J. Bhosle

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

Mechanism and non-mechanism based imaging biomarkers for assessing biological response to treatment in non-small cell lung cancer

Therapeutic options in locally advanced non-small cell lung cancer (NSCLC) have expanded in the past decade to include a palate of targeted interventions such as high dose targeted thermal ablations, radiotherapy and growing platform of antibody and small molecule therapies and immunotherapies. Although these therapies have varied mechanisms of action, they often induce changes in tumour architecture and microenvironment such that response is not always accompanied by early reduction in tumour mass, and evaluation by criteria other than size is needed to report more effectively on response. Functional imaging techniques, which probe the tumour and its microenvironment through novel positron emission tomography and magnetic resonance imaging techniques, offer more detailed insights into and quantitation of tumour response than is available on anatomical imaging alone. Use of these biomarkers, or other rational combinations as readouts of pathological response in NSCLC have potential to provide more accurate predictors of treatment outcomes. In this article, the robustness of the more commonly available positron emission tomography and magnetic resonance imaging biomarker indices is examined and the evidence for their application in NSCLC is reviewed.

AKT inhibition synergistically enhances growth-inhibitory effects of gefitinib and increases apoptosis in non-small cell lung cancer cell lines.

OBJECTIVES EGFR inhibitors are ineffective against most EGFR wild-type non-small cell lung cancer, for which novel treatment strategies are needed. AKT signalling is essential for mediating EGFR survival signals in NSCLC. We evaluated the combination of gefitinib and two different AKT inhibitors, the allosteric inhibitor AKTi-1/2 and the ATP-competitive pan-AKT inhibitor AZD5363, in EGFR-mutant (HCC-827 and PC-9) and -wild-type (NCI-H522, NCI-H1651), non-small cell lung cancer cell lines. MATERIALS AND METHODS Drug interaction was studied in two EGFR mutant and two EGFR wild-type non-small cell lung cancer cell lines by calculating combination index (CI) using median effect analysis. The effects on p-EGFR, p-ERK, p-AKT, p-S6 and apoptosis were studied by Western blot analysis. RESULTS The combination of gefitinib and AKTi-1/2 or AZD5363 showed synergistic growth inhibition in all cell lines. CI values for the combination of gefitinib and AKTi-1/2 were 0.35 (p=0.0048), 0.56 (p=0.036), 0.75 (p=0.13) and 0.64 (p=0.0003) in NCI-H522, NCI-H1651, HCC-827 and PC-9 cell lines, respectively; CI values of 0.45 (p=0.0087) and 0.22 (p<0.0001) were observed in NCI-H522 and PC-9 cells, respectively, when gefitinib was combined with AZD5363. Additive inhibition of signalling output through AKT and key downstream proteins (S6) and increased apoptosis were demonstrated. CONCLUSION Dual inhibition of EGFR and AKT may be a useful up-front strategy for patients with EGFR-mutant and -wild-type non-small cell lung cancer.

Epidermal Growth Factor Mutation as a Diagnostic and Therapeutic Target in Metastatic Poorly Differentiated Thyroid Carcinoma: A Case Report and Review of the Literature

Poorly differentiated cancers are a diagnostic and therapeutic challenge in oncology. New therapies are needed for patients with poorly differentiated thyroid carcinoma (PDTC) or anaplastic thyroid cancer, as these patients often present with advanced disease and effective systemic treatment options are currently limited. Epidermal growth factor (EGFR) mutations may occur in PDTC more often than previously thought. However, there are fewer than 6 cases reported in the literature where EGFR tyrosine kinase inhibitors (TKIs) (such as erlotinib or gefitinib) were used to target EGFR mutations in PDTC. Here, we present the case of a 79-year-old male with metastatic PDTC with an EGFR mutation who responded to treatment with the selective EGFR TKI erlotinib, with a progression-free survival of more than 11 months. A lung primary rather than a thyroid primary was initially detected. We suggest that the EGFR status should be analysed at diagnosis in any patient with a poorly differentiated tumour. The presence of an EGFR mutation may provide an effective therapeutic pathway for these patients. This pathway requires further investigation and consideration in the future.

