Tianhong Li

Genotyping and Genomic Profiling of Non–Small-Cell Lung Cancer: Implications for Current and Future Therapies

Substantial advances have been made in understanding critical molecular and cellular mechanisms driving tumor initiation, maintenance, and progression in non-small-cell lung cancer (NSCLC). Over the last decade, these findings have led to the discovery of a variety of novel drug targets and the development of new treatment strategies. Already, the standard of care for patients with advanced-stage NSCLC is shifting from selecting therapy empirically based on a patients clinicopathologic features to using biomarker-driven treatment algorithms based on the molecular profile of a patients tumor. This approach is currently best exemplified by treating patients with NSCLC with first-line tyrosine kinase inhibitors when their cancers harbor gain-of-function hotspot mutations in the epidermal growth factor receptor (EGFR) gene or anaplastic lymphoma kinase (ALK) gene rearrangements. These genotype-based targeted therapies represent the first step toward personalizing NSCLC therapy. Recent technology advances in multiplex genotyping and high-throughput genomic profiling by next-generation sequencing technologies now offer the possibility of rapidly and comprehensively interrogating the cancer genome of individual patients from small tumor biopsies. This advance provides the basis for categorizing molecular-defined subsets of patients with NSCLC in whom a growing list of novel molecularly targeted therapeutics are clinically evaluable and additional novel drug targets can be discovered. Increasingly, practicing oncologists are facing the challenge of determining how to select, interpret, and apply these new genetic and genomic assays. This review summarizes the evolution, early success, current status, challenges, and opportunities for clinical application of genotyping and genomic tests in therapeutic decision making for NSCLC.

Association of Epidermal Growth Factor Receptor Activating Mutations with Low ERCC1 Gene Expression in Non-small Cell Lung Cancer

Introduction: Patients with non-small cell lung cancer (NSCLC) with cancers harboring activating mutations in the epidermal growth factor receptor (EGFR) show improved efficacy from EGFR tyrosine kinase inhibitors. Some clinical studies also suggest enhanced efficacy of platinum-based chemotherapy in patients with EGFR-mutant cancers. We investigated the relationship of EGFR mutation status and DNA repair capacity, as exemplified by excision repair cross-complementing 1 (ERCC1) gene expression, as a potential explanation for this observation. Methods: Microdissected formalin-fixed paraffin-embedded tumors from 1207 patients with NSCLC were analyzed by real-time polymerase chain reaction for mRNA expression levels of ERCC1 and for EGFR mutation status by an allele-specific polymerase chain reaction assay. Results: NSCLC subtype was adenocarcinoma (AC) in 712 patients, squamous in 175, and not otherwise specified or other in 320. EGFR activating mutations were detected in 183/1207 patients (15.2%). Median ERCC1 expression overall was 1.82 (range, 0.22-27.31) and was histology related: AC, median = 1.68 (0.22-11.33) and squamous, median = 2.42 (0.51-14.28) (p < 0.001). Using a previously defined reference level of <1.7, ERCC1 expression was categorized as low in 556 of 1207 patients (46.1%). The presence of EGFR mutations was highly associated with ERCC1 expression (p < 0.001). This association was retained when adjusting for AC histologic subtype (p = 0.001). Conclusions: NSCLC specimens harboring EGFR activating mutations are more likely to express low ERCC1 mRNA levels. Whether these findings translate into enhanced clinical efficacy of EGFR-mutant cancers to platinum-based chemotherapy remains to be determined.

Large-Scale Screening and Molecular Characterization of EML4-ALK Fusion Variants in Archival Non–Small-Cell Lung Cancer Tumor Specimens Using Quantitative Reverse Transcription Polymerase Chain Reaction Assays

Introduction: The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non–small-cell lung cancer (NSCLC) patients. Methods: We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients. Results: Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23–89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001). Conclusions: This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.

Acquired resistance to targeted therapies against oncogene-driven non-small-cell lung cancer: Approach to subtyping progressive disease and clinical implications

In the emerging era of targeted therapy for advanced-stage non-small-cell lung cancer, it is becoming increasingly important to anticipate underlying driver oncogene alterations at the time of initial diagnosis and tumor-tissue acquisition, so that patients can be selected in a timely fashion for first-line tyrosine kinase inhibitor (TKI) therapy if their cancers are found to harbor tyrosine-kinase-activating mutations in the epidermal growth factor receptor gene or gain-of-function rearrangements in the anaplastic lymphoma kinase gene. However, despite the clear benefits of TKI therapy over chemotherapy in these settings, the eventual emergence of acquired resistance and progressive disease (PD) is universal. How to best approach oncogene-driven non-small-cell lung cancer at the time of acquired resistance to initial TKI therapy is an increasingly complex question because of variability in mechanisms of resistance, extent of PD, and inter- and intrapatient tumor heterogeneity. Here we propose an approach to subtyping PD in the setting of acquired resistance as well as subsequent clinical implications.

