proliferation, motility, survival, and angiogenesis. Dasatinib inhibits SFKs, which leads to cytotoxicity, cell cycle arrest, apoptosis, and decreased invasion of cancer cells. Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor that regulates survival and proliferation. Dasatinib results in rapid and durable inhibition of c-Src, whereas STAT3 undergoes only transient inactivation. We hypothesized that the reactivation of STAT3 after dasatinib treatment represents the engagement of a compensatory signal for cell survival that blocks the antitumor effects of SFK inhibition. Experimental Design: The effects of upstream inhibitors on STAT3 activation were assessed with western blotting and a quantitative bioplex phosphoprotein assay. We used the 3–(4,5-dimethylthiazol–2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the cytotoxicity and propidium iodine/annexin V staining with flourescence-activated cell sorting (FACS) analysis to evaluate cell cycle change and apoptosis. The combination index was calculated by the Chou-Talalay equation. Cytokines were quantitated using a multiplexed, particle-based FACS analysis. Results: C-Src and several downstream molecules were rapidly and durably inhibited by dasatinib. However, STAT3 was reactivated by 24 h. The addition of JAK inhibitors during dasatinib incubation resulted in sustained inhibition of STAT3, although JAK activation by dasatinib was not shown. Combined SFK and JAK inhibition resulted in synergistic cytotoxicity due to increased apoptosis. Conclusions: The reactivation of STAT3 during dasatinib treatment is caused by the engagement of a compensatory pathway that suppresses the antitumor effects of SFK inhibition and allows cancer cell survival. Abrogation of this pathway resulted in synergistic cytotoxicity. Given that STAT3 reactivation occurred in 14 of 15 solid tumor cell lines, dasatinib combined with Janus-activated kinase inhibitors may have widespread application in cancer treatment.

Background: HNSCC is the sixth most common cancer worldwide. To date, Cetuximab is the only approved targeted therapy for HNSCC treatment. Thus,there is an immediate need to discover effective targets. Two pharmacogenomic HTS studies, Cancer Genome Project (CGP) and Cancer Cell Line Encyclopedia (CCLE) provide a large repository of drug sensitivity data. The PI3K/mTOR pathway is one of the frequently activated signaling cascades in HNSCC. However, it is unclear which class of PI3K/mTOR inhibitors is most promising and which biomarkers may be used to predict sensitivity. The rationale for this study is to identify novel biomarkers to targets such as PI3K/mTOR pathway by data mining these public databases. The potential biomarkers will be characterized in vitro in 68 HNSCC cell lines.Methods:The landscape of drug sensitivity profiles in 23 HNSCC cell lines (CGP) was analyzed by boxplot illustrations. Drugs that induce growth inhibition at low doses (median≤10 μM) were considered “effective”. Chemotherapy drugs, drugs with missing values and unknown targets were excluded from analyses. Hierarchical clustering of cell lines was performed based on drug sensitivity using GOWER distance metric and Ward's linkage after normalization. Clustering of 140 drugs based on their sensitivity profiles was also done. In vitro, drug response to PI3K pathway inhibitors in 28 HNSCC cell lines was assessed by ATP based cell viability assay (CellTiter-Glo). Results: In the CGP datasets, we identified a set of effective drugs with median IC75 Citation Format: Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Integrative drug sensitivity analysis of PI3K /mTOR pathway inhibitors in Head and Neck Squamous Cell Carcinoma (HNSCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 417. doi:10.1158/1538-7445.AM2015-417

Background:Head and neck squamous cell carcinoma (HNSCC) is the 6th most frequently diagnosed non-skin cancer in the world. Risk factors associated with HNSCC include smoking, alcohol use and human papillomavirus (HPV) infection. With the decline in tobacco use, there has been an increase in the incidence of HPV-associated head and neck cancers. Our increased understanding of the mutational landscape demonstrates that HPV-positive and HPV-negative HNSCC are molecularly distinct. Additionally, patients with HPV-positive tumors respond better to therapy and have better prognosis overall. However, there are no targeted therapies for HPV+ HNSCC. Human cell lines can serve as preclinical models for studying cancer progression and identifying possible therapeutic targets. Our aim was to determine if HNSCC tumors and cell lines derived from head and neck tumors display similar proteomic profiles. Methods: Reverse phase protein array (RPPA) analysis was performed on 66 HNSCC cell lines. The HPV-status of these cell lines was determined by quantitative and reverse transcriptase polymerase chain reaction (PCR). Results: Real-time PCR analysis confirmed that 9 of the 66 cell lines were HPV-positive. Differential expression of 156 proteins was analyzed and proteomic pathway scores were determined by t-test in HPV-positive and HPV-negative HNSCC cell lines. We identified significant proteomic differences between HPV-positive and negative HNSCC cell lines. As observed in HPV-positive head and neck tumors, the clinical biomarker p16 was overexpressed in HPV-positive cell lines (p-value Citation Format: Nene N. Kalu, Tuhina Mazumdar, Lixia Diao, Patrick Kwok Shing Ng, Jing Wang, Jeffery Myers, Faye M. Johnson, Lauren Averett Byers. Characterization of HPV-positive head and neck cancer cell lines as preclinical models for targeted therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1826. doi:10.1158/1538-7445.AM2015-1826

Objectives: Infection with the human papillomavirus (HPV) is an important risk factor for development of head and neck squamous cell carcinoma (HNSCC). Strikingly, HPV-positive HNSCCs carry a more favorable prognosis and are a biologically distinct subgroup when compared with their HPV-negative counterparts. We aimed to discover novel therapeutic targets by conducting a high-throughput drug screening platform in vitro with both FDA approved and investigational drugs. Based on our prior proteomic analysis we tested inhibitors of pathways activated in HPV+ HNSCC tumors. Methods: Cell viability assays were performed by the Cell Titer Glo method in a panel of 66 fingerprinted HNSCC cell lines using 13 drugs at concentrations of 0.011 to 9.613 μM. This panel includes 9 HPV+ lines and 57 HPV- lines (5 oropharynx, 15 oral cavity, and 37 others). The IC50 values were calculated. Western Blot assay was used to confirm that the drugs inhibited their targets. Results: We observed a wide range of sensitivities to all 13 drugs with the exception of BKM120 which was effective in all tested lines (Table 1). In contrast, nearly all cell lines were resistant to BMN673, ruxolitinib, LEE011, and selicilib. The HPV+ cell lines were more sensitive to MEK162 (p Conclusions: For most agents tested, HNSCC cell lines displayed similar drug sensitivity regardless of the tumor site. However, HPV+ lines were more sensitive to MEK162 and more resistant to Bosutinib. Future analysis will include comparing drug sensitivity to mutation, gene and protein expression in these lines. Taken together, the results may provide a rationale for the clinical evaluation of MEK inhibitors as a molecular targeted approach for the treatment of HPV+ HNSCC. Table 1. IC50 values in 66 HNSCC cell lines DrugDrug's targetMedian IC50 (μM)Range of IC50(μM)Cmax (μM)ErlotinibEGFR5.970.12- >9.614LEE011CDK4/69.610.01- >9.61NABKM120PI3K0.640.01- >1.434MEK162MEK9.610.01- >9.611BosutinibSrc2.320.19- >9.610.8SeliciclibCDK2/59.610.01- >9.619.02VolasertibPLK1.120.01- >9.611.2AZD7762CHK1/20.120.02- >3.02NADocetaxelChemotherapy9.610.01- >9.610.08CisplatinChemotherapy8.620.01- >9.617.33RuxolitinibJAK9.610.01- >9.611.48AZD1775Wee10.250.03- >7.521.65BMN673PARP9.610.20- >9.610.07 Citation Format: Ming Zhang, Tuhina Mazumdar, Shaohua Peng, Pan Tong, Vaishnavi Sambandam, Lauren A. Byers, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. The difference of drug sensitivity between HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2015-2427

6001 Background: We designed a randomized phase II trial to determine the efficacy of two induction chemotherapy (ICT) cetuximab (C) containing regimens with demonstrated efficacy and acceptable to...

2597 Background: Although C and D both demonstrate activity resistance can develop. Preclinical studies demonstrated that activation of MET requires c-SRC. We hypothesized that a c-SRC inhibitor (D...

Non-small cell lung cancer (NSCLC) is a common and rapidly fatal cancer for which targeted therapies have been markedly effective in only about 15% of patients - specifically those with EGFR mutations or EML4-ALK translocations. During a clinical trial of the multi-targeted tyrosine kinase inhibitor dasatinib we identified a patient with stage IV NSCLC who harbored a novel BRAF mutation (Y472C), had a near complete radiographic response to the multitargeted kinase inhibitor dasatinib as sole therapy and remained without active cancer 7 years following treatment. We discovered that Y472CBRAF is a kinase-inactivating BRAF mutation (KIBRAF). BRAF mutations can result in increased or decreased BRAF kinase activity, as well as kinase-neutral mutations, and occur in 8% of NSCLC and many other tumor types. We found that NSCLC cells that harbor KIBRAF undergo senescence when exposed to the dasatinib whereas NSCLC patients and cell lines with wild type BRAF are resistant to dasatinib. Transfection of other NSCLC cells with KIBRAF mutations also increased their dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. NSCLC cells transfected with Y472CBRAF exhibited CRAF, MEK, and ERK activation. Currently there are no well-defined, canonical pathways that explain the observed dasatinib-induced senescence in NSCLC cells with KIBRAF. Dasatinib inhibits the activity of 40 distinct kinase targets and can induce BRAF-CRAF dimerization and CRAF activation in cells with activated RAS or KIBRAF mutations. To investigate the mechanism leading to senescence we performed reverse phase protein arrays to simultaneously examine the expression of 137 proteins and phosphoproteins and gene expression arrays. This approach was limited by the existence of only two NSCLC cell lines with KIBRAF but nonetheless we determined that dasatinib induced DNA damage and subsequently activated DNA repair pathways and the HIPPO signaling pathway only in NSCLC cells with KIBRAF. We are currently conducting a clinical trial for NSCLC patients with KIBRAF mutations to determine the efficacy of dasatinib in these patients prospectively. Citation Format: Faye Johnson, Banibrata Sen, Tuhina Mazumdar, Shaohua Peng, Humam Kadara, Lixia Diao, Lauren Byers. Kinase-impaired BRAF mutations as predictors of resistance and sensitivity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA10.

