Suyu Liu

Effect of KRAS Oncogene Substitutions on Protein Behavior: Implications for Signaling and Clinical Outcome

BACKGROUND Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) play a critical role in cancer cell growth and resistance to therapy. Most mutations occur at codons 12 and 13. In colorectal cancer, the presence of any mutant KRas amino acid substitution is a negative predictor of patient response to targeted therapy. However, in non-small cell lung cancer (NSCLC), the evidence that KRAS mutation is a predictive factor is conflicting. METHODS We used data from a molecularly targeted clinical trial for 215 patients with tissues available out of 268 evaluable patients with refractory NSCLC to examine associations between specific mutant KRas proteins and progression-free survival and tumor gene expression. Transcriptome microarray studies of patient tumor samples and reverse-phase protein array studies of a panel of 67 NSCLC cell lines with known substitutions in KRas and in immortalized human bronchial epithelial cells stably expressing different mutant KRas proteins were used to investigate signaling pathway activation. Molecular modeling was used to study the conformations of wild-type and mutant KRas proteins. Kaplan-Meier curves and Cox regression were used to analyze survival data. All statistical tests were two-sided. RESULTS Patients whose tumors had either mutant KRas-Gly12Cys or mutant KRas-Gly12Val had worse progression-free survival compared with patients whose tumors had other mutant KRas proteins or wild-type KRas (P = .046, median survival = 1.84 months) compared with all other mutant KRas (median survival = 3.35 months) or wild-type KRas (median survival = 1.95 months). NSCLC cell lines with mutant KRas-Gly12Asp had activated phosphatidylinositol 3-kinase (PI-3-K) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling, whereas those with mutant KRas-Gly12Cys or mutant KRas-Gly12Val had activated Ral signaling and decreased growth factor-dependent Akt activation. Molecular modeling studies showed that different conformations imposed by mutant KRas may lead to altered association with downstream signaling transducers. CONCLUSIONS Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.

Inhibition of c-Src expression and activation in malignant pleural mesothelioma tissues leads to apoptosis, cell cycle arrest, and decreased migration and invasion

Malignant pleural mesothelioma (MPM) is a deadly disease with few systemic treatment options. One potential therapeutic target, the non–receptor tyrosine kinase c-Src, causes changes in proliferation, motility, invasion, survival, and angiogenesis in cancer cells and may be a valid therapeutic target in MPM. To test this hypothesis, we determined the effects of c-Src inhibition in MPM cell lines and examined c-Src expression and activation in tissue samples. We analyzed four MPM cell lines and found that all expressed total and activated c-Src. Three of the four cell lines were sensitive by in vitro cytotoxicity assays to the c-Src inhibitor dasatinib, which led to cell cycle arrest and increased apoptosis. Dasatinib also inhibited migration and invasion independent of the cytotoxic effects, and led to the rapid and durable inhibition of c-Src and its downstream pathways. We used immunohistochemical analysis to determine the levels of c-Src expression and activation in 46 archived MPM tumor specimens. The Src protein was highly expressed in tumor cells, but expression did not correlate with survival. However, expression of activated Src (p-Src Y419) on the tumor cell membrane was higher in patients with advanced-stage disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Y419. Lower levels of membrane expression of inactive c-Src (p-Src Y530) correlated with advanced N stage (P = 0.02). Activated c-Src may play a role in survival, metastasis, and invasion of MPM, and targeting c-Src may be an important therapeutic strategy. [Mol Cancer Ther 2007;6(7):1962–72]

Bayesian adaptive randomization designs for targeted agent development.