Intercalated Erlotinib-Docetaxel Dosing Schedules Designed to Achieve Pharmacodynamic Separation: Results of a Phase I/II Trial

Introduction: Epidermal growth factor receptor tyrosine kinase inhibitors given concurrently with chemotherapy do not improve patient outcomes compared with chemotherapy alone in advanced non-small cell lung cancer (NSCLC). On the basis of preclinical models, we hypothesized pharmacodynamic separation, achieved by intermittent delivery of epidermal growth factor receptor tyrosine kinase inhibitors intercalated with chemotherapy, as a reasonable strategy to deliver combination therapy. Methods: A phase I dose-escalating trial using two scheduling strategies (arms A and B) was conducted in patients with advanced solid tumors to determine the feasibility of intermittent erlotinib and docetaxel. Phase II efficacy evaluation was conducted in an expanded cohort of patients with previously treated advanced NSCLC using arm B scheduling. Docetaxel was given every 21 days (70–75 mg/m2 intravenously) in both arms. In arm A, erlotinib was administered on days 2, 9, and 16 (600–1000 mg); in arm B, erlotinib was delivered on days 2 through 16 (150–300 mg). Patients without progression or unacceptable toxicity after six cycles continued erlotinib alone. Results: Eighty-one patients were enrolled in this study (17 arm A; 25 arm B; and 39 at phase II dose). Phase I patients had advanced solid tumors and 22 with NSCLC (10 and 12 patients for arms A and B, respectively). Treatment was well tolerated for both arms, with dose-limiting toxicities including grade 3 infection and febrile neutropenia in arm A (maximum tolerated dose [MTD] of erlotinib 600 mg/docetaxel 70 mg/m2) and grade 4 rash, febrile neutropenia, grade 3 mucositis, and grade 3 diarrhea in arm B (MTD of erlotinib 200 mg/docetaxel 70 mg/m2). The MTD for arm B was chosen for phase II evaluation given the feasibility of administration, number of responses (one complete response and three partial responses), and achievement of pharmacodynamic separation. The response rate for patients treated at the phase II dose was 28.2%, and the disease control rate was 64.1%. Median progression-free and overall survival were 4.1 and 18.2 months, respectively. Common grade ≥3 toxicities were neutropenia (36%) and diarrhea (18%). Conclusions: Pharmacodynamic separation using intercalated schedules of erlotinib delivered on an intermittent basis together with docetaxel chemotherapy is feasible and tolerable. Further studies using this approach together with interrogation of relevant molecular pathways are ongoing.

Phase I Trial of Arginine Deprivation Therapy with ADI-PEG 20 Plus Docetaxel in Patients with Advanced Malignant Solid Tumors

Purpose: This phase I study examined the toxicity and tolerability of pegylated arginine deiminase (ADI-PEG 20) in combination with docetaxel in patients with advanced solid malignancies. Experimental Design: Eligible patients had histologically proven advanced solid malignancies, with any number of prior therapies, Zubrod performance status 0–2, and adequate organ function. Patients received ADI-PEG 20 weekly intramuscular injection ranging from 4.5 to 36 mg/m2 and up to 10 doses of docetaxel (75 mg/m2) every 3 weeks. Primary endpoints were safety, toxicity, and a recommended phase II dose. Circulating arginine levels were measured before each cycle. Tumor response was measured as a secondary endpoint every 6 weeks on study. Results: Eighteen patients received a total of 116 cycles of therapy through four dose levels of ADI-PEG 20. A single dose-limiting toxicity (grade 3 urticarial rash) was observed at the 1st dose level, with no additional dose-limiting toxicities observed. Hematologic toxicities were common with 14 patients experiencing at least one grade 3 to 4 leukopenia. Fatigue was the most prevalent toxicity reported by 16 patients. Arginine was variably suppressed with 10 patients achieving at least a 50% reduction in baseline values. In 14 patients with evaluable disease, four partial responses (including 2 patients with PSA response) were documented, and 7 patients had stable disease. Conclusions: ADI-PEG 20 demonstrated reasonable toxicity in combination with docetaxel. Promising clinical activity was noted, and expansion cohorts are now accruing for both castrate-resistant prostate cancer and non–small cell lung cancer at a recommended phase II dose of 36 mg/m2. Clin Cancer Res; 21(11); 2480–6. ©2015 AACR.

