The evolution of non-small cell lung cancer metastases in TRACERx.

Metastatic disease is responsible for the majority of cancer-related deaths1. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse.

A cross sectional study to determine the prevalence of cough and its impact in patients with lung cancer: a patient unmet need.

BACKGROUND: There is absence of literature related to cough prevalence and its characteristics in lung cancer patients, with information deriving only from broader symptoms occurrence studies. The aims of this study were to provide a snapshot of the prevalence of all-cause-cough in lung cancer patients and to characterise cough in terms of its impact and severity. METHODS: A cross-sectional study recruiting consecutive lung cancer patients over a pre-defined period of time and using cough-specific validated tools in a tertiary referral centre in the UK, including a cough severity VAS and the Manchester Cough in Lung Cancer scale (MCLCS). RESULTS: Data was collected from 202 patients. All-cause cough prevalence was 57% (through VAS) both in the screened (N = 223) and research (N = 202) population or 67% (through the MCLCS), and cough severity was moderate at a mean of 32 mm (in a 100 mm VAS). Age, sex, smoking status, lung cancer histology, stage and comorbidities were not associated with cough prevalence. The only variable associated with lower cough reports was being 'on anticancer treatment'; fewer patients on treatment reported a cough (40%) compared to those off treatment (54%) (p = 0.04). The impact of cough (as measured by MCLCS) was also significant (mean score = 22). About 18% of patients felt moderate/severe distress from their cough and about 15% often or always reported disturbed sleep due to coughing. Half the patients felt their cough warranted treatment. CONCLUSIONS: Cough is a common symptom in lung cancer with considerable impact on patients' lives. Cough presence and severity should regularly be assessed in clinical practice. There is an urgent need to focus on developing more potent antitussive treatments and improve the management of this complex and distressing symptom.

Direct Ras G12C inhibitors: crossing the rubicon.

Despite its status as the most commonly mutated oncogene in cancer, Ras has long been considered 'undruggable'. In 2019, we will see the first clinical trial results for direct mutant Ras inhibitors, a result of persistent cross-disciplinary research that has been informed by a number of previous clinical and biological failures.

KRAS-mutant non-small cell lung cancer: Converging small molecules and immune checkpoint inhibition.

KRAS is the most frequent oncogene in non-small cell lung cancer (NSCLC), a molecular subset characterized by historical disappointments in targeted treatment approaches such as farnesyl transferase inhibition, downstream MEK inhibition, and synthetic lethality screens. Unlike other important mutational subtypes of NSCLC, preclinical work supports the hypothesis that KRAS mutations may be vulnerable to immunotherapy approaches, an efficacy associated in particular with TP53 co-mutation. In this review we detail reasons for previous failures in KRAS-mutant NSCLC, evidence to suggest that KRAS mutation is a genetic marker of benefit from immune checkpoint inhibition, and emerging direct inhibitors of K-Ras which will soon be combined with immunotherapy during clinical development. With signs of real progress in this subgroup of unmet need, we anticipate that KRAS mutant NSCLC will be the most important molecular subset of cancer to evaluate the combination of small molecules and immune checkpoint inhibitors (CPI).

Cough in Patients With Lung Cancer: A Longitudinal Observational Study of Characterization and Clinical Associations.

BACKGROUND: Cough is common in patients with lung cancer, and current antitussive treatments are suboptimal. There are little published data describing cough in patients with lung cancer or work assessing clinical associations. The aim of this study is to fill that gap. METHODS: This was a longitudinal prospective observational single-cohort study over 60 days. Patients were assessed through self-reported validated scales and, in a subsample, ambulatory cough monitoring at study entry (day 0), day 30, and day 60. RESULTS: At study entry, 177 patients were included and 153 provided data at day 60. The median duration of cough was 52 weeks (interquartile range, 8.5-260). Cough was described as severe enough to warrant treatment in 62% of the patients. Depending on the scale used, performance status was associated with both cough severity and cough impact (P < .001) at study entry, whereas higher cough severity at study entry was associated with female sex (P = .02), asthma (P = .035), and reflux disease (P < .001). Cough impact at study entry was additionally associated with experiencing nausea (P = .018). Cancer characteristics (ie, cancer stage, histology) were not associated with cough severity nor cough impact; neither was smoking or COPD. CONCLUSIONS: This is the first study to describe characteristics of cough in patients with lung cancer and to identify clinical associations that may be relevant for its treatment. Our data suggest that cough is a frequent and distressing symptom and an unmet clinical need. Its association with gastrointestinal symptoms in this study may improve our understanding of pathophysiology and therapeutic options for cough occurring in patients with lung cancer.

