Maya Ilouze

Next-Generation Sequencing Identifies and Immunohistochemistry Confirms a Novel Crizotinib- Sensitive ALK Rearrangement in a Patient with Metastatic Non-Small-Cell Lung Cancer

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) gene fusion occurs in 2–7% of NSCLC cases[1,2] and is typically identified by Vysis ALK Break-Apart fluorescent in situ hybridization (FISH) assay. Tumors expressing this fusion respond to treatment crizotinib[1].

Fluorescence In Situ Hybridization, Immunohistochemistry, and Next-Generation Sequencing for Detection of EML4-ALK Rearrangement in Lung Cancer

BACKGROUND The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next-generation sequencing (NGS) analysis. MATERIALS AND METHODS We studied FISH and IHC (D5F3 antibody) systematically for EML4-ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance. RESULTS Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression-free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC. CONCLUSION The FISH-based method of detecting EML4-ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy.

Volatile fingerprints of cancer specific genetic mutations

UNLABELLED We report on a new concept for profiling genetic mutations of (lung) cancer cells, based on the detection of patterns of volatile organic compounds (VOCs) emitted from cell membranes, using an array of nanomaterial-based sensors. In this in-vitro pilot study we have derived a volatile fingerprint assay for representative genetic mutations in cancer cells that are known to be associated with targeted cancer therapy. Five VOCs were associated with the studied oncogenes, using complementary chemical analysis, and were discussed in terms of possible metabolic pathways. The reported approach could lead to the development of novel methods for guiding treatments, so that patients could benefit from safer, more timely and effective interventions that improve survival and quality of life while avoiding unnecessary invasive procedures. Studying clinical samples (tissue/blood/breath) will be required as next step in order to determine whether this cell-line study can be translated into a clinically useful tool. FROM THE CLINICAL EDITOR In this novel study, a new concept for profiling genetic mutations of (lung) cancer cells is described, based on the detection of patterns of volatile organic compounds emitted from cell membranes, using an array of nano-gold based sensors.

Unique volatolomic signatures of TP53 and KRAS in lung cells

Background:Volatile organic compounds (VOCs) are potential biomarkers for cancer detection in breath, but it is unclear if they reflect specific mutations. To test this, we have compared human bronchial epithelial cell (HBEC) cell lines carrying the KRASV12 mutation, knockdown of TP53 or both with parental HBEC cells.Methods:VOC from headspace above cultured cells were collected by passive sampling and analysed by thermal desorption gas chromatography mass spectrometry (TD-GC–MS) or sensor array with discriminant factor analysis (DFA).Results:In TD-GC–MS analysis, individual compounds had limited ability to discriminate between cell lines, but by applying DFA analysis combinations of 20 VOCs successfully discriminated between all cell types (accuracies 80–100%, with leave-one-out cross validation). Sensor array detection DFA demonstrated the ability to discriminate samples based on their cell type for all comparisons with accuracies varying between 77% and 93%.Conclusions:Our results demonstrate that minimal genetic changes in bronchial airway cells lead to detectable differences in levels of specific VOCs identified by TD-GC–MS or of patterns of VOCs identified by sensor array output. From the clinical aspect, these results suggest the possibility of breath analysis for detection of minimal genetic changes for earlier diagnosis or for genetic typing of lung cancers.

Effective Crizotinib Schedule for Brain Metastases in ALK Rearrangement Metastatic Non–Small-Cell Lung Cancer

Journal of Thoracic Oncology ® • Volume 8, Number 12, December 2013 The echinoderm microtubule–associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) gene fusion occurs in 2% to 7% of non–small-cell lung cancer cases. Tumors expressing this fusion respond to treatment with crizotinib, an ALK tyrosine kinase inhibitor. However, brain metastases frequently occur, even in the presence of systemic response to therapy.