Background With better understanding of the disease’s etiology and mechanism, many targeted agents are being developed to tackle the root cause of problems, hoping to offer more effective and less toxic therapies. Targeted agents, however, do not work for everyone. Hence, the development of target agents requires the evaluation of prognostic and predictive markers. In addition, upon the identification of each patient’s marker profile, it is desirable to treat patients with best available treatments in the clinical trial accordingly. Methods Many designs have recently been proposed for the development of targeted agents. These include the simple randomization design, marker stratified design, marker strategy design, efficient targeted design, etc. In contrast to the frequentist designs with equal randomization, we propose novel Bayesian adaptive randomization designs that allow evaluating treatments and markers simultaneously, while providing more patients with effective treatments according to the patients’ marker profiles. Early stopping rules can be implemented to increase the efficiency of the designs. Results Through simulations, the operating characteristics of different designs are compared and contrasted. By carefully choosing the design parameters, types I and II errors can be controlled for Bayesian designs. By incorporating adaptive randomization and early stopping rules, the proposed designs incorporate rational learning from the interim data to make informed decisions. Bayesian design also provides a formal way to incorporate relevant prior information. Compared with previously published designs, the proposed design can be more efficient, more ethical, and is also more flexible in the study conduct. Limitations Response adaptive randomization requires the response to be assessed in a relatively short time period. The infrastructure must be set up to allow timely and more frequent monitoring of interim results. Conclusion Bayesian adaptive randomization designs are distinctively suitable for the development of multiple targeted agents with multiple biomarkers. Clinical Trials 2010; 7: 584—596. http://ctj.sagepub.com

Comprehensive Biomarker Analysis and Final Efficacy Results of Sorafenib in the BATTLE Trial

Purpose: To report the clinical efficacy of sorafenib and to evaluate biomarkers associated with sorafenib clinical benefit in the BATTLE (Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination) program. Patients and Methods: Patients with previously treated non–small cell lung cancer (NSCLC) received sorafenib until progression or unacceptable toxicity. Eight-week disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Prespecified biomarkers included K-RAS, EGFR, and B-RAF mutations, and EGFR gene copy number. Gene expression profiles from NSCLC cell lines and patient tumor biopsies with wild-type EGFR were used to develop a sorafenib sensitivity signature (SSS). Results: A total of 105 patients were eligible and randomized to receive sorafenib. Among 98 patients evaluable for eight-week DCR, the observed DCR was 58.2%. The median PFS and OS were 2.83 [95% confidence interval (CI), 2.04–3.58] and 8.48 months (95% CI, 5.78–10.97), respectively. Eight-week DCR was higher in patients with wild-type EGFR than patients with EGFR mutation (P = 0.012), and in patients with EGFR gene copy number gain (FISH-positive) versus patients FISH-negative (P = 0.048). In wild-type EGFR tumors, the SSS was associated with improved PFS (median PFS 3.61 months in high SSS vs. 1.84 months in low SSS; P = 0.026) but not with eight-week DCR. Increased expression of fibroblast growth factor-1, NF-κB, and hypoxia pathways were identified potential drivers of sorafenib resistance. Conclusion: Sorafenib demonstrates clinical activity in NSCLC, especially with wild-type EGFR. SSS was associated with improved PFS. These data identify subgroups that may derive clinical benefit from sorafenib and merit investigation in future trials. Clin Cancer Res; 19(24); 6967–75. ©2013 AACR.

Clinical Outcomes and Biomarker Profiles of Elderly Pretreated NSCLC Patients from the BATTLE Trial