Vasculogenic mimicry in small cell lung cancer.

Small cell lung cancer (SCLC) is characterized by prevalent circulating tumour cells (CTCs), early metastasis and poor prognosis. We show that SCLC patients (37/38) have rare CTC subpopulations co-expressing vascular endothelial-cadherin (VE-cadherin) and cytokeratins consistent with vasculogenic mimicry (VM), a process whereby tumour cells form 'endothelial-like' vessels. Single-cell genomic analysis reveals characteristic SCLC genomic changes in both VE-cadherin-positive and -negative CTCs. Higher levels of VM are associated with worse overall survival in 41 limited-stage patients' biopsies (P<0.025). VM vessels are also observed in 9/10 CTC patient-derived explants (CDX), where molecular analysis of fractionated VE-cadherin-positive cells uncovered copy-number alterations and mutated TP53, confirming human tumour origin. VE-cadherin is required for VM in NCI-H446 SCLC xenografts, where VM decreases tumour latency and, despite increased cisplatin intra-tumour delivery, decreases cisplatin efficacy. The functional significance of VM in SCLC suggests VM regulation may provide new targets for therapeutic intervention.

Intracranial Efficacy of Crizotinib Versus Chemotherapy in Patients With Advanced ALK-Positive Non-Small-Cell Lung Cancer: Results From PROFILE 1014.

PURPOSE: Intracranial efficacy of first-line crizotinib versus chemotherapy was compared prospectively in the phase III PROFILE 1014 study in ALK-positive non-small-cell lung cancer. PATIENTS AND METHODS: Patients were randomly assigned to receive crizotinib (250 mg twice daily; n = 172) or chemotherapy (pemetrexed 500 mg/m(2) plus cisplatin 75 mg/m(2) or carboplatin at area under the curve 5 to 6, every 3 weeks for ≤ six cycles; n = 171). Patients with stable treated brain metastases (tBM) were eligible. Intracranial efficacy was assessed at baseline and every 6 or 12 weeks in patients with or without known brain metastases (BM), respectively; intracranial time to tumor progression (IC-TTP; per protocol) and intracranial disease control rate (IC-DCR; post hoc) were measured. The intent-to-treat population was also assessed. RESULTS: Of 343 patients in the intent-to-treat population, 23% had tBM at baseline. A nonsignificant IC-TTP improvement was observed with crizotinib in the intent-to-treat population (hazard ratio [HR], 0.60; P = .069), patients with tBM (HR, 0.45; P = .063), and patients without BM (HR, 0.69; P = .323). Among patients with tBM, IC-DCR was significantly higher with crizotinib versus chemotherapy at 12 weeks (85% v 45%, respectively; P < .001) and 24 weeks (56% v 25%, respectively; P = .006). Progression-free survival was significantly longer with crizotinib versus chemotherapy in both subgroups (tBM present: HR, 0.40; P < .001; median, 9.0 v 4.0 months, respectively; BM absent: HR, 0.51; P < .001; median, 11.1 v 7.2 months, respectively) and in the intent-to-treat population (HR, 0.45; P < .001; median, 10.9 v 7.0 months, respectively). CONCLUSION: Compared with chemotherapy, crizotinib demonstrated a significantly higher IC-DCR in patients with tBM. Improvements in IC-TTP were not statistically significant in patients with or without tBM, although sensitivity to detect treatment differences in or between the two subgroups was low.

Discovery and Validation of Predictive Biomarkers of Survival for Non-small Cell Lung Cancer Patients Undergoing Radical Radiotherapy: Two Proteins With Predictive Value.