BACKGROUND Small cell lung carcinoma (SCLC) cell lines commonly express KIT and its ligand, stem cell factor, suggesting an autocrine loop promoting cell growth. Imatinib inhibits KIT kinase activity. SCLC cells treated with imatinib in vitro undergo cell cycle arrest. Imatinib reduces resistance to irinotecan in vitro. Common metabolic pathways suggest there may be drug interactions between imatinib and irinotecan or cisplatin. In the current study, the authors investigated the feasibility of combining these drugs in the treatment of patients with SCLC. METHODS Two Phase I studies were conducted independently at two institutions. Patients with extensive-disease SCLC underwent therapy with cisplatin, irinotecan, and imatinib using two similar regimens. In one study, immunohistochemical analysis of the expression of potential imatinib targets was performed on pretreatment biopsy specimens, and blood specimens were collected and analyzed for imatinib, irinotecan, and cisplatin pharmacokinetic parameters. RESULTS Nine patients were enrolled and were evaluable for toxicity. A high incidence of neutropenia, diarrhea, and thrombosis was observed that precluded dose escalation. Six patients were evaluable for response after four cycles; five patients experienced a partial response and the other patient had developed progressive disease. Four of six tumor specimens tested expressed platelet-derived growth factor receptor-α and two expressed KIT. Irinotecan clearance was found to be significantly decreased by imatinib (P < 0.04). No significant alteration in the disposition of cisplatin was observed. CONCLUSIONS The maximum tolerated dose for this combination with granulocyte–colony-stimulating factor support was identified as imatinib at a dose of 300 mg/day with irinotecan (at a dose of 65 mg/m2) and cisplatin (at a dose of 30 mg/m2) given on Days 1 and 8, every 21 days. The decreased irinotecan clearance may explain the high incidence of diarrhea and neutropenia noted in the current study. Cancer 2006. © 2005 American Cancer Society.

Imatinib mesylate is a tyrosine kinase inhibitor of the ABL, platelet-derived growth factor receptor (PDGFR), and c-kit kinases. Inhibition of BCR-ABL and c-kit accounts for its clinical activity in leukemia and sarcoma, respectively. In this report, we describe other cellular targets for imatinib. Treatment of head and neck squamous carcinoma cells with clinically relevant concentrations of imatinib-induced changes in cell morphology and growth similar to changes associated with epidermal growth factor receptor (EGFR) activation. Imatinib-induced changes were blocked with the EGFR antagonist cetuximab, which suggested direct involvement of EGFR in this process. Western blot analysis of cells incubated with imatinib demonstrated activation of EGFR and downstream signaling that was reduced by inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1) and EGFR, but not Her2/ErbB2. An in vitro kinase assay showed that imatinib did not directly affect EGFR kinase activity, suggesting involvement of EGFR-activating molecules. Inhibitors and neutralizing antibodies against heparin-binding epidermal growth factor-like growth factor (HB-EGF), and to a lesser extent transforming growth factor-α, reduced imatinib-mediated mitogen activated protein kinase (MAPK) activation. Imatinib stimulated the rapid release of soluble HB-EGF and the subsequent induction of membrane-bound HB-EGF, which correlated with biphasic MAPK activation. Together, these results suggested that imatinib affects EGFR activation and signaling pathways through rapid release and increased expression of endogenous EGFR-activating ligands. Although, imatinib primarily inhibits tyrosine kinases, it also stimulates the activity of EGFR tyrosine kinase in head and neck squamous tumors. This finding demonstrates the need for careful use of this drug in cancer patients. © 2005 Wiley-Liss, Inc.

Imatinib mesylate is a novel anti-tumor agent useful in the clinical management of chronic myelogenous leukemia and gastrointestinal stromal tumors with minimal toxicity relative to other forms of cancer therapy. Its clinical activity and minimal toxicity are related to specific inhibition of cellular targets including BCR-ABL, platelet-derived growth factor receptor and c-kit kinases, resulting in the collapse of downstream signaling cascades important for transformation. In some patients, unexpected toxicities arise that are not associated with inhibition of any known cellular imatinib target. In this report, we investigated the effects of imatinib on squamous carcinoma cell signaling. Imatinib induced expression of COX-2 in a dose-dependent manner with concomitant accumulation of prostaglandin E2. COX-2 induction by imatinib was initiated through epidermal growth factor (EGF) receptor kinase activation and downstream signaling through mitogenic-activated protein kinase. COX-2 induction by imatinib was blocked by MEK1 or EGF receptor inhibition. Imatinib did not activate stressor cytokine-signaling pathways (p38 kinase, nuclear factor-kB nuclear translocation) or affect COX-1 expression. Imatinib failed to activate EGF receptor signals in other tumor types, suggesting that COX-2 induction in imatinib-treated cells is mediated through release of autocrine factors expressed or activated in squamous tumors. COX-2 induction by imatinib in squamous tumors derived from the head and neck region is unique with respect to other target-specific agents and may represent one of the unintended toxic effects of imatinib described in some patients.

Purpose: Local recurrence is the most common site of failure for locally advanced nasopharyngeal carcinoma (NPC) treated with neoadjuvant cisplatin/5-fluorouracil (PF) and definitive radiation at our center. Based on this, we studied the addition of chemotherapy during the boost phase of radiation after neoadjuvant PF for advanced T-stage (T3–T4) NPC. This strategy was based on theoretical radiosensitization with chemotherapy during accelerated repopulation of the tumor with relatively radioresistant clonogens. Methods and Materials: Three cycles of neoadjuvant PF was followed by conventionally fractionated radiation with additional PF during the boost portion of the radiation course. An initial Phase I study was done to establish the maximum tolerated dose of concurrent PF. Results: Forty-four patients were enrolled. Six patients in Phase I defined the MTD for concurrent PF as: cisplatin 10 mg/m 2 /day and PF 320 mg/m 2 /day, on Days 1–5 during Weeks 6 and 7 of radiation therapy based on dose-limiting toxicities of mucositis, neutropenia, and thrombocytopenia. Forty-one patients were treated with concurrent therapy per protocol: complete, partial, and minor responses were seen in 23, 16, and 2 patients, respectively. Progression-free and overall survival rates at 5 years were 55% (95% CI, 41–75%) and 66% (95% CI, 52–85%), respectively. Seven of 11 tumor-related deaths were due to local recurrence. Nine of 10 patients with local recurrence had T4-stage disease at presentation. Local control of T4 disease was achieved in 74% of patients overall, and in 25% (1/4) with World Health Organization (WHO) type 1, 76% (16/21) with WHO type 2, and 90% (9/10) with WHO type 3 histology. Common toxicities included mucositis, dermatitis, fatigue, vomiting, and weight loss. Conclusions: This regimen was feasible and associated with promising overall survival. Local recurrence remains the major reason for treatment failure in advanced T-stage NPC, especially WHO types 1 and 2. Other strategies to improve local control in these patients should be investigated.

BACKGROUND Promising results from a Phase II trial of induction chemotherapy and sequential radiotherapy for advanced nasopharyngeal carcinoma (NPC) at The University of Texas M. D. Anderson Cancer Center (Houston, TX) and two retrospective reviews of the authors' historical experience with NPC demonstrated that distant failure was directly correlated with advanced lymph node status. Furthermore, local control was excellent for patients with T1–3 disease that was managed with radiation alone or with a sequential approach involving chemotherapy. Neoadjuvant chemotherapy (primarily with cisplatin + 5-fluorouracil) was associated with a significantly decreased risk of distant metastasis and with improved survival. Based on these findings, the authors evaluated a novel induction regimen involving docetaxel and carboplatin for patients with previously untreated T1–2N2–3M0 NPC. METHODS Docetaxel (80 mg/m2 on Day 1) and carboplatin (to an area under the time-concentration curve of 6 on Day 1) were administered every 21 days for 3 cycles, after which radiotherapy was administered. NPC was restaged with magnetic resonance imaging and nasopharyngoscopy 3 weeks after the completion of chemotherapy and 6 weeks after the completion of radiotherapy. RESULTS Over 5 years, 18 patients were enrolled in the study. Grade 4 neutropenia and Grade 2 fatigue were observed in 51% and 28% of chemotherapy courses, respectively. After chemotherapy, 2 patients had complete responses, 14 had partial responses, 1 had a minor response, and 1 had progressive disease. The latter two patients and one patient who had a partial response underwent off-study chemoradiotherapy. After radiotherapy or chemoradiotherapy, 12 patients had complete responses and 6 had partial responses. Seven patients had recurrent disease; two had local recurrences, and five had distant metastases. CONCLUSIONS Although unlikely to be superior to cisplatin + 5-fluorouracil, the trial regimen could be administered quickly in the outpatient setting, was logistically more convenient for the patient, and was devoid of serious nonhematologic toxic effects. We believe that the risk-based approach examined in the current study merits further investigation. Cancer 2004;100:991–8. © 2004 American Cancer Society.