Background: Treating elderly non–small-cell lung cancer (NSCLC) patients in the salvage setting is challenging because of concerns of intolerance to therapy. Here we report outcomes (survival and toxicity) of elderly patients on the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial. Methods: Two hundred and fifty-five chemorefractory NSCLC patients received tumor molecular analysis, and were randomized to erlotinib, erlotinib-bexarotene, vandetanib, or sorafenib. Retrospective subgroup analyses were conducted comparing outcomes among age groups (< 65 versus ≥ 65 years; < 70 versus ≥ 70 years; < 75 versus ≥ 75 years), treatments, and sex. Results: Median age was 62 years (range, 26–84); 38% were aged 65 years or more. No significant differences among age groups were seen in rates of biopsy-related pneumothorax, treatment-related death, compliance, grade 3 to 4 hematologic toxicities, response rate, nor overall survival. However, older women aged 65 years or more had more grade 3 to 4 nonhematologic toxicities (p = 0.05). Elderly men aged 65 years or more (p = 0.008) had a higher disease-control rate at 8 weeks and a better progression-free survival (PFS) (p = 0.0068). Elderly women aged 70 years or more had a trend toward higher 8-week disease-control rate (p = 0.06). Older men aged 65 years or more treated with vandetanib had a better median PFS (p = 0.03) whereas PFS of older women aged 70 years or more was worse (p = 0.03) compared with younger patients. Elderly men aged 70 years or more treated with sorafenib had a higher overall survival compared with younger men (p = 0.04). Tumor tissue biomarkers show distinct differences by sex and age. Conclusion: Fit elderly NSCLC patients should be considered for salvage targeted therapy. In this subset of patients, older men seem to have significant clinical benefit from certain agents. Tumor biomarker analysis demonstrates sex and age variations, and is hypothesis-generating.

Phase II Trials of Imatinib Mesylate and Docetaxel in Patients with Metastatic Non-small Cell Lung Cancer and Head and Neck Squamous Cell Carcinoma

Purpose: Two phase II clinical trials in the aerodigestive tumors were undertaken to evaluate the efficacy of imatinib mesylate-docetaxel. We hypothesized that imatinib mesylate would inhibit platelet-derived growth factor receptor (PDGFR) on pericytes and increase docetaxel uptake into tumor cells for an additive antitumor effect. Baseline tumor specimens, serum, and perfusion computed tomography (CT) scans were obtained for supportive evaluation. Materials and Methods: Eligible patients with metastatic non-small cell lung cancer (NSCLC) treated with 1 prior therapy and chemonaive patients with head and neck squamous cell carcinoma (HNSCC) were enrolled in separate trials, which administered both docetaxel (60 mg/m2 every 3 weeks) and oral imatinib mesylate (400 mg daily). Both trials used interim analyses for efficacy and safety. Results: Twenty-two patients with NSCLC and seven patients with HNSCC were enrolled. Both trials were closed early due to lack of efficacy, significant toxicity, and a potential antagonistic effect. In the NSCLC study, the response rate was 4.5%, median progression-free survival (PFS) 7.9 weeks, and overall survival 35.6 weeks. The HNSCC trial yielded a response rate 0%, PFS 8.8 weeks, and overall survival 34.7 weeks. Baseline NSCLC tumor immunohistochemical biomarker analyses indicated that lower expression of stromal PDGFR&bgr; correlated with a better PFS, whereas stromal PDGFR&agr; and tumor cell PDGFR&bgr; were associated with a worse clinical outcome when treated with imatinib mesylate-docetaxel. Conclusion: We do not recommend further investigation of this regimen in the aerodigestive tumors. Future investigations in PDGFR tyrosine kinase inhibitors should be used with caution in combination with taxanes and validation of the potential predictive or prognostic biomarkers stromal PDGFR&agr;/&bgr;, and tumor cell PDGFR&bgr; are needed.

Clinical and Biomarker Outcomes of the Phase II Vandetanib Study from the BATTLE Trial