A study of PD-L1 expression in KRAS mutant non-small cell lung cancer cell lines exposed to relevant targeted treatments

We investigated PD-L1 changes in response to MEK and AKT inhibitors in KRAS mutant lung adenocarcinoma (adeno-NSCLC). PD-L1 expression was quantified using immunofluorescence and co-culture with a jurkat cell-line transfected with NFAT-luciferase was used to study if changes in PD-L1 expression in cancer cell lines were functionally relevant. Five KRAS mutant cell lines with high PD-L1 expression (H441, H2291, H23, H2030 and A549) were exposed to GI50 inhibitor concentrations of a MEK inhibitor (trametinib) and an AKT inhibitor (AZD5363) for 3 weeks. Only 3/5 (H23, H2030 and A549) and 2/5 cell lines (H441 and H23) showed functionally significant increases in PD-L1 expression when exposed to trametinib or AZD5363 respectively. PD-L1 overexpression is not consistent and is unlikely to be an early mechanism of resistance to KRAS mutant adeno-NSCLC treated with MEK or AKT inhibitors.

Characterisation of the Phosphatidylinositol 3-Kinase Pathway in Non-Small Cell Lung Cancer Cells Isolated from Pleural Effusions

Objectives: We hypothesised that it was possible to quantify phosphorylation of important nodes in the phosphatidylinositol 3-kinase (PI3K) pathway in cancer cells isolated from pleural effusions of patients with non-small cell lung cancer (NSCLC) and study their correlation to somatic mutations and clinical outcomes. Materials and Methods: Cells were immunomagnetically separated from samples of pleural effusion in patients with NSCLC. p-AKT, p-S6K and p-GSK3β levels were quantified by ELISA; targeted next-generation sequencing was used to characterise mutations in 26 genes. Results: It was possible to quantify phosphoproteins in cells isolated from 38/43 pleural effusions. There was a significant correlation between p-AKT and p-S6K levels [r = 0.85 (95% confidence interval 0.73-0.92), p < 0.0001], but not p-AKT and p-GSK3β levels [r = 0.19 (95% confidence interval -0.16 to 0.5), p = 0.3]. A wide range of mutations was described and p-S6K was higher in samples that harboured at least one mutation compared to those that did not (p = 0.03). On multivariate analysis, p-S6K levels were significantly associated with poor survival (p < 0.01). Conclusion: Our study has shown a correlation between p-AKT levels and p-S6K, but not GSK3β, suggesting differences in regulation of the distal PI3K pathway by AKT. Higher p-S6K levels were associated with adverse survival, making it a critically important target in NSCLC.

Phase I clinical trials in patients with advanced non-small cell lung cancer treated within a Drug Development Unit: What have we learnt?

OBJECTIVES Despite advances in novel drug development for patients with advanced non-small cell lung cancer (NSCLC), there are still only a limited number of approved treatments. We therefore evaluated the clinical outcomes of patients with advanced NSCLC referred to a dedicated phase I clinical trials unit assessed baseline clinical factors associated with successful enrollment onto phase I trials. MATERIAL AND METHODS We conducted a retrospective study involving patients with advanced NSCLC referred to the Drug Development Unit at the RMH between January 2005 and December 2013. RESULTS 257 patients with advanced NSCLC were referred for consideration of phase I trials, of which only 89 (35%) patients successfully commenced phase I trials. The commonest reasons for not entering study included poor ECOG performance status and rapid disease progression. A multivariate analysis identified that ECOG performance status (0-1) and RMH prognostic score (0-1) were associated with successful enrollment onto phase I trials (p<0.001). Single agent therapies included novel agents against the phosphatidylinositol-3 kinase pathway, insulin growth factor-1 receptor and pan-HER family tyrosine kinases. These trial therapies were well tolerated and mainly associated with grade 1-2 adverse events, with a minority experiencing grade 3 toxicities. Nine (10%) patients, 4 with known EGFR or KRAS mutations, achieved RECIST partial responses. Median time to progression was 2.6 months and median overall survival was 8.1 months for patients enrolled. CONCLUSIONS Phase I trial therapies were generally well tolerated with potential antitumor benefit for patients with advanced NSCLC. Early referral to drug development units at time of disease progression should be considered to enhance the odds of patient participation in these studies.