Irinotecan (CPT-11, Camptosar) has significant activity in small-cell lung cancer. In addition, preclinical models have demonstrated synergy between irinotecan and two standard front-line drugs for small-cell lung cancer: cisplatin and etoposide; phase III data also show significant survival benefit for irinotecan/cisplatin compared to standard treatment. These data suggest a potential clinical advantage to combining etoposide, cisplatin, and irinotecan as first-line therapy for small-cell lung cancer. The purpose of this phase I study was to establish if alternating weekly therapy with irinotecan/cisplatin and etoposide/cisplatin was tolerated and to determine the maximum tolerated dose of these agents in patients with small-cell lung cancer. Patients generally tolerated the weekly alternating chemotherapy with irinotecan/cisplatin and etoposide/cisplatin well, and this combination possessed significant antitumor activity in small-cell lung cancer.

Head and neck Squamous cell carcinoma (HNSCC) is a lethal, disabling and disfiguring cancer with no biomarker-directed therapeutic options. We are in a desperate need for novel targeted therapies. PI3K/AKT/mTOR is one of the most often altered pathways in HNSCC. This pathway is important for cancer cell signaling and several pharmacological inhibitors are already in clinical trials for other cancer types. Our lab previously found that HNSCC cell lines with PIK3CA mutations were more sensitive to pathway inhibitors compared to other PI3K pathway alterations such as PIK3CA amplification or PTEN loss and that activation of ERK is one mechanism responsible for PI3K inhibitor resistance. In this report we extended our study to further investigate the predictive biomarkers, biological effects of PI3K inhibition, the potential pathways of resistance and the use of rational drug combination in a diverse panel of HNSCC cell lines. We tested 64 HNSCC cell lines with the PI3K inhibitor GDC0941 and correlated drug sensitivity with basal PI3K pathway activation, PI3K copy number or PIK3CA mutation. We confirmed that cell lines with PIK3CA mutations were more sensitive to GDC0941 (P= 6.35X 10 -13) and those with amplification were not. We tested another four PI3K pathway inhibitors, including one PI3K inhibitor GSK1059615, one dual PI3K/mTOR inhibitor GDC0980, one AKT inhibitor GDC690693 and one mTOR inhibitor AZD8055 in a panel of 18 cell lines and found that the trend of drug sensitivity was similar (P < 0.05) in two PI3K inhibitors and one dual inhibitor where as AKT and mTOR inhibitors had a distinct trend. We evaluated the basal activation of PI3K/AKT/mTOR pathway using reverse phase protein array (RPPA) with two proteomic scores: PI3K/AKT and TSC/mTOR. Neither score predicted sensitivity to GDC0941. We also analyzed the PI3K/AKT and TSC/mTOR scores in 200 HNSCC tumors from the TCGA . In the same way PIK3CA amplification did not correlate with the scores whereas the PI3K/AKT score was higher in the PIK3CA mutants (P < 0.039) compared to WT PIK3CA. The RPPA did identify several biomarkers, including VEGFR2, pCRAF and PCNA, whose expression correlated with GDC0941 sensitivity. Treatment with GDC0941 caused cell cycle arrest in sensitive cell lines, but not in resistant cell lines. To identify potential resistance pathways following GDC0941 treatment in resistant lines, we tested 39 phosphoproteins using phosphoscan analysis and found that EGFR, IGF-1R, RET, FGFR3, and M-CSFR were activated in resistant cell lines after incubation with GDC0941. Combination therapy with GDC0941 and the EGFR inhibitor erlotinib was synergistic in resistant cell lines. Our data will be useful to conduct biomarker-based clinical trials in HNSCC in the near future. Citation Format: Tuhina Mazumdar, Lauren A. Byers, Patrick Ng, Gordon B. Mills, Shaohua Peng, Lixia Diao, Youhong Fan, Katherine Stemke-Hale, John V. Heymach, Jeffrey N. Myers, Bonnie S. Glisson, Faye M. Johnson. Evaluation of predictive biomarkers and resistance mechanisms of PI3K pathway inhibition in head and neck squamous cell carcinoma. [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 903. doi:10.1158/1538-7445.AM2014-903

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death. Therapies that target specific genetic aberrations are very effective in a subset on NSCLC. An effective approach for discovering novel therapeutic targets is to characterize responsive tumors. In this regard, we discovered a novel, kinase-inactivating BRAF mutation in the tumor of a patient with a marked and durable response to the multi-targeted kinase inhibitor dasatinib. Likewise, dasatinib induces senescence in NSCLC cells with endogenous kinase-inactivating BRAF mutations (KIBRAF). To identify the mechanism underlying this effect, we used reverse phase protein array, Western blotting and gene expression analysis on NSCLC cell lines with KIBRAF or WTBRAF incubated with dasatinib. We found that ATM, ATR, Rad 51, pH2AX, CDC6, CHEK1 (Chk1), pLATS and TAZ, were differentially regulated in mutant vs. WT cell lines. Quantitative real-time PCR analysis revealed that CHEK1, TAZ and TAZ's downstream targets were decreased following incubation with dasatinib in KIBRAF cells. Overexpression of CHEK1 or TAZ in KIBRAF NSCLC cells resulted in dasatinib resistance and decreased dasatinib-induced senescence. Given the differential effect on CHEK1 and pH2AX, we studied the effect of dasatinib on DNA damage using the COMET assay and found that dasatinib induces a significant increase in COMET-positive KIBRAF NSCLC cells. These data suggest that the inhibition of Chk1 along with inhibition of Hippo pathway results in the dasatinib sensitivity in NSCLC with KIBRAF. Citation Format: Banibrata Sen, Shaohua Peng, Tuhina Mazumdar, Lauren A. Byers, Humam Kadara, Faye M. Johnson. The multitargeted kinase inhibitor dasatinib induces DNA damage, Hippo pathway engagement and senescence in lung cancer cell lines that possess kinase-inactivating BRAF mutations. [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 3707. doi:10.1158/1538-7445.AM2014-3707

6033 Background: The epidermal growth factor receptor (EGFR) and Src family kinases are upregulated in HNSCC. EGFR interacts with cSrc to activate oncogenic STAT3 signaling; dual targeting is syner...

6030 Background: The incidence of oropharyngeal cancer (OPC) has increased 5% per year over the past decade due to a rise in human papilloma virus (HPV)-driven OPC. HPV+ OPC is clinically and molecularly distinct from HPV- head and neck squamous cell carcinomas (HNSCC). However, there are currently no validated therapeutic targets for HPV+ HNSCC. Methods: Reverse phase protein array (RPPA) was performed on HNSCC tumors from The Cancer Genome Atlas to measure cancer-associated pathways and targets. Differences in individual markers and proteomic pathway scores between HPV+ and HPV– tumors were assessed by t-test in the overall group and for the subset of OPC. Results: Significant proteomic differences were observed in 14 HPV+ and 186 HPV- HNSCC. Of 160 proteins, the top marker overexpressed in HPV+ HNSCC was p16, an established clinical biomarker of HPV status (p

The results of a large, randomized, North American trial (SWOG S0124) that compared etoposide and cisplatin with irinotecan and cisplatin for patients with extensive small-cell lung cancer have failed to confirm the benefit observed with irinotecan that was demonstrated in an earlier Japan Clinical Oncology Group study (JCOG 9511).

Beta1,4-Galactosyltransferase I (GalT I) is localized on the leading lamellipodia of migrating cells, where it associates with the cytoskeleton and facilitates cell spreading and migration on basal lamina matrices. It has previously been reported that a variety of highly metastatic murine and human cell lines are characterized by elevated levels of cell surface GalT I, although the intracellular biosynthetic pool is similar between cells of high and low metastatic potential. In this study, we examined whether the elevated expression of surface GalT I characteristic of metastatic cells is instructive or incidental to their metastatic behavior by altering the expression of surface GalT I and by the use of GalT I-specific perturbants. Surface GalT I levels were positively and negatively altered on murine melanoma cells by either overexpressing full-length GalT I or by homologous recombination, respectively. The consequences of altered surface GalT I expression on cell invasion in vitro and lung colonization in vivo were determined. Increasing surface GalT I expression on cells of low metastatic potential to levels characteristic of highly metastatic cells recapitulated the highly invasive phenotype in vitro. Alternatively, decreasing surface GalT I expression on highly metastatic cells to levels characteristic of low metastatic cells reduced their invasive behavior in vitro and metastatic activity in vivo. Within the physiological range of surface GalT I expression, the invasive potential of each clonal cell line correlated strongly with the level of surface GalT I expressed. As an independent means to assess the involvement of surface GalT I in metastatic behavior, cells were pretreated with two different classes of surface GalT I perturbants, a competitive oligosaccharide substrate and a substrate modifier protein. Both perturbants inhibited metastatic colonization of the lung, whereas control reagents did not. Finally, as reported by others, surface GalT I on metastatic cells selectively interacted with one glycoprotein substrate, or ligand, of approximately 100 kDa, the identity of which remains obscure. These results show that the elevated expression of surface GalT I characteristic of highly metastatic cells contributes to their invasive phenotype in vitro and to their metastatic phenotype in vivo.