Lung cancer is the most frequent cause of cancer-related death world-wide. Radiotherapy alone or in conjunction with chemotherapy is the standard treatment for locally advanced non-small cell lung cancer (NSCLC). Currently there is no predictive marker with clinical utility to guide treatment decisions in NSCLC patients undergoing radiotherapy. Identification of such markers would allow treatment options to be considered for more effective therapy. To enable the identification of appropriate protein biomarkers, plasma samples were collected from patients with non-small cell lung cancer before and during radiotherapy for longitudinal comparison following a protocol that carries sufficient power for effective discovery proteomics. Plasma samples from patients pre- and during radiotherapy who had survived > 18 mo were compared to the same time points from patients who survived < 14 mo using an 8 channel isobaric tagging tandem mass spectrometry discovery proteomics platform. Over 650 proteins were detected and relatively quantified. Proteins which showed a change during radiotherapy were selected for validation using an orthogonal antibody-based approach. Two of these proteins were verified in a separate patient cohort: values of CRP and LRG1 combined gave a highly significant indication of extended survival post one week of radiotherapy treatment.

Diagnostic Mutation Profiling and Validation of Non-Small-Cell Lung Cancer Small Biopsy Samples using a High Throughput Platform.

BACKGROUND: A single platform designed for the synchronous screening of multiple mutations can potentially enable molecular profiling in samples of limited tumor tissue. This approach is ideal for the assessment of advanced non-small-cell lung cancer (NSCLC) diagnostic specimens, which often comprise small biopsies. Therefore, we aimed in this study to validate the mass spectrometry-based Sequenom LungCarta panel and MassARRAY platform using DNA extracted from a single 5 µM formalin-fixed paraffin-embedded tissue section. METHODS: Mutations, including those with an equivocal spectrum, detected in 90 cases of NSCLC (72 lung biopsies, 13 metastatic tissue biopsies, three resections, and two cytology samples) were validated by a combination of standard sequencing techniques, immunohistochemical staining for p53 protein, and next-generation sequencing with the TruSight Tumor panel. RESULTS: Fifty-five mutations were diagnosed in 47 cases (52%) in the following genes: TP53 (22), KRAS (15), EGFR (5), MET (3), PIK3CA (3), STK11 (2), NRF-2 (2), EPHA5 (1), EPHA3 (1), and MAP2K1 (1). Of the 90 samples, one failed testing due to poor quality DNA. An additional 7 TP53 mutations were detected by next-generation sequencing, which facilitated the interpretation of p53 immunohistochemistry but required 5 × 10 µM tumor sections per sample tested. CONCLUSIONS: The LungCarta panel is a sensitive method of screening for multiple alterations (214 mutations across 26 genes) and which optimizes the use of limited amounts of tumor DNA isolated from small specimens.

Lungscape: resected non-small-cell lung cancer outcome by clinical and pathological parameters.

INTRODUCTION: The Lungscape project was designed to address the impact of clinical, pathological, and molecular characteristics on outcome in resected non-small- cell lung cancer (NSCLC). MATERIALS AND METHODS: A decentralized biobank with fully annotated tissue samples was established. Selection criteria for participating centers included sufficient number of cases, tissue microarray building capability, and documented ethical approval. Patient selection was based on availability of comprehensive clinical data, radical resection between 2003 and 2009 with adequate follow-up, and adequate quantity and quality of formalin-fixed tissue. RESULTS: Fifteen centers contributed 2449 cases. The 5-year overall survival (OS) was 69.6% and 63.6% for stages IA and IB, 51.6% and 47.7% for stages IIA and IIB, and 29.0% and 13.0% for stages IIIA and IIIB, respectively (p < 0.001). Median and 5-year relapse-free survival (RFS) were 52.8 months and 47.3%, respectively. Distant relapse was recorded for 44.4%, local for 26.0%, and both for 16.9% of patients. Based on multivariate analysis for the OS, RFS, and time to relapse, the factors significantly associated with all of them are performance status and pathological stage. CONCLUSION: The aim of this report is to present the results from Lungscape, the first large series reporting on NSCLC surgical outcome measured not only by OS but also by RFS and time to relapse and including multivariate analysis by significant clinical and pathological prognostic parameters. As tissue from all patients is preserved locally and is available for detailed molecular investigations, Lungscape provides an excellent basis to evaluate the influence of molecular parameters on the disease outcome after radical resection, besides providing an overview of the molecular landscape of stage I to III NSCLC.