The Phosphatase Inhibitor Menadione (Vitamin K3) Protects Cells from EGFR Inhibition by Erlotinib and Cetuximab

Purpose: Skin toxicity is the main side effect of epidermal growth factor receptor (EGFR) inhibitors, often leading to dose reduction or discontinuation. We hypothesized that phosphatase inhibition in the skin keratinocytes may prevent receptor dephosphorylation caused by EGFR inhibitors and be used as a new potential strategy for the prevention or treatment of this side effect. Experimental Design: Menadione (Vitamin K3) was used as the prototype compound to test our hypothesis. HaCat human skin keratinocyte cells and A431 human squamous carcinoma cells were used. EGFR inhibition was measured by Western blotting and immunofluorescence. Phosphatase inhibition and reactive oxygen species (ROS) generation were measured by standard ELISA and fluorescence assays. Results: Menadione caused significant and reversible EGFR activation in a dose-dependent manner starting at nontoxic concentrations. EGFR activation by menadione was associated with reversible protein tyrosine phosphatase inhibition, which seemed to be mediated by ROS generation as exposure to antioxidants prevented both menadione-induced ROS generation and phosphatase inhibition. Short-term coincubation of cells with nontoxic concentrations of menadione and the EGFR inhibitors erlotinib or cetuximab prevented EGFR dephosphorylation. Seventy-two–hour coincubation of cells with the highest nontoxic concentration of menadione and erlotinib provided for a fourfold cell growth inhibitory protection in HaCat human keratinocyte cells. Conclusions: Menadione at nontoxic concentrations causes EGFR activation and prevents EGFR dephosphorylation by erlotinib and cetuximab. This effect seems to be mediated by ROS generation and secondary phosphatase inhibition. Mild oxidative stress in skin keratinocytes by topical menadione may protect the skin from the toxicity secondary to EGFR inhibitors without causing cytotoxicity. Clin Cancer Res; 17(21); 6766–77. ©2011 AACR.

Evolving Treatment Algorithms for Advanced Non–Small-Cell Lung Cancer: 2009 Looking Toward 2012

Evolving Treatment Algorithms for Advanced Non–Small-Cell Lung Cancer: 2009 Looking Toward 2012 David R. GANDARA, Philip C. MACK, Tianhong LI, Primo N. LARA, JR, Roy S. HERBST Division of Hematology and Oncology, University of California Davis Cancer Center, Sacramento, CA; Department of Oncology, Montefiore-Einstein Cancer Center, Bronx, NY; Department of Thoracic, Head and Neck Medical Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX

Phase II Study of the AKT Inhibitor MK-2206 plus Erlotinib in Patients with Advanced Non–Small Cell Lung Cancer Who Previously Progressed on Erlotinib

Purpose: Preclinical modeling in non–small cell lung cancer (NSCLC) showed that stimulation with hepatocyte growth factor (HGF), the ligand for MET, could reverse the cytostatic and cytotoxic effects of the EGFR inhibitor erlotinib in erlotinib-sensitive cell lines. Inhibitors of AKT signaling mitigated this HGF-mediated resistance, partially restoring erlotinib activity. We conducted a phase II trial of erlotinib plus MK-2206, a highly selective inhibitor of AKT, in NSCLC patients. Experimental Design: Eligible patients must have progressed following prior benefit from erlotinib, defined as response or stable disease > 12 weeks. Treatment consisted of erlotinib 150 mg orally every day + MK-2206 45 mg orally every alternate day on a 28-day cycle. Primary endpoints were RECIST response rate > 30% (stratum 1: EGFR mutant) and disease control rate (DCR) > 20% at 12 weeks (stratum 2: EGFR wild-type). Results: Eighty patients were enrolled, 45 and 35 in stratum 1 and 2, respectively. Most common attributable adverse events (all grade 3) were rash, diarrhea, fatigue, and mucositis. Response and DCR were, respectively, 9% and 40% in stratum 1; 3% and 47% in stratum 2. Median progression-free survival was 4.4 months in stratum 1 and 4.6 months in stratum 2. Conclusions: Combination MK-2206 and erlotinib met its primary endpoint in erlotinib-pretreated patients with EGFR wild-type NSCLC. Although activity was seen in EGFR-mutated NSCLC, this did not exceed a priori estimates. AKT pathway inhibition merits further clinical evaluation in EGFR wild-type NSCLC. Clin Cancer Res; 21(19); 4321–6. ©2015 AACR.

Histology-Related Associations of ERCC1, RRM1, and TS Biomarkers in Patients with Non–Small-Cell Lung Cancer: Implications for Therapy

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