Background: The Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination trial1 prospectively obtained serum and tumor core biopsies and randomized 255 chemorefractory non–small-cell lung cancer (NSCLC) patients into four phase II trials: erlotinib, erlotinib-bexarotene, vandetanib, or sorafenib. Herein, we report the clinical and biomarker results of the phase II vandetanib trial. Results: Fifty-four patients received vandetanib. The 8-week disease control rate was 33%, median progression-free survival (PFS) 1.81 months, and median overall survival (OS) 6.5 months. No demographic subgroups had PFS or OS benefit. Eight patients with EGFR mutations had a trend for higher 8-week disease control rate (63% versus 31%; p = 0.12) but worse OS (5.9 months versus 9 months; p = 0.8). Patients with EGFR gene amplification (n = 6) had a worse OS (3.9 months versus 9.5 months; p = 0.04). KRAS mutation patients (3.9 months versus 9.5 months; p = 0.23) also had a worse OS. For the serum biomarker analysis, patients with below the median serum expression of interleukin 9c (p = 0.019) and eotaxin (p = 0.007) had a shorter PFS. A trend toward a shorter PFS was also seen in patients with higher than the median neutrophil gelatinase-associated lipocalin (p = 0.079) and lower than the median TNF-related apoptosis-inducing ligand (p = 0.087). Conclusion: Our trial results are largely consistent with the literature in unselected pretreated NSCLC patients. Although vandetanib improved median PFS in EGFR mutation patients with epidermal growth factor receptor tyrosine kinase inhibitor–resistance compared with EGFR wild-type, there was no OS advantage. Although vandetanib is no longer in development in NSCLC, identification of a molecular phenotype that responds to dual epidermal growth factor receptor and vascular endothelial growth factor receptor inhibition would contribute to the field.

Phase I Trial of Weekly Topotecan and Gemcitabine in Patients With Solid Tumors

Objective:This phase I trial was designed to determine the maximal tolerated dose (MTD) of the combination of topotecan and gemcitabine given in a weekly schedule. Materials and Methods:In this single-arm, open label, dose-escalation study, we administered topotecan (0.75–1.5 mg/m2) and gemcitabine (1000 mg/m2) on days 1, 8, and 15 every 4 weeks to 25 patients with advanced solid tumors. Results:The topotecan MTD, when combined with gemcitabine, was 1.25 mg/m2/wk. Dose-limiting toxicities consisted of febrile granulocytopenia in 2 patients at the highest dose level. At the MTD, no episodes of granulocytopenia were observed, whereas 2/9 patients exhibited grade 3 thrombocytopenia. Other common grades 3–4 adverse events across all cohorts included non-neutropenic infections, fatigue, skin reactions, vomiting, and fever. One partial response and 2 stable diseases were observed in patients with nasopharyngeal carcinoma. Disease stabilization was also observed in patients with squamous cell carcinoma of the head and neck (3), nonsmall cell lung cancer (1), and thymoma (1). Conclusions:Topotecan and gemcitabine combined in a weekly schedule exhibit a favorable toxicity profile. Efficacy results support the further evaluation of this regimen in patients with head and neck cancer (particularly nasopharyngeal carcinoma).

Abstract 4819: Gene-expression profiles predict sorafenib efficacy in wild-type EGFR non-small cell lung cancer (NSCLC)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Results from our Biomarkers-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) program suggest that patients with chemorefractory wild-type (wt) EGFR NSCLC including those with mutant KRAS may benefit from sorafenib. Using 3 different approaches, we tested the hypothesis that gene expression profiles from wild-type (wt) EGFR tumors may predict sorafenib efficacy by capturing effects on multiple targets. Material and Methods: Baseline tumor biopsies from 37 BATTLE patients (pts) with EGFR wt tumors and treated with sorafenib were profiled (Affymetrix Human Gene 1.ST), as well as 68 EGFR wt NSCLC cell lines with available IC50 to sorafenib (Illumina HumanWG-6 v3.0 expression beadchip). (i) We first developed an In vitro Sorafenib Signature (ISS). Correlation of IC50 with each individual probe expression level was computed. Most significant probes were summarized by the first principal component (PC), and correlated with IC50 of sorafenib. To validate the signature, the first PC was computed in BATTLE samples, and progression-free survival (PFS) of pts with high- vs. low-sensitivity signature was compared based on the median of the first PC. (ii) Alternatively, we developed a Clinical Sorafenib Signature (CSS) using BATTLE samples. We compared 23 (62%) pts who achieved 8-week disease control with 14 (38%) who did not (t-test). Most significant probesets were summarized by the first PC and PFS of pts with a high- vs. low-sensitivity signature were compared. To validate the signature, the first PC was computed in cell lines and correlated with IC50 of sorafenib. (iii) Finally, we tested a previously reported KRAS mutation gene expression signature derived by comparing genes differentially expressed in mutant vs. wt KRAS early stage resected lung adenocarcinomas, in 124 BATTLE samples including 24 mutant KRAS. Results: (i) The ISS included 50 probes. The first PC was correlated with the IC50 of sorafenib (rho = –0.71, P < 0.0001). The ISS was then tested in BATTLE and PFS was significantly different in pts with the high- (median PFS 3.61 months) vs. the low-sensitivity signature (median PFS 1.84 months, log-rank P = 0.0263). (ii) The CSS developed in BATTLE included 80 probesets summarized using the first PC. PFS was significantly different in pts with the high- vs. the low-sensitivity signature (log-rank P < 0.0001). The CSS was then tested in cell lines and the first PC was signicantly correlated with IC50 of sorafenib (rho = 0.24, P = 0.0483). (iii) Finally, the KRAS signature was significantly associated with KRAS mutation, but no association was observed with outcome in pts treated with sorafenib in BATTLE. Conclusion: We report 2 gene expression signatures, ISS and CSS, that predicted benefit from sorafenib in patients with chemorefractory NSCLC and in vitro sensitivity to sorafenib respectively. Further validation is planned in our ongoing BATTLE-2 program. 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 4819. doi:1538-7445.AM2012-4819