Exhaled Breath Analysis for Monitoring Response to Treatment in Advanced Lung Cancer

Introduction: The Response Evaluation Criteria in Solid Tumors (RECIST) serve as the accepted standard to monitor treatment efficacy in lung cancer. However, the time intervals between consecutive computerized tomography scans might be too long to allow early identification of treatment failure. This study examines the use of breath sampling to monitor responses to anticancer treatments in patients with advanced lung cancer. Methods: A total of 143 breath samples were collected from 39 patients with advanced lung cancer. The exhaled breath signature, determined by gas chromatography/mass spectrometry and a nanomaterial‐based array of sensors, was correlated with the response to therapy assessed by RECIST: complete response, partial response, stable disease, or progressive disease. Results: Gas chromatography/mass spectrometry analysis identified three volatile organic compounds as significantly indicating disease control (PR/stable disease), with one of them also significantly discriminating PR/stable disease from progressive disease. The nanoarray had the ability to monitor changes in tumor response across therapy, also indicating any lack of further response to therapy. When one‐sensor analysis was used, 59% of the follow‐up samples were identified correctly. There was 85% success in monitoring disease control (stable disease/partial response). Conclusion: Breath analysis, using mainly the nanoarray, may serve as a surrogate marker for the response to systemic therapy in lung cancer. As a monitoring tool, it can provide the oncologist with a quick bedside method of identifying a lack of response to an anticancer treatment. This may allow quicker recognition than does the current RECIST analysis. Early recognition of treatment failure could improve patient care.

Clinical Impact of Hybrid Capture-Based Next-Generation Sequencing on Changes in Treatment Decisions in Lung Cancer.

Introduction: Targeted therapy significantly prolongs survival in lung adenocarcinoma. Current diagnostic guidelines include only EGFR and anaplastic lymphoma receptor tyrosine kinase gene (ALK) testing. Next‐generation sequencing (NGS) reveals more actionable genomic alterations than do standard diagnostic methods. Data on the influence of hybrid capture (HC)‐based NGS on treatment are limited, and we investigated its impact on treatment decisions and clinical outcomes. Methods: This retrospective study included patients with advanced lung cancer on whom HC‐based NGS was performed between November 2011 and October 2015. Demographic and clinicopathologic characteristics, treatments, and outcome data were collected. Results: A total of 101 patients were included (median age 63 years [53% females, 45% never‐smokers, and 85% with adenocarcinoma]). HC‐based NGS was performed upfront and after EGFR/ALK testing yielded negative or inconclusive results in 15% and 85% of patients, respectively. In 51.5% of patients, HC‐based NGS was performed before first‐line therapy, and in 48.5%, it was performed after treatment failure. HC‐based NGS identified clinically actionable genomic alterations in 50% of patients, most frequently in EGFR (18%), Ret proto‐oncogene (RET) (9%), ALK (8%), Mesenchymal‐epithelial transition factor (MET) receptor tyrosine kinase gene (6%), and erb‐b2 receptor tyrosine kinase 2 gene (ERBB2) (5%). In 15 patients, it identified EGFR/ALK aberrations after negative results of prior standard testing. Treatment strategy was changed for 43 patients (42.6%). The overall response rate in these patients was 65% (complete response 14.7%, partial response 50%). Median survival was not reached. Immunotherapy was administered in 33 patients, mostly without an actionable driver, with a presenting disease control rate of 32%, and with an association with tumor mutation burden. Conclusions: HC‐based NGS influenced treatment decisions in close to half of the patients with lung adenocarcinoma and was associated with an overall response rate of 65%, which may translate into a survival benefit.

Detection of Lung Cancer and EGFR Mutation by Electronic Nose System

Introduction: Early detection of lung cancer (LC) has been well established as a significant key point in patient survival and prognosis. New highly sensitive nanoarray sensors for exhaled volatile organic compounds that have been developed and coupled with powerful statistical programs may be used when diseases such as LC are suspected. Detection of genetic aberration mutation by nanoarray sensors is the next target. Methods: Breath samples were taken from patients who were evaluated for suspicious pulmonary lesions. Patients were classified as those with benign nodules, as patients with LC with or without the EGFR mutation, and according to their smoking status. Breath prints were recognized by nanomaterial‐based sensor array, and pattern recognition methods were used. Results: A total of 119 patients participated in this study, 30 patients with benign nodules and 89 patients with LC (16 with early disease and 73 with advanced disease). Patients with LC who harbored the EGFR mutation (n = 19) could be discriminated from those with wild‐type EGFR (n = 34) with an accuracy of 83%, sensitivity of 79%, and specificity of 85%. Discrimination of early LC from benign nodules had 87% accuracy and positive and negative predictive values of 87.7 and 87.5% respectively. Moderate discrimination (accuracy of 76%) was found between LC of heavy smokers and that of never‐smokers or distant past light smokers. Conclusions: Breath analysis could discriminate patients with LC who harbor the EGFR mutation from those with wild‐type EGFR and those with benign pulmonary nodules from those patients with early LC. A positive breath print for the EGFR mutation may be used in treatment decisions if tissue sampling does not provide adequate material for definitive mutation analysis.