Background: NSCLC is a deadly disease. Targeted therapy is very effective; however, only ∼ 20% of patients are candidates for this approach. In a search for new therapeutic targets in NSCLC, we have identified Polo like kinase (Plk). Plk1 regulates centrosomes at the G2/M transition and orchestrates cell cycle progression. Clinical trials have shown that a subgroup of refractory NSCLC patients responded to Plki as single agents. Our aim is to validate and identify biomarkers that predict sensitivity to Plki. Methods: To identify therapeutic targets, we evaluated 2 databases with 123 NSCLC cell lines tested with 130 drugs incorporating genomic and gene expression data seeking agents that were markedly effective in a subgroup of cell lines. To identify association between sensitivity to Plki and gene expression, we acquired the datasets from the databases and tested the correlation between IC50 and gene expression. Spearman rank was used to test the association between mutation in key genes and IC50. MTT assay was used to test drug sensitivity in NSCLC cell lines in our laboratory. Cell cycle was evaluated by flow cytometry using BrdU incorporation. Immunofluorescence with anti-phospho-H2yAX antibody was used to identify mitotic catastrophe. Results: In the databases, 12/19 NSCLC lines had IC50 < Cmax of 1.6 µM for the PLKi BI2536 and 6/19 had IC50 < 30 nM. The correlation analysis found mRNA overexpression of 20 genes to be significantly associated with sensitivity to both Plki. Based upon the likelihood of their expression influencing Plk1 function, 4 genes were selected for further study: CEP250, which encodes a core centrosomal protein, and TGFA, RHBDF1 and FOSL2, which are involved with epithelial cell proliferation. KRAS mutation was also correlated with sensitivity to Plki. Our preliminary cell line screening found that 10/37 NSCLC cell lines are exquisitely sensitive to BI2536 with IC70 < 40 nM. The Plki's BI2536 and BI6727 cause complete Plk substrate (TCTP) inhibition with 25 and 100 nM respectively at 12 h as measured by Western blotting. However, in a resistant cell line, TCTP reactivation was observed following 24 h Plk inhibition, while in a sensitive cell line, there was sustained TCTP inhibition up to 120 h. Cell cycle analysis of sensitive cell lines showed a significant arrest in G2/M. Drug treatment caused mitotic catastrophe in sensitive lines. Conclusion: Our statistical analysis utilizing data from 2 databases demonstrated an association between CEP250, TGFA, RHBDF1 and FOSL2 overexpression and KRAS mutation and sensitivity to Plki. These results are being validated in a larger cell line screen that may identify additional biomarkers. Substrate reactivation was observed in one resistant cell line following 24h of Plk inhibition, demonstrating one potential mechanism for resistance. Screening of additional cell lines and mechanistic studies are ongoing to extend these findings. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A182. Citation Format: Renata Ferrarotto, Manasi Suryavanshi, Suk Young Yoo, Jing Wang, Lauren Byers, Bonnie S. Glisson, John Heymach, Faye M. Johnson. Identifying biomarkers of sensitivity to polo-like kinase inhibitors (Plki) in non small cell lung cancer (NSCLC). [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 A182.

Background: A variety of methods for capture of rare CTCs of epithelial origin are available; most employ antibodies to epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). Using a classic phenotypic definition, a CTC is nucleated, CK(+), CD45(-) cell. However, some CTCs may elude capture as they originate from primary tumor cells which have undergone epithelial-mesenchymal transition (EMT). We report here the use of ApoStream™, a novel dielectrophoresis field-flow-assisted, antibody-free method to isolate CTCs from blood. Methods: Blood was collected from consented NSCLC patients and processed using ApoStream™. For CTC enumeration comparison, the CellSearch® FDA-approved kit was used. Isolated cells were evaluated with a multiplexed immunofluorescent assay and laser scanning cytometry was applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI and EpCAM. To determine specific EGFR mutations from captured CTCs, samples were analyzed using Improved and Complete Enrichment with CO-amplification at Lower Denaturation temperature (ICE COLD-PCR). Results: Blood samples from 37 NSCLC patients and 8 healthy volunteers were processed and the results of the cell populations isolated using ApoStream™ are summarized in table. EGFR mutations [exon 19 deletion and exon 21 L858R] were determined and found to be concordant when compared to tumor tissue analysis by Sanger sequencing. Conclusions: The ApoStream™ platform enriched EpCAM(+) and EpCAM(-) CTCs from the blood of NSCLC patients demonstrating utility in recovering cancer cells with multiple phenotypes. From recovered CTCs, detection of EGFR mutations was possible indicating the clinical relevance and potential utility of CTCs as an alternative to tissue biopsy. Additional sensitivity and complete mutation analysis will be presented. (a portion of this data was presented at 2013 ASCO Annual Meeting, Chicago, IL, USA) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C29. Citation Format: Hai T. Tran, Vladislava Melnikova, Anne S. Tsao, Frank V. Fossella, Faye M. Johnson, Vali Papadimitrakoupoulou, Katherine Richardson, Marcia E. Lewis, Ben Legendre, Kenna L. Anderes, Darren W. Davis, John V. Heymach. Characterization and identification of specific EGFR mutations in circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients using an antibody independent method, ApoStream™: An update report. [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 C29.

11044 Background: A variety of methods for isolation of CTCs of epithelial origin are available; most employ antibodies to epithelial cell adhesion molecule (EpCAM). Using classic phenotypic definition, a CTC is nucleated, cytokeratin CK(+), CD45(-) cell. However, some CTCs may elude capture as they originate from primary tumor cells which have undergone epithelial-mesenchymal transition (EMT). We report here the use of ApoStream, a novel dielectrophoresis field-flow-assisted, antibody-free method to isolate CTCs from blood. Methods: Blood was collected from consented NSCLC patients and processed using ApoStrea. For CTC enumeration comparison, CellSearch FDA-approved kit was used. Isolated cells were evaluated with multiplexed immunofluorescent assay and laser scanning cytometry analysis were applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI and EpCam. To determine specific EGFR mutations from captured CTCs, samples were analyzed using Improved and Complete Enrichment with CO-amplification at Lower Denaturation temperature (ICE COLD-PCR). Results: Blood samples from 32 NSCLC patients and 3 healthy volunteers were processed. ApoStream isolated 0 to 65 CK(+)/CD45(-) CTCs(n=32) and CellSearch isolated 0 to 13 EpCAM(+)/CK(+)/CD45(-) CTCs(n=7). Additionally, ApoStream™ recovered 37-3536 CK(-)/CD45(-) and 4-10702 CK(+)/CD45(+) cells. EpCAM expression was detected in 7-100% of CK(+)/CD45(-) and 0-5% of CK(-)/CD45(-) cells, and 18-100% of CK(+)/CD45(+) cells. EGFR mutations [exon 19 deletion and exon 21 L858R] were determined and found to be concordant when compared to tumor tissue analysis by Sanger sequencing. Conclusions: The ApoStream platform enriched EpCAM(+) and EpCAM(-) CTCs from the blood of NSCLC patients demonstrating utility in recovering cancer cells with multiple phenotypes. From recovered CTCs, detection of EGFR mutations was possible indicating the clinical relevance and potential utility of CTCs as an alternative to tissue biopsy. Complete mutation analysis will be presented.

8102 Background: In preclinical models, combining EGFR and Src inhibition has been shown to have at least additive and possibly synergistic effects. In this phase I/II (phI/II) trial, we combined EGFR inhibitor E with D, a multi-targeted tyrosine kinase inhibitor with activity against Src. Primary endpoint of PhI was to determine maximum tolerated dose (MTD) of the combination of E and D; primary endpoint of PhII was progression free survival (PFS) at 12 weeks. Methods: In preclinical models, combining EGFR and Src inhibition has been shown to have at least additive and possibly synergistic effects. In this phase I/II (phI/II) trial, we combined EGFR inhibitor E with D, a multi-targeted tyrosine kinase inhibitor with activity against Src. Primary endpoint of PhI was to determine maximum tolerated dose (MTD) of the combination of E and D; primary endpoint of PhII was progression free survival (PFS) at 12 weeks. Results: 53 pts were enrolled between 2/09 and 4/12. 6 were not eligible. Of the 47 eligible pts (12 in PhI, 35 in PhII): median (range) age was 62 yrs (44-88), 26 pts (45%) were female. 6 pts in PhI and all 35 pts in PhII had NSCLC: adeno 25 pts, squamous 11, NSCLC NOS 5. Other tumor types enrolled in PhI: head and neck cancer (2), adenoid cystic carcinoma (1), mesothelioma (2), small cell lung cancer (1). 47% of pts were chemo-naïve. 8 pts had activating EGFR mutations (6 exon 19 deletions, 2 L858R mutations). The PhI starting dose E150/D100 was not well tolerated as 2 of 3 pts experienced grade 3 hypophosphatemia as a dose limiting toxicity. MTD was found to be E150/D70. 11% achieved PR and 44% SD. All observed responders had EGFR mutations. 9 pts (19%) stopped treatment early and were unevaluable. Median time on treatment was 43 days (range 1-262). Median time on treatment for the 8 pts with activating EGFR mutations was 144 days. Conclusions: Combination of E and D is safe and feasible in NSCLC, with MTD E 150 mg PO daily and D 70 mg PO daily. Rate of PR and SD were 11%,and 44%, respectively. Biomarker and pharmacokinetic studies will be reported. Clinical trial information: NCT00826449.