Prevalence and clinical outcomes for patients with ALK-positive resected stage I to III adenocarcinoma: results from the European Thoracic Oncology Platform Lungscape Project.

PURPOSE: The prevalence of anaplastic lymphoma kinase (ALK) gene fusion (ALK positivity) in early-stage non-small-cell lung cancer (NSCLC) varies by population examined and detection method used. The Lungscape ALK project was designed to address the prevalence and prognostic impact of ALK positivity in resected lung adenocarcinoma in a primarily European population. METHODS: Analysis of ALK status was performed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) in tissue sections of 1,281 patients with adenocarcinoma in the European Thoracic Oncology Platform Lungscape iBiobank. Positive patients were matched with negative patients in a 1:2 ratio, both for IHC and for FISH testing. Testing was performed in 16 participating centers, using the same protocol after passing external quality assessment. RESULTS: Positive ALK IHC staining was present in 80 patients (prevalence of 6.2%; 95% CI, 4.9% to 7.6%). Of these, 28 patients were ALK FISH positive, corresponding to a lower bound for the prevalence of FISH positivity of 2.2%. FISH specificity was 100%, and FISH sensitivity was 35.0% (95% CI, 24.7% to 46.5%), with a sensitivity value of 81.3% (95% CI, 63.6% to 92.8%) for IHC 2+/3+ patients. The hazard of death for FISH-positive patients was lower than for IHC-negative patients (P = .022). Multivariable models, adjusted for patient, tumor, and treatment characteristics, and matched cohort analysis confirmed that ALK FISH positivity is a predictor for better overall survival (OS). CONCLUSION: In this large cohort of surgically resected lung adenocarcinomas, the prevalence of ALK positivity was 6.2% using IHC and at least 2.2% using FISH. A screening strategy based on IHC or H-score could be envisaged. ALK positivity (by either IHC or FISH) was related to better OS.

Molecular analysis of circulating tumour cells-biology and biomarkers.

Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test-and readily amenable to serial sampling-have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch(®) system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial-mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.

Variant Ciz1 is a circulating biomarker for early-stage lung cancer.

There is an unmet need for circulating biomarkers that can detect early-stage lung cancer. Here we show that a variant form of the nuclear matrix-associated DNA replication factor Ciz1 is present in 34/35 lung tumors but not in adjacent tissue, giving rise to stable protein quantifiable by Western blot in less than a microliter of plasma from lung cancer patients. In two independent sets, with 170 and 160 samples, respectively, variant Ciz1 correctly identified patients who had stage 1 lung cancer with clinically useful accuracy. For set 1, mean variant Ciz1 level in individuals without diagnosed tumors established a threshold that correctly classified 98% of small cell lung cancers (SCLC) and non-SCLC patients [receiver operator characteristic area under the curve (AUC) 0.958]. Within set 2, comparison of patients with stage 1 non-SCLC with asymptomatic age-matched smokers or individuals with benign lung nodules correctly classified 95% of patients (AUCs 0.913 and 0.905), with overall specificity of 76% and 71%, respectively. Moreover, using the mean of controls in set 1, we achieved 95% sensitivity among patients with stage 1 non-SCLC patients in set 2 with 74% specificity, demonstrating the robustness of the classification. RNAi-mediated selective depletion of variant Ciz1 is sufficient to restrain the growth of tumor cells that express it, identifying variant Ciz1 as a functionally relevant driver of cell proliferation in vitro and in vivo. The data show that variant Ciz1 is a strong candidate for a cancer-specific single marker capable of identifying early-stage lung cancer within at-risk groups without resort to invasive procedures.

Liquid chromatography-mass spectrometry calibration transfer and metabolomics data fusion.

Metabolic profiling is routinely performed on multiple analytical platforms to increase the coverage of detected metabolites, and it is often necessary to distribute biological and clinical samples from a study between instruments of the same type to share the workload between different laboratories. The ability to combine metabolomics data arising from different sources is therefore of great interest, particularly for large-scale or long-term studies, where samples must be analyzed in separate blocks. This is not a trivial task, however, due to differing data structures, temporal variability, and instrumental drift. In this study, we employed blood serum and plasma samples collected from 29 subjects diagnosed with small cell lung cancer and analyzed each sample on two liquid chromatography-mass spectrometry (LC-MS) platforms. We describe a method for mapping retention times and matching metabolite features between platforms and approaches for fusing data acquired from both instruments. Calibration transfer models were developed and shown to be successful at mapping the response of one LC-MS instrument to another (Procrustes dissimilarity = 0.04; Mantel correlation = 0.95), allowing us to merge the data from different samples analyzed on different instruments. Data fusion was assessed in a clinical context by comparing the correlation of each metabolite with subject survival time in both the original and fused data sets: a simple autoscaling procedure (Pearson's R = 0.99) was found to improve upon a calibration transfer method based on partial least-squares regression (R = 0.94).