Abstract 955: Specific forms of mutant KRAS predict patient benefit from targeted therapy in the BATTLE-1 clinical trial in advanced non-small cell lung cancer

Mutant KRAS (mut-KRAS) is present in 17-25% of all human cancers, where it plays a critical role in driving cancer cell growth and resistance to therapy. Despite numerous attempts, there is still no effective therapy for mut-KRAS tumors. Understanding the signaling mechanisms activated by mut-KRAS and finding agents to inhibit mut-KRAS signaling are important unmet needs in cancer therapy today. The recently completed BATTLE-1 clinical trial, a prospective, multi-arm, biopsy-mandated, biomarker-driven, clinical trial in advanced refractory non-small cell lung cancer (NSCLC), found that mut-KRAS did not accurately predict patient outcome (progression-free survival) to targeted intervention. This finding contradicted published evidence for such a relationship from colon cancer and some previous NSCLC studies. We explored more specifically the nature of the KRAS mutations, which are primarily found at codons 12 and 13, where different base substitutions lead to alternate amino acid (aa) substitutions. NSCLC has a much higher proportion of mut-KRAS G12C(cysteine) aa substitutions (47%) due to carcinogens in tobacco smoke, and only 15% mut-KRAS have G12D(aspartate). These data contrast those in other solid tumors, such as colon or pancreas, which predominantly manifest mut-KRAS G12D (50%) and only 9% mut-KRAS G12C. In a subset analysis of the BATTLE-1 data, we showed significantly worse progression-free survival in patients with mut-KRAS G12C, versus other mut-KRAS including G12D (p=.041) and who were treated with erlotinib, vandetanib or sorafenib. In a panel of NSCLC cell lines with known mut-KRAS aa substitutions to identify pathways activated by the different mut-KRAS genotypes, we found that mut-KRAS G12D activates both PI-3-K and MEK signaling, while mut-KRAS G12C does not and alternatively activates PKCζ and RAL signaling. This finding was confirmed in immortalized human bronchial epithelial (HBEC) cells stably transfected with wt-KRAS or different forms of mut-KRAS. Our molecular modeling studies show that the different conformation imposed by mut-KRAS G12C could lead to altered association with downstream signaling transducers, compared to mut-KRAS G12D. The significance of the findings for developing mut-KRAS therapies is profound, since it suggests that not all mut-KRAS may be addictive; and that different combinations of inhibitors of downstream signaling may be needed for different mut-KRAS. 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 955. doi:10.1158/1538-7445.AM2011-955