HNSCC is a common, deadly, and disfiguring disease. While combinations of radiotherapy, surgery, and chemotherapy are highly effective in HNSCC, there is significant morbidity associated with the disease and recurrence is common. There is great interest in identifying molecular events and pathways which drive HNSCC progression in order that targeted therapies may be developed. The loss of the tumor suppressor function such as p53 and NOTCH1, as well as activation of the STAT3 and STAT5 pathways, has been implicated in HNSCC progression. We have identified interactions between STAT3, STAT5, and the novel tumor suppressor SOCS2 to be important in HNSCC. To extend these findings to humans, we performed experiments to identify prognostic markers in HNSCC and to investigate the expression of STAT3, STAT5, and SOCS2 in HNSCC tissue. Immunohistochemistry was performed on tissue microarrays from resected tumor specimens of 123 stage I-IVB oral cavity SCC patients (treated with surgery +/- adjuvant radiation therapy) with annotated clinical outcome information from a median follow up of 76 months collected at the UT/MD Anderson Cancer Center. The array was screened with antibodies against STAT5, STAT3, and SOCS2, and scored in a blinded fashion by a pathologist. SOCS2 expression (present vs. absent) correlated significantly with recurrence-free survival in both the univariate (hazard ratio 0.24, p=0.0003; Cox proportional hazards model) and multivariate (hazard ratio 0.24, p=0.0004) analyses. Notably all patients who lacked SOCS2 tumor expression recurred within 45 months. There was a trend towards a correlation of SOCS2 expression with overall survival (HR 0.50, p=0.11) and disease-specific survival (HR 0.52, p=0.28). SOCS2 expression did not significantly correlate with tumor stage and extracapsular extension emphasizing that its absence is an independent marker of poor prognosis. Our prior published work demonstrates that STAT5 drives SOCS2 expression. The expression of SOCS2 in these patient samples positively correlated with total and phosphoSTAT5 (r=0.29 and 0.21 respectively; p This work was supported The University of Texas SPORE in Head and Neck Cancer (P50 CA097007), The Cancer Center Support Grant, and the ASCO Cancer Foundation Young Investigator Award (WW). Citation Format: Courtney Nicholas, Maria I. Nunez, Nusrat Harun, J. Jack Lee, Jeffrey Myers, Ignacio I. Wistuba, Banibrata Sen, Adel K. El-Naggar, Stephen Y. Lai, Faye M. Johnson, William N. William. SOCS2 (suppressor of cytokine signaling protein 2) is a prognostic indicator of progression-free survival in head and neck squamous cell carcinoma (HNSCC) patients. [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 5578. doi:10.1158/1538-7445.AM2013-5578

Head and neck squamous cell carcinoma (HNSCC) is a deadly and disfiguring disease for which better systemic therapy is desperately needed. The development of new therapies for HNSCC and the understanding of its biology both depend upon clinically relevant animal models. An increasingly promising xenograft model, the heterotransplant model, is developed by surgically implanting tumor tissue directly from a patient into an immunocompromised mouse. We have transplanted 30 HNSCC primary tumors from untreated patients directly into mice that included 27 tongue tumors, one maxillary gingival tumor, and two from the floor of the mouth. Five of 30 (17%) transplanted tumors could be serially passaged and used for therapeutic and mechanistic studies. One cell line has been established from a tongue primary. Histopathologic characterization of our panel showed a concordance between xenografts (up to the tenth generation) and the corresponding patients’ tumors in terms of tumor differentiation, cancer cell appearance, necrosis, and keratin production. To further characterize our model, we conducted gene expression analysis with Affymetrix U133A-microarrays on 20 patient tumors; 5 third generation and one tenth generation tumor in mice; one tumor-derived cell line; and 2 established cell lines (OSC19, Tu167). Although unsupervised clustering clearly separates patient tumors, xenografts, and cell lines into distinct groups, the early and late xenografts that were derived from the same patient clustered together. From the >54,000 probes tested, there were 1417 probes that were distinct between the tumors growing in mice vs. the corresponding human tumors from which they were derived (p,0.01, fold change 1.4). There were 992 probes that were distinct between the human tumors that subsequently grew in mice vs. those that did not - suggesting that this model enriches for cancers with distinct biological features (p,0.01, fold change 1.4). We have successfully used one heterotransplant model for multiple studies of cancer drug efficacy. Our results demonstrate the feasibility of a heterotransplant model of HNSCC. Although the gene expression patterns are distinct, the histology of the tumors in mice is similar to that of the same tumor in humans. Additionally, gene expression patterns and histology are relatively stable over multiple generations. Biologic gene pathways analysis for similarities and differences in this model will further refine the promise of heterotransplants as an in vivo model to test the efficacy of anti-cancer drugs. This work was supported by R01-CA143369-01 Citation Format: Shaohua Peng, Chad Creighton, Yiqun Zhang, Banibrata Sen, Tuhina Mazumdar, Jeffrey Myers, Diana Bell, Adel El-Naggar, Michelle Williams, Faye Johnson. Molecular and histologic characterization a patient-derived heterotransplant mouse model of head and neck squamous carcinoma. [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 333. doi:10.1158/1538-7445.AM2013-333

Cancers of the head and neck are difficult to treat because both the therapy and the tumor impact essential functions such as speech and swallowing and they can significantly alter facial appearance. The RAS/RAF/MEK/ERK cascade is one of the major signaling pathways that leads to head and neck squamous cell carcinoma (HNSCC) cell survival. There are three RAS genes (HRAS, KRAS, and NRAS) and together, they are mutated in about 30% of human cancers. RAS can also be activated by upstream signaling. Paradoxically RAF inhibition induces RAF-dimerization and causes subsequent activation of MEK/ERK pathway in presence of activated RAS in melanoma. Similarly the ABL inhibitor nilotinib acts as weak inhibitor of RAF kinase and also induces RAF-dimerization and subsequent activation of MEK/ERK in the presence of activated RAS in leukemia and pancreatic cancer cells. Nilotinib synergizes with MEK inhibitition to kill nilotinib-resistant leukemia cells (Abl T351I). We hypothesized that nilotinib and the MEK inhibitor MEK162 would be synergistic in HNSCC due to the frequent activation of RAS via upstream growth factor receptors. RAS mutations are relatively uncommon in HNSCC. We screened 60 validated HNSCC cell lines through sequenome analysis and we found three HNSCC cell lines with activating HRAS mutations (G12D and Q61L) [∼5%]. We found that nilotinib induces significant RAF dimerization and subsequent activation of MEK-ERK pathway irrespective of RAS mutational status. We next investigated how paradoxical MEK/ERK pathway activation affected the growth of HNSCC cells. In this context we treated a panel of HNSCC cell lines harboring WT and mutated HRAS with nilotinib and MEK162. Nilotinib alone had nearly no effect on cell viability and MEK162 had a moderate effect on some HNSCC lines. The combination of nilotinib and MEK162 synergized to inhibit the growth of HNSCC cells independent of RAS mutational status. The drug combination led to an accumulation of cells in G1 and a significant reduction in cell proliferation as measured with BrDU incorporation and colony formation. HNSCC cells with mutant HRAS undergo apoptosis in presence of both the drugs. In contrast none of these drugs induce senescence in HNSCC cell lines. Here we show that in HNSCC, nilotinib as single agent drives the paradoxical activation of RAF-MEK-ERK pathway in RAS mutation-independent manner and we uncovered a synthetic lethal pathway that can be used treat HNSCC. Animal studies are planned. This work was supported by University of Texas SPORE in Head and Neck Cancer P50 CA097007. Citation Format: Banibrata Sen, Tuhina Mazumdar, Shaohua Peng, Katherine S. Hale, Bonnie S. Glisson, Faye M. Johnson. Paradoxical activation of the RAF-MEK-ERK pathway in response to nilotinib induces synthetic lethality with MEK inhibition in head and neck cancer cells. [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 4095. doi:10.1158/1538-7445.AM2013-4095