Understanding cough and its management in lung cancer.

PURPOSE OF REVIEW: This article summarizes the current understanding of cough in lung cancer, strategies for its management and highlights areas where further research is warranted.Cough is common, severe and distressing for many lung cancer patients. Currently few effective cough interventions exist for lung cancer patients. This review proposes some of the mechanisms that may underlie cough in lung cancer and presents the existing data on antitussive therapy in cancer patients. A greater focus on the cough mechanisms may enable effective antitussives to be developed in the future for lung cancer patients. A brief overview of the validated cough assessment tools is provided. The use of such tools will enable robust clinical endpoints to be determined for future cough intervention studies. A 'cough treatment pyramid' is presented to provide a pragmatic approach to the management of cough in lung cancer patients. RECENT FINDINGS: Despite the small number of publications on cough in lung cancer, some recent research has characterized cough for the first time in lung cancer patients. Its impact on quality of life domains such as psychological, social and physical is significant. The lung cancer symptom cluster of cough, breathlessness and fatigue has also been described. A recently developed cough severity assessment tool has provided researchers with a short well validated seven-item questionnaire to determine this important cough characteristic. In addition, a Cochrane Database Systematic Review on Interventions for Cough in Cancer has also been published. These studies are all described in the present review. Understanding the impact of a symptom such as cough, the assessment of cough and its potential underlying mechanisms is crucial if we are to manage this symptom effectively. SUMMARY: Current cough management in lung cancer patients is lagging behind the management of other cancer symptoms. There is now an increasing need to diagnose and treat cough more effectively, as lung cancer patients are living longer with chronic symptoms such as cough.

Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer.

PURPOSE: Circulating tumor cells (CTCs) may have utility as surrogate biomarkers and "virtual" biopsies. We report the clinical significance and molecular characteristics of CTCs and CTC clusters, termed circulating tumor microemboli (CTM), detected in patients with small-cell lung cancer (SCLC) undergoing standard treatment. PATIENTS AND METHODS: Serial blood samples from 97 patients receiving chemotherapy were analyzed using EpCam-based immunomagnetic detection and a filtration-based technique. Proliferation status (Ki67) and apoptotic morphology were examined. Associations of CTC and CTM number with clinical factors and prognosis were determined. RESULTS: CTCs were present in 85% of patients (77 of 97 patients) and were abundant (mean ± standard deviation = 1,589 ± 5,565). CTM and apoptotic CTCs were correlated with total CTC number and were detected in 32% and 57% of patients, respectively. Pretreatment CTCs, change in CTC number after one cycle of chemotherapy, CTM, and apoptotic CTCs were independent prognostic factors. Overall survival was 5.4 months for patients with ≥ 50 CTCs/7.5 mL of blood and 11.5 months (P < .0001) for patients with less than 50 CTCs/7.5 mL of blood before chemotherapy (hazard ratio = 2.45; 95% CI, 1.39 to 4.30; P = .002). Subpopulations of apoptotic and of proliferating solitary CTCs were detected, whereas neither were observed within cell clusters (CTM), implicating both protection from anoikis and relative resistance to cytotoxic drugs for cells within CTM. CONCLUSION: Both baseline CTC number and change in CTC number after one cycle of chemotherapy are independent prognostic factors for SCLC. Molecular comparison of CTCs to cells in CTM may provide novel insights into SCLC biology.

Circulating tumour cells, their role in metastasis and their clinical utility in lung cancer.