RET Fusion Lung Carcinoma: Response to Therapy and Clinical Features in a Case Series of 14 Patients

Background RET (rearranged during transfection) fusions have been reported in 1% to 2% of lung adenocarcinoma (LADC) cases. In contrast, KIF5B‐RET and CCDC6‐RET fusion genes have been identified in 70% to 90% and 10% to 25% of tumors, respectively. The natural history and management of RET‐rearranged LADC are still being delineated. Materials and Methods We present a series of 14 patients with RET‐rearranged LADC. The response to therapy was assessed by the clinical response and an avatar model in 2 cases. Patients underwent chemotherapy, targeted therapy, and immunotherapy. Results A total of 14 patients (8 women; 10 never smokers; 4 light smokers; mean age, 57 years) were included. KIF5B‐RET and CCDC6‐RET variants were diagnosed in 10 and 4 cases, respectively. Eight patients had an early disseminated manifestation, seven with KIF5B‐RET rearranged tumor. The features of this subset included bilateral miliary lung metastases, bone metastases, and unusual early visceral abdominal involvement. One such patient demonstrated an early and durable complete response to cabozantinib for 7 months. Another 2 patients treated with cabozantinib experienced a partial response, with rapid significant clinical improvement. Four patients with tumors harboring CCDC6‐RET and KIF5B‐RET fusions showed pronounced and durable responses to platinum‐based chemotherapy that lasted for 8 to 15 months. Two patients’ tumors showed programmed cell death ligand 1‐positive staining but did not respond to pembrolizumab. The median overall survival was 22.8 months. Conclusion RET‐rearranged LADC in our series tended to occur as bilateral disease with early visceral involvement, especially with KIF5B fusion. Treatment with cabozantinib achieved responses, including 1 complete response. However, further studies are required in this group of patients. Micro‐Abstract Data are increasing regarding RET (rearranged during transfection) fusions in lung cancer. We present our experience with the natural history of this disease and its response to targeted therapy and standard chemotherapy in 14 patients. In our series, RET‐rearranged lung adenocarcinoma had an early disseminated presentation, especially with KIF5B fusion. Treatment with cabozantinib achieved responses, including 1 complete response.

Cancer metabolism: the volatile signature of glycolysis—in vitro model in lung cancer cells*

Discovering the volatile signature of cancer cells is an emerging approach in cancer research, as it may contribute to a fast and simple diagnosis of tumors in vivo and in vitro. One of the main contributors to such a volatile signature is hyperglycolysis, which characterizes the cancerous cell. The metabolic perturbation in cancer cells is known as the Warburg effect; glycolysis is preferred over oxidative phosphorylation (OXPHOS), even in the presence of oxygen. The precise mitochondrial alterations that underlie the increased dependence of cancer cells on aerobic glycolysis for energy generation have remained a mystery. We aimed to profile the volatile signature of the glycolysis activity in lung cancer cells. For that an in vitro model, using lung cancer cell line cultures (A549, H2030, H358, H322), was developed. The volatile signature was measured by proton transfer reaction mass spectrometry under normal conditions and glycolysis inhibition. Glycolysis inhibition and mitochondrial activity were also assessed by mitochondrial respiration capacity measurements. Cells were divided into two groups upon their glycolytic profile (PET positive and PET negative). Glycolysis blockade had a unique characteristic that was shared by all cells. Furthermore, each group had a characteristic volatile signature that enabled us to discriminate between those sub-groups of cells. In conclusion, lung cancer cells may have different subpopulations of cells upon low and high mitochondrial capacity. In both groups, glycolysis blockade induced a unique volatile signature.