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with 600,000 new cases and 350,000 cancer deaths each year worldwide. It is a disabling and disfiguring cancer with a profound impact eating, speaking, and appearance. We need better systemic treatment for HNSCC. Targeted therapy can be remarkably effective if it inhibits key oncogenic drivers but no such targets have yet been identified in HNSCC. To identify potential targets, we screened sixty validated HNSCC cell lines using Sequenom for a panel of sixty potentially targetable mutations commonly found in squamous cancers. In addition, we used SNP CHIP to identify gene copy number gain or loss. Alterations in the PI3K pathway were the most commonly identified molecular targets in HNSCC cell lines. Likewise, the PI3K signaling pathway is commonly altered in multiple cancers driving cancer cell growth and survival. We identified 5 cell lines with PIK3CA mutations, 5 cell lines with PIK3CA amplification, and 2 cell lines with PTEN loss. We tested these cell lines along with those lacking identified PI3K pathway alterations for sensitivity to two PI3K inhibitors (GDC0941, GSK1059615), one dual PI3K/mTOR inhibitor (GDC0980), one AKT inhibitor (GSK690693) and one mTOR inhibitor (AZD8055). The 5 cell lines with PIK3CA mutations were almost universally sensitive to all inhibitors whereas those with PI3KCA gene amplification were almost universally resistant to all inhibitors. Among two cell lines with PTEN loss, one was sensitive and other resistant to the inhibitors. Cell lines lacking identified PI3K pathway alterations had mixed sensitivity. We measured basal activation of several pathway components including pAKT, pS6, p4EBP1 and found that only pS6 predicted response to PI3K pathway inhibitors. Following treatment with PI3K inhibitors, we discovered an increase in pERK and insufficient inhibition of cMYC in resistant cell lines, but not in sensitive cell lines. The combination of PI3K and MEK inhibition was synergistic in resistant cell lines. Our study identified that among the different types genetic lesions in the PI3K pathway, PIK3CA mutations are the best predictors of sensitivity to pathway inhibitors. In resistant HNSCC combinations with MEK inhibitors may be effective. Multiple inhibitors of the PI3K and MEK/ERK pathways are in clinical development making the clinical application of our data possible in the near future. This work was supported by University of Texas SPORE in Head and Neck Cancer P50 CA097007. Citation Format: Tuhina Mazumdar, Lauren A. Byers, Shaohua Peng, Lixia Diao, Katherine S. Hale, Bonnie S. Glisson, Faye M. Johnson. Genetic determinants of sensitivity and resistance to PI3K inhibitors in head and neck cancers for efficient targeted 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 2388. doi:10.1158/1538-7445.AM2013-2388

The type 1 insulin-like growth factor receptor (IGF-1R) signaling is dysregulated in many cancers, thus being a target of cancer therapy. Several preclinical trials have reported the efficacy of IGF-1R targeted therapies, mostly using tyrosine kinase inhibitors or monoclonal antibodies (mAbs); however many of clinical trials have shown limited therapeutic efficacy. In this study, we investigated the resistance mechanism to IGF-1R mAb-based therapies using cixutumumab, a fully humanized mAb against IGF-1R. We tested the efficacy of cixutumumab using OSC19 and 686LN head and neck squamous cell carcinoma (HNSCC) and MDA-MB-231 breast cancer orthotopic and H1299 non-small cell lung cancer (NSCLC) xenograft tumor models. We observed that cixutumumab treatment induced either no change or increased tumor growth and decreased the survival duration of the animals bearing those tumors. Treatment with cixutumumab upregulated migration and invasion of HNSCC and NSCLC cells without any effects on cell proliferation. We further examined the effects of cixutumumab on tumor angiogenesis and found that conditioned media from cancer cells treated with cixutumumab increased tube formation and migration of human umbilical endothelial cells (HUVECs). The angiogenic activity of cixutumumab was also confirmed by matrigel plug assay with HNSCC cell lines. Collectively, these data suggest that cixutumumab-induced angiogenesis-stimulating activities and migration and invasion potential of cancer cells are major factors of resistance to IGF-1R mAbs. These data further suggest that inhibitors of angiogenesis are useful strategy to overcome the resistance against the IGF-1R mAb-based therapy. Citation Format: Ji-Sun Lee, Ju-Hee Kang, Hye-Jin Boo, Jeffrey N. Myers, Faye M. Johnson, Bonnie S. Glisson, Seoung-Hyun Oh, Ho-Young Lee. Angiogenic activity leads failure of antitumor activity of cixutumumab, an anti-Insulin like growth factor receptor monoclonal antibody. [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 5645. doi:10.1158/1538-7445.AM2013-5645

NSCLC is a lethal disease for which personalized therapies that target specific genetic aberrations have been markedly effective in subsets of patients. An important approach for discovering effective cancer therapeutic targets is to characterize responsive tumors. We conducted a phase II trial of the tyrosine kinase inhibitor dasatinib in stage IV NSCLC. The dramatic response of one patient, who remains cancer free four years later, led us to examine the molecular characteristics of his tumor. We performed a comprehensive analysis of this NSCLC patient's tumor including mutational analysis of 40 genes, array comparative genomic hybridization, and immunohistochemistry. We discovered a novel, inactivating BRAF mutation (Y472CBRAF) in the patient's tumor; no inactivating BRAF mutations were found in the non-responding patients. Cells transfected with Y472CBRAF exhibited CRAF, MEK, and ERK activation, which were identical to signaling changes that occur with previously known inactivating BRAF mutants. Dasatinib induced senescence in NSCLC cells with endogenous inactivating BRAF mutations. Transfection of cells with inactivating BRAF mutations led to increased dasatinib sensitivity; conversely, cells transfected with an activating BRAF mutation were more resistant. Likewise, BRAF inhibition in NSCLC cells expressing wild-type BRAF enhanced dasatinib sensitivity. Dasatinib sensitivity may depend upon CRAF since dasatinib led to decreased CRAF activity and only NSCLC cells with inactivating BRAF mutations were sensitive to CRAF inhibition. We hypothesize that patient's BRAF mutation was likely responsible for his marked response to dasatinib and suggests that tumors bearing inactive BRAF mutations will be exquisitely sensitive to dasatinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3667. doi:1538-7445.AM2012-3667

There are approximately 47,000 new cases of head and neck cancer diagnosed in the United States each year. The survival rate has not improved significantly in the past 30 years. The development of new therapies for HNSCC and the understanding of its biology both depend upon clinically relevant animal models. There are several existing animal models for HNSCC that include chemically-induced cancer models, syngeneic models using animal cancer cells, transgenic mice, and xenograft models using cultured human HNSCC cell lines injected into immunocompromised mice, either subcutaneously or orthotopically. An increasingly promising xenograft model, the heterotransplant model, is developed by surgically implanting tumor tissue directly from a patient into an immunocompromised mouse. The resulting tumor is serially passaged in vivo and never cultured on plastic. We have transplanted 20 HNSCC primary tumors from untreated patients directly into mice that included 17 tongue tumors, one maxillary gingival tumor, and two from the floor of the mouth. Although several tumors grew initially in mice, only 2 of 20 (10%) established tumors that could be serially passaged and used for therapeutic and mechanistic studies. One cell line has been established from a tongue heterotransplant (HOSC1). The two established heterotransplants maintained the histological appearance of the original tumors. To test the biological and molecular signaling response of HNSCC to targeted therapy, we utilized one heterotransplant that was passaged into 40 mice. Both the Src inhibitor dasatinib and the JAK inhibitor INCB16562 modestly inhibited tumor growth; the combination was significantly more effective than the single agents. Likewise, the tumors treated with the combination had significantly more apoptosis and less proliferation than control tumors. Consistent with our published in vitro results, Src inhibition did not result in STAT3 inhibition, but JAK inhibition abrogated STAT3 activation. Src was inhibited in vivo by dasatinib. Our results demonstrate that the heterotransplant model of HNSCC has promise as an in vivo model to test the efficacy of anti-cancer drugs. The low “take rate” is problematic but will be addressed in the future by intra-operative biopsies and larger tumor specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2361. doi:1538-7445.AM2012-2361

OBJECTIVE To quantify physicians' preferences among possible outcomes associated with chronic hepatitis B treatments and to determine which outcomes are most important to physicians in making treatment decisions. METHODS Physicians in five countries who treat chronic hepatitis B patients completed a web-enabled, choice-format, conjoint-analysis survey. The survey presented physicians with four treatment-choice questions for three different patient types. Each treatment-choice question included a pair of hypothetical medication profiles. Medication outcomes included how long the medication has been studied (weight of evidence); the probability that a patient's viral load remains undetectable for 5 years, with a possible histological improvement or reversal of disease progression (long-term efficacy); the 5-year treatment-related risk of fracture; the 5-year treatment-related risk of renal dysfunction; and patient cost. Treatment-choice questions were derived from a predetermined experimental design with known statistical properties. For each country, the random-parameters logit was used to estimate preference weights for all outcome levels and the mean relative importance of each outcome. RESULTS Long-term efficacy and risk of renal dysfunction were the most important outcomes for the 788 physicians completing the survey, whereas weight of evidence was the least important. However, physicians perceived significant differences in weight of evidence timeframes. Physicians in Germany and France ranked efficacy above side-effect risk, whereas physicians in Spain, Italy, and Turkey ranked side-effect risk above efficacy in importance. CONCLUSION Physician preferences among treatment profiles indicate systematic differences in the relative importance of treatment outcomes. Physicians require higher efficacy for treatments with higher side-effect risk but somewhat less efficacy for treatments with longer evidence.