Circulating tumour cells (CTCs) have attracted much recent interest in cancer research as a potential biomarker and as a means of studying the process of metastasis. It has long been understood that metastasis is a hallmark of malignancy, and conceptual theories on the basis of metastasis from the nineteenth century foretold the existence of a tumour "seed" which is capable of establishing discrete tumours in the "soil" of distant organs. This prescient "seed and soil" hypothesis accurately predicted the existence of CTCs; microscopic tumour fragments in the blood, at least some of which are capable of forming metastases. However, it is only in recent years that reliable, reproducible methods of CTC detection and analysis have been developed. To date, the majority of studies have employed the CellSearch™ system (Veridex LLC), which is an immunomagnetic purification method. Other promising techniques include microfluidic filters, isolation of tumour cells by size using microporous polycarbonate filters and flow cytometry-based approaches. While many challenges still exist, the detection of CTCs in blood is becoming increasingly feasible, giving rise to some tantalizing questions about the use of CTCs as a potential biomarker. CTC enumeration has been used to guide prognosis in patients with metastatic disease, and to act as a surrogate marker for disease response during therapy. Other possible uses for CTC detection include prognostication in early stage patients, identifying patients requiring adjuvant therapy, or in surveillance, for the detection of relapsing disease. Another exciting possible use for CTC detection assays is the molecular and genetic characterization of CTCs to act as a "liquid biopsy" representative of the primary tumour. Indeed it has already been demonstrated that it is possible to detect HER2, KRAS and EGFR mutation status in breast, colon and lung cancer CTCs respectively. In the course of this review, we shall discuss the biology of CTCs and their role in metastagenesis, the most commonly used techniques for their detection and the evidence to date of their clinical utility, with particular reference to lung cancer.

Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches.

INTRODUCTION: Epithelial circulating tumor cells (CTCs) are detectable in patients with non-small cell lung cancer (NSCLC). However, epithelial to mesenchymal transition, a widely reported prerequisite for metastasis, may lead to an underestimation of CTC number. We compared directly an epithelial marker-dependent (CellSearch) and a marker-independent (isolation by size of epithelial tumor cells [ISET]) technology platform for the ability to identify CTCs. Molecular characteristics of CTCs were also explored. METHODS: Paired peripheral blood samples were collected from 40 chemonäive, stages IIIA to IV NSCLC patients. CTCs were enumerated by Epithelial Cell Adhesion Molecule-based immunomagnetic capture (CellSearch, Veridex) and by filtration (ISET, RareCell Diagnostics). CTCs isolated by filtration were assessed by immunohistochemistry for epithelial marker expression (cytokeratins, Epithelial Cell Adhesion Molecule, epidermal growth factor receptor) and for proliferation status (Ki67). RESULTS: CTCs were detected using ISET in 32 of 40 (80%) patients compared with 9 of 40 (23%) patients using CellSearch. A subpopulation of CTCs isolated by ISET did not express epithelial markers. Circulating tumor microemboli (CTM, clusters of ≥ 3 CTCs) were observed in 43% patients using ISET but were undetectable by CellSearch. Up to 62% of single CTCs were positive for the proliferation marker Ki67, whereas cells within CTM were nonproliferative. CONCLUSIONS: Both technology platforms detected NSCLC CTCs. ISET detected higher numbers of CTCs including epithelial marker negative tumor cells. ISET also isolated CTM and permitted molecular characterization. Combined with our previous CellSearch data confirming CTC number as an independent prognostic biomarker for NSCLC, we propose that this complementary dual technology approach to CTC analysis allows more complete exploration of CTCs in patients with NSCLC.

Targeted agents in non-small cell lung cancer (NSCLC): clinical developments and rationale for the combination with thoracic radiotherapy.

In recent years there has been undoubted progress in the evaluation and development of targeted agents for non-small cell lung cancer (NSCLC). A major contributor has been the discovery of molecular subtypes harbouring a critical oncogenic driver mutation, specifically sensitizing mutations in the epidermal growth factor receptor (EGFR) gene and the EML4-ALK gene translocation. Radiotherapy is a cornerstone of therapy for the curative intent treatment of early stage, localized disease; and for the palliation of symptoms in advanced, metastatic disease. In this molecular targeted era there is limited understanding of how best to combine targeted agents with radiotherapy and in general clinical studies with radiotherapy have lagged behind studies of targeted agents with chemotherapy. Here we summarise the progress made to date and highlight future directions.