Molecular Adequacy of Image-Guided Rebiopsies for Molecular Retesting in Advanced Non–Small Cell Lung Cancer: A Single-Center Experience

Introduction In the era of biomarker‐driven systemic therapy for advanced NSCLC, the role of routine repeated biopsies for decision making outside EGFR‐mutant disease remains unproven. We report our center’s experience of safety and adequacy for molecular retesting of tumor material obtained from image‐guided lung rebiopsies in NSCLC. Methods We performed a retrospective case note analysis of patients undergoing image‐guided lung rebiopsies at a single cancer center between 2011 and 2014. The primary objective was to determine the pathological success rate. Secondary and exploratory objectives were to determine technical success rate, histological concordance, molecular adequacy, genotypes identified, and complication rate. Results In all, 103 patients underwent transthoracic image‐guided procedures. A total of 66 rebiopsies in NSCLC were identified and analyzed. The pathological success rate was 87.1%. A high histological discordance rate was observed (12 of 52 evaluable cases [23.1%]). Pretest molecular adequacy as determined by the lung pathologist was 78.8% (52 of 66). Of 52 adequate samples 51 were sent for molecular analysis, with a total of 209 genes analyzed (including EGFR, ALK receptor tyrosine kinase gene [ALK], KRAS, BRAF, dicoidin domain receptor tyrosine kinase 2 gene [DDR2], NRAS, ROS1, and rearranged during transfection proto‐oncogene gene [RET]). The rate of postgenotyping molecular adequacy was 87.1% (182 of 209). Overall, 20 new potentially actionable mutations were identified, with 13 of 66 patients (19.7%) starting to receive new targeted treatment as a result. Overall, rebiopsies informed clinical decision making in 63.6% of cases. The rates of complications were 15% for pneumothorax, 3% for pneumothorax requiring chest drain, and 8% for hemoptysis. Conclusions We have validated the pathological and molecular adequacy rates of rebiopsies and demonstrated clinical utility in routine decision making.

The diagnosis and treatment of brain metastases in EGFR mutant lung cancer.

The epidemiology of non-small-cell lung cancer (NSCLC) has changed with a new pattern of disease emerging - a form of adenocarcinoma in mostly younger female patients, who are never or light smokers and more frequently in East Asian populations. Description of EGF receptor (EGFR) mutations has allowed new management strategies to evolve. Oral targeted therapies have broadened the treatment options in the advanced setting with the potential for periods of long term response. The brain is a common site of metastases with EGFR mutated lung cancer typically displaying asymptomatic, small volume, multiple lesions that respond to treatment. We explore the role of local and system therapies for brain metastases in this disease including the role of EGFR inhibitors.

33 Outcomes of patients undergoing adjuvant platinum–vinorelbine chemotherapy for resected non-small cell lung cancer (NSCLC)

Introduction: Cisplatin vinorelbine adjuvant chemotherapy significantly improves survival in resected NSCLC. We evaluated outcomes of patients receiving adjuvant chemotherapy in our institution between 2006 2011. Methods: Outcomes of stage IB IIIA NSCLC patients who received platinum vinorelbine following radical lung surgery were collected and analysed to assess overall survival (OS), progression-free survival (PFS), and treatment intensity. Results: 53 patients were identified (M:F, 23:30), mean age 62, and 35% were adenocarcinoma. Resected stages were IB-IIIA, with 33% staged IIIA. When tested, EGFR mutation prevalence was 33% (39% never smokers; 61% ever smokers). Median chemotherapy cycles given was 4 with no toxicity related deaths. There was no difference in PFS or OS in patients having carboplatin compared with cisplatin. Significantly improved OS in patients that received 3 cycles of chemotherapy was observed (HR = 0.25, 0.07 0.97, p = 0.04). Updated results will be presented at the Annual Meeting. Conclusion: Our small dataset indicates that four cycles of adjuvant platinum vinorelbine chemotherapy is deliverable in the real world setting, and that less than 3 chemotherapy cycles is associated with poorer outcomes.