Cancers of the head and neck are difficult to treat because both the therapy and the tumor impact essential functions such as speech and swallowing and they can also significantly alter facial appearance. One promising molecular target for which new agents have been developed is the Src family kinases (SFKs). SFK inhibition in cancer cells leads to an universal abrogation of invasion but a variable and weak effects on apoptosis and proliferation. The pathways downstream of c-Src promoting survival are not well-characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal for head and neck cancers, we sought to characterize the mechanisms of resistance to SFK inhibition in Head and Neck squamous cell carcinoma (HNSCC). SFKs were inhibited in a panel of oral cancer cell lines and the effects on subsequent survival and signaling were measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and their intrinsic kinase activity by in vitro kinase assay. Cytotoxicity was measured using an MTT assay and the Chou-Talalay combination index calculated. The in vivo effects of c-Met and SFK inhibitors were assessed using an orthotopic model of oral cancer. SFK inhibition resulted in c-Met inhibition in cell lines that were sensitive to SFK inhibitors, but not in resistant cell lines. Isolated c-Met act as a c-Src substrate in both sensitive and resistant cells, whereas distinct difference exists in c-Src and c-Met interaction in intact sensitive and resistant cells. The epidermal growth factor receptor contributed to c-Met activation in resistant cells whereas in sensitive cells c-Met acts as a direct downstream target for c-Src. We demonstrated the biological consequences of this mechanism in vitro with synergistic cytotoxicity and enhanced apoptosis when SFK and c-Met inhibitors were combined. Likewise, the combination resulted in decreased tumor size in vivo. In conclusion, we demonstrate that sustained c-Met activation can mediate resistance to SFK inhibition. The differences between c-Src and c-Met signaling in sensitive and resistant cells are not due to intrinsic structural changes in c-Src or c-Met, but rather to distinct interactions within the intact cells. The synergistic cytotoxic effects of SFK and c-Met inhibition may be important for the treatment of head and neck cancers and should be tested in future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2011-1046

2530 Background: OSI-906 is a potent inhibitor of IGF-1R and IR tyrosine kinase activity. Increased IGF-1R activity is observed in human malignancies and implicated in resistance to chemotherapy. M...

Systemic therapy that results in both decreased invasion and significant cytotoxicity would be ideal to treat head and neck squamous cell carcinoma (HNSCC). One promising therapeutic target is Src due to its well-defined roles in both invasion and survival. STAT3 is a key mediator of the oncogenic effects of Src. However we previously found that sustained Src inhibition resulted in only transient inhibition of STAT3. Likewise, Src inhibition resulted in a universal reduction in invasion, but a more variable effect on apoptosis. Restoring STAT3 inhibition enhanced apoptosis. Previously we showed that STAT3 reactivation was mediated by JAK2 kinase activation and altered JAK2-STAT3 binding. Reactivation of JAK2/STAT3 suggests a feedback loop, possibly via the loss of a negative regulator. Of the three known feedback loops that regulate JAK/STAT function, we considered the most likely candidate to be the suppressors of cytokine signaling (SOCS) proteins, which can compete for STAT binding and inhibit JAK kinase activity. To determine if SOCS proteins are involved in JAK/STAT3 reactivation, we measured RNA levels of all 8 SOCS family members in 5 HNSCC cell lines using quantitative PCR. The expression of most of the SOCS decrease after initial Src inhibition, but recover, consistent with their regulation by STAT3. However, the expression of SOCS2 decreased in all 5 lines. Likewise, we found that the addition of a molar stoichiometric amount of recombinant SOCS2 inhibits Jak2 kinase activity in vitro. To confirm that SOCS2 regulates STAT3 activity, we knocked-down SOCS2 by siRNA which resulted in the activation of STAT3. Similarly, transient overexpression of SOCS2 resulted in significant downregulation of activated STAT3. Others have reported that STAT5 can regulate SOCS2 expression. Consistent with this, we found that Src inhibition led to durable STAT5 inhibition. Moreover, the depletion of both isoforms of STAT5 (a and b) with siRNA resulted in a dramatic downregulation of the SOCS2 expression. To determine the biological significance of SOCS2 expression, we discovered that specific SOCS2 depletion reduced HNSCC cytotoxicity following Src inhibition. Our results demonstrate that sustained Src inhibition leads to the loss of SOCS2 expression via STAT5 inhibition. The loss of SOCS2 allows the recovery of JAK2 kinase activity, JAK2/STAT3 binding, STAT3 activation, and cell survival despite durable Src inhibition. To our knowledge, this feedback pathway has never been described previously. Given that pharmacologic Src and JAK inhibitors are currently being evaluated in clinical trials, these results have significant implications for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4005.

e19069 Background: Early determination of therapeutic failure can potentially spare a pt ineffective and toxic treatment. We previously reported in a retrospective study the use of CT within 4 weeks of initiation of chemotherapy in advanced NSCLC to assess response and progression by RECIST (Bruzzi et al. JTO 2006). Here, we prospectively assess whether CT imaging after the first cycle of pemetrexed in advanced NSCLC has a role in evaluating response and management. Methods: We accrued pts with PS 2 or 3 advanced NSCLC receiving at least one dose of 1st or 2nd line pemetrexed. A repeat CT prior to a 2nd course was required. Pts with progression by RECIST were to come off study. CT scans were done using multislice CT technology (GE Lightspeed Plus), and images were reconstructed with slice thicknesses of 3.75mm or less. All images were reviewed using a PACs workstation (Stentor iSite) and measurements were done with electronic calipers. RECIST criteria: progression, an increase in the tumor's longest dimension by 20%, response, a decrease by 30%. Results: Thirty pts had a median age of 68 years (45 - 81). PS 2/3, 1st/2nd line, and F/M were 16/14, 17/13, and 12/18 respectively. Pts received 1–8 cycles (median 2). Twelve pts received only 1 course of whom 7 pts had f/u CTs at a median of 20 days (12–25) after 1st chemo dose. Of these pts, 5/7 had progression by RECIST and the other 2/7 pts had stable disease with 1 pt who came off due to serial PEs and 1 pt who opted off for reasons of travel. Two of the 5 pts who had progression by CT had no detectable change by CXR. Of 5/12 pts treated with only 1 course without f/u CT, 2 pts died, 2 pts had progression by CXR, and 1 pt stopped after treatment for pneumonia. All 18 pts receiving ≥2 cycles had a repeat CT prior to their 2nd course. Conclusions: The results of this prospective trial support earlier retrospective findings that short-term follow-up using CT in pts with advanced NSCLC can detect tumor progression and impact patient management. We will also present f/u CTs in pts who received ≥ 1course to determine whether early signs of progression predict later RECIST determined progression. [Table: see text]

e19015 Background: Lung cancer is the leading cause of cancer-related deaths. Better systemic therapies are needed. One potential therapeutic target is c-Src which is expressed and activated in NSCLC patient tumors where it can mediate invasion, angiogenesis, and proliferation. Additionally, epidermal growth factor receptor (EGFR) and c-Src cooperate to promote NSCLC survival. We are conducting a phase II study of dasatinib, a tyrosine kinase inhibitor of c-Src, Abl, c-Kit, PDGFR, Btk, and EphA2. The primary objective is to determine the rate of progression free survival at 12 weeks in patients with metastatic NSCLC treated with dasatinib as front line therapy. Methods: Patients with metastatic NSCLC were treated with dasatinib (100 mg BID), with PET/CT scans every 6 weeks. KRAS and EGFR mutations, EGFR copy number, and pSrc expression were measured in pre-treatment biopsies. Blood was collected pretreatment and on day 21 to measure drug exposure (PK), pharmacodynamics (PD), and serum cytokine levels. Results: Twenty five patients have enrolled on study. Of the 16 patients evaluable for response: 1 had a partial response (PR) with no evidence of recurrence for at least 18 months (male smoker with adenocarcinoma and KRAS mutation); 6 patients had stable disease (SD) which includes 3 patients with prolonged stable disease for 4, 6, and 18 months; 9 had progressive disease (PD). Only 4 patients had a significant change in SUV (>25%): decreased in 2 with PR and SD and increased in 2 with PD and SD. There is currently no clear association between EGFR and KRAS mutational analysis and response, although the sample number is small. Only 2 patients have activating EGFR mutations: one with SD and one with PD. PK, PD, and cytokine data will be presented. The most common grade 3/4 toxicity is dyspnea/pleural effusion that has led to a reduced dasatinib starting dose in subsequent patients. Conclusions: Dasatinib as a single agent has activity in a subset of patients with NSCLC. Planned correlative studies may lead to the discovery of biomarkers that predict response. Toxicities observed were consistent with prior dasatinib phase I studies in solid tumor patients. Supported by NCI/CTEP (NIH contract N01-CM-62202), The Commonwealth Foundation for Cancer Research and Bristol-Myers Squibb. [Table: see text]

14042 Background: SPRYCEL™ is a potent, orally active, multi-targeted kinase inhibitor, active against BCR-ABL and SRC family kinases. We report the results of a Phase I dose-escalation study evaluating safety, tolerability, pharmacokinetics, and biomarkers of dasatinib in pts with advanced solid tumors. Methods: Pts with adequate hematologic, renal, cardiac and liver function received oral dasatinib once daily for 7 days per week. Three doses and schedules were examined: 90 mg BID; 140 mg QD; and 180 mg QD. Pharmacokinetics and pharmacodynamic biomarkers were collected on Days 1 and 26 of Cycle 1. Tissue biomarkers were assessed at screening. CT was performed at least every 8 weeks, and FDG-PET at weeks 4, and 8. Results: 26 pts [M=15, F=11] ECOG PS ≤ 2 with epithelial tumors (n=14) or other solid tumors (n=12) have been treated in escalating dose levels. Toxicity was generally mild; most patients came off study for progressive disease. DLTs of pleural effusions were seen in 3/9 subjects on the 180 mg cohort, 2 of whom had pre-existing effusions. Patients with pleural effusion have been excluded from future enrollment. The maximum tolerated dose has not been identified. There have been no objective responses on CT. Six patients have had stable disease with continued study treatment for 2–10 months. Conclusions: Dasatinib can be safely administered at doses of 140 and 180 mg QD. Clinical efficacy, pharmacokinetics, and correlative studies of tumor biopsy material and early FDG-PET results will be reported. No significant financial relationships to disclose.

6026 Background: The role of the epithelial-mesenchymal transition (EMT) is crucial to invasive and progression of several epithelial malignancies. The role of this process in head and neck squamous cell carcinoma (HNSCC) remains poorly defined. Methods: Eleven head and neck squamous cancer cell lines and 50 primary tumor specimens formed the materials for this study. We used Western blotting, immunohistochemistry, RT-PCR cell motility assay to determine the expression and the functional implications of several cell adhesions and singling molecular associated with EMT in these tumors. Results: Our results showed Src and E-Cadherin to be paradoxically overexpressed in the majority of cell lines and in primary tumor tissues relative to normal squamous mucosa. In both cell lines and tumor tissues elevation of activated Src was accompanied by down-regulation of E-Cadherin and vimentin detection. In-vitro inhibition of Src led to the increased E-Cadherin of expression and cell proliferation in squamous carcinoma cell lines. Immunophenotypic analysis of the protein products of these genes in primary tumor tissues demonstrated a spectrum of changes that correlated significantly with the differentiation, invasive patterns, sarcomatoid transformation and lymph node status. Our results show that high activated Src and low E-Cadherin expression were correlated with finger-like invasive pattern, poor differentiation, sarcomatoid transformation and lymph node metastasis in HNSC. Conclusions: Our study indicates that Src and E-Cadherin play a major role in EMT invasion and progression of head and neck squamous carcinoma. These proteins may be targeted in the therapeutic intervention of patients with HNSCC. No significant financial relationships to disclose.

18119 Background: Irinotecan has significant activity in small-cell lung cancer. Randomized trials comparing IP to EP have yielded conflicting results. Our phase I study of alternating weekly therapy with IP and EP with granulocyte colony-stimulating factor (G-CSF) support found this regimen to be tolerated and active in untreated small cell lung cancer.(Johnson et al Clin Lung Cancer 2003) Methods: Based on these findings we conducted a phase II study to estimate the efficacy of weekly combination therapy in previously untreated patients with extensive disease small-cell lung cancer (ED-SCLC). Patients received 12 weeks of therapy with cisplatin (20 mg/m2) on day 1 and irinotecan (100 mg/m2) on day 1 and G-CSF days 2–5 (IP) alternating with cisplatin (20 mg/m2) on day 1 and etoposide 60 mg/m2 on days 1–3 with G-CSF on days 4–7 (EP). Patients with asymptomatic brain metastases were included. The primary end point was overall survival. Results: 38 patients were enrolled from 7/2001 to 7/2006. The median age was 60 years (range 37–73) and 55% were female. ECOG performance was 0 in 11 patients, 1 in 25 patients and 2 in 2 patients. 87% of patients completed 12 weekly cycles of chemotherapy (range 1–12) with 90% of the planned dose intensity of irinotecan delivered. The objective response rate in 33 evaluable patients was 87% (0 CR, 30 PR). Median follow-up time was 43.7 months. Median progression free survival was 6.4 months (95% CI 5.16–7.59 months). The median survival was 10.9 months (95% CI 8.61–13.5 months). The 2 year survival rate was 19% (95% CI 5–31%). Grade 3–4 non-hematologic toxicities included diarrhea (8%), hyponatremia (5%), dehydration (3%), and neutropenic fever (5%).Grade 3–4 hematologic toxicities included anemia (13%), neutropenia (5%), and thrombocytopenia (3%). There was 1 therapy related death due to sepsis. Conclusions: Weekly therapy with IP alternating with EP with growth factor support was well tolerated in patients with untreated ED- SCLC, but did not show improved progression-free or overall survival when compared with historical controls at our institution. No significant financial relationships to disclose.

6084 More effective systemic treatment is needed for HNSCC. The taxoids are the most efficacious single agents identified. Epidermal growth factor receptor (EGFR) is almost universally expressed by HNSCC tumors but inhibitors have only modest activity as single agents in clinical studies. However, the combination of the taxoids with EGFR inhibition has additive anti-tumor effects in HNSCC in vitro and in animals. Despite similar pre-clinical data in lung cancer, clinical trials showed no benefit when EGFR inhibitors were added to chemotherapy. In vitro studies in lung, breast and colon cancer cell lines demonstrate that the separation of EGFR TKI from chemotherapy and the order in which these agents are administered influences efficacy. We sought to determine if a similar sequence-dependent effect exists in HNSCC in order to help design future clinical trials with these agents. We chose three human HNSCC cell lines with diverse levels of basal EGFR expression and activation. None had EGFR mutations, which are very rare in HNSCC. Cells were treated with single agent docetaxel or erlotinib or combinations for 72 h and cytotoxicity was measured with an MTT assay. Concurrent treatment with docetaxel and erlotinib resulted in more cytotoxicity than single agents and calculated combination indices demonstrated additive effects. When erlotinib was added 4 to 24 h prior to docetaxel, the efficacy was similar to or less than that of concurrent therapy regardless of whether the erlotinib was washed off prior to the docetaxel or left in the cell medium. In contrast, when docetaxel was added ≥ 10 h prior to erlotinib, cytotoxicity was enhanced as compared to concurrent therapy. The most efficacious combination was docetaxel treatment for 24 h followed by erlotinib for 48 h. This combination was chosen for further analysis. Consistent with previously published studies in other cell types, erlotinib resulted in G1 cell cycle arrest and docetaxel in G2-M arrest. On-going studies to investigate the mechanism for the sequence-dependent cytotoxicity include examination of the effects of the sequence on cell cycle and apoptosis, tubulin expression, and cell signaling molecules that mediate pro-survival signals from EGFR. [Table: see text]

7713 Background: Malignant pleural mesothelioma (MPM) is a lethal disease with few effective therapeutic options. We sought to determine whether Src, a non-receptor tyrosine kinase, could be a new therapeutic target in MPM and to establish the potential therapeutic use of pharmacologic Src inhibitors in this disease. Methods: We analyzed four MPM cell lines (MSTO-211H, NCI-H28, NCI-H2052, and NCI- H2452) for immunohistochemical (IHC) expression of total and phosphorylated Src (Tyr 419, Tyr 530). These cell lines were treated with dasatinib, a Src inhibitor, and evaluated for apoptosis, cell cycle analysis, and migration and invasion. Downstream signaling events were studied by Western blot analysis. We also conducted IHC analyses with total Src, phosphorylated Src Tyr 419 (p-Src Tyr 419), and phosphorylated Src Tyr 530 on 46 archived MPM tumor specimens and correlated the biomarker results with the clinical outcome. Results: All four MPM cell lines expressed total and activated Src (p-Src Tyr 419). Three of the four cell lines were sensitive in vitro to cytotoxicity by dasatinib with inhibition of migration and invasion, cell cycle inhibition, and apoptosis. Treatment with dasatinib inhibited several pathways downstream of Src. In the archived MPM tumor specimens, Src protein was highly expressed on IHC analysis in tumor cells, but that expression did not correlate with overall or progression-free survival. However, expression of activated Src (p-Src Tyr 419) on the tumor cell membrane was higher in patients with stage 4 disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Tyr 419. MPM nodal involvement at N1 was associated with the highest membrane expression of inactive Src (p-Src Tyr 530) (P = 0.02), whereas N2 disease was associated with the lowest expression. No gene mutations in Src exon 12 were found in the cell lines or tumor specimens. Conclusions: Activated Src may have an important role in survival, metastasis, and invasion in MPM, and targeting Src may be an important therapeutic strategy. No significant financial relationships to disclose.

7257 Background: SCLC cell lines commonly express c-kit and its ligand, suggesting an autocrine loop promoting cell growth. IM inhibits c-kit kinase activity. SCLC cells treated with IM in vitro undergo cell cycle arrest. In this study we investigated the feasibility of combining IM with cytotoxic chemotherapy for extensive disease SCLC. Methods: Six patients received P (30 mg/m2) and I (65 mg/m2) on days 1 and 8 every 21 days for four cycles. IM (300 mg) was given daily during chemotherapy and then as maintenance until progression of disease. Immunohistochemistry (IHC) for potential IM targets was done on patients' pre-treatment biopsies. Results: The first 3 patients experienced significant neutropenia that required GCSF support. One patient died during treatment from neutropenic fever. GCSF was added prophylactically to the subsequent 3 patients who did not experience significant neutropenia. Five patients had grade 2 diarrhea and one had grade 3 diarrhea. One patient had a deep vein thrombosis. This p...

The first Meeting on “Molecular Origins of Lung Cancer - Prospects for Personalized Prevention and Therapy” was held from January 10 to 14, 2010 in San Diego, California. The purpose of the meeting was to discuss important basic, translational, and clinical work aimed at improving lung cancer prevention, detection, and treatment. Topics included drug design, target identification, early detection, cancer stem cells, microRNAs, genome wide approaches to determining risk and outcome, mouse models, and tumor microenvironment. The role of cancer advocates in supporting research was an important component of the meeting. Meeting presentations demonstrated that emerging technologies can molecularly dissect lung cancers that have important relevance for clinical utility. This includes molecular based strategies not only for treatment of established cancers but also individualized and molecularly-based strategies for cancer risk reduction and chemoprevention.