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Supplementary Materials and Methods; Supplementary Tables S1-S3.

Purpose: Blood-based surrogate markers would be attractive biomarkers for early detection, diagnosis, prognosis, and prediction of therapeutic outcome in cancer. Disease-associated gene expression signatures in peripheral blood mononuclear cells (PBMC) have been described for several cancer types. However, RNA-stabilized whole blood–based technologies would be clinically more applicable and robust. We evaluated the applicability of whole blood–based gene expression profiling for the detection of non–small cell lung cancer (NSCLC).Experimental Design: Expression profiles were generated from PAXgene-stabilized blood samples from three independent groups consisting of NSCLC cases and controls (n = 77, 54, and 102), using the Illumina WG6-VS2 system.Results: Several genes are consistently differentially expressed in whole blood of NSCLC patients and controls. These expression profiles were used to build a diagnostic classifier for NSCLC, which was validated in an independent validation set of NSCLC patients (stages I–IV) and hospital-based controls. The area under the receiver operator curve was calculated to be 0.824 (P < 0.001). In a further independent dataset of stage I NSCLC patients and healthy controls the AUC was 0.977 (P < 0.001). Specificity of the classifier was validated by permutation analysis in both validation cohorts. Genes within the classifier are enriched in immune-associated genes and show specificity for NSCLC.Conclusions: Our results show that gene expression profiles of whole blood allow for detection of manifest NSCLC. These results prompt further development of gene expression–based biomarker tests in peripheral blood for the diagnosis and early detection of NSCLC. Clin Cancer Res; 17(10); 3360–7. ©2011 AACR.

Dual blockade of the EGFR and VEGF pathways in EGFR-mutated metastatic non-small-cell lung cancer (NSCLC) is supported by preclinical and clinical data, yet the approach is not widely implemented. RELAY assessed erlotinib, an EGFR tyrosine kinase inhibitor (TKI) standard of care, plus ramucirumab, a human IgG1 VEGFR2 antagonist, or placebo in patients with untreated EGFR-mutated metastatic NSCLC.This is a worldwide, double-blind, phase 3 trial done in 100 hospitals, clinics, and medical centres in 13 countries. Eligible patients were aged 18 years or older (20 years or older in Japan and Taiwan) at the time of study entry, had stage IV NSCLC, with an EGFR exon 19 deletion (ex19del) or exon 21 substitution (Leu858Arg) mutation, an Eastern Cooperative Oncology Group performance status of 0 or 1, and no CNS metastases. We randomly assigned eligible patients in a 1:1 ratio to receive oral erlotinib (150 mg/day) plus either intravenous ramucirumab (10 mg/kg) or matching placebo once every 2 weeks. Randomisation was done by an interactive web response system with a computer-generated sequence and stratified by sex, geographical region, EGFR mutation type, and EGFR testing method. The primary endpoint was investigator-assessed progression-free survival in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study treatment. This trial is registered at ClinicalTrials.gov, NCT02411448, and is ongoing for long-term survival follow-up.Between Jan 28, 2016, and Feb 1, 2018, 449 eligible patients were enrolled and randomly assigned to treatment with ramucirumab plus erlotinib (n=224) or placebo plus erlotinib (n=225). Median duration of follow-up was 20·7 months (IQR 15·8-27·2). At the time of primary analysis, progression-free survival was significantly longer in the ramucirumab plus erlotinib group (19·4 months [95% CI 15·4-21·6]) than in the placebo plus erlotinib group (12·4 months [11·0-13·5]), with a stratified hazard ratio of 0·59 (95% CI 0·46-0·76; p0·0001). Grade 3-4 treatment-emergent adverse events were reported in 159 (72%) of 221 patients in the ramucirumab plus erlotinib group versus 121 (54%) of 225 in the placebo plus erlotinib group. The most common grade 3-4 treatment-emergent adverse events in the ramucirumab plus erlotinib group were hypertension (52 [24%]; grade 3 only) and dermatitis acneiform (33 [15%]), and in the placebo plus erlotinib group were dermatitis acneiform (20 [9%]) and increased alanine aminotransferase (17 [8%]). Treatment-emergent serious adverse events were reported in 65 (29%) of 221 patients in the ramucirumab plus erlotinib group and 47 (21%) of 225 in the placebo plus erlotinib group. The most common serious adverse events of any grade in the ramucirumab plus erlotinib group were pneumonia (seven [3%]) and cellulitis and pneumothorax (four [2%], each); the most common in the placebo plus erlotinib group were pyrexia (four [2%]) and pneumothorax (three [1%]). One on-study treatment-related death due to an adverse event occurred (haemothorax after a thoracic drainage procedure for a pleural empyema) in the ramucirumab plus erlotinib group.Ramucirumab plus erlotinib demonstrated superior progression-free survival compared with placebo plus erlotinib in patients with untreated EGFR-mutated metastatic NSCLC. Safety was consistent with the safety profiles of the individual compounds in advanced lung cancer. The RELAY regimen is a viable new treatment option for the initial treatment of EGFR-mutated metastatic NSCLC.Eli Lilly.

PURPOSE Third-generation epidermal growth factor receptor ( EGFR) tyrosine kinase inhibitors (TKIs) are effective in acquired resistance (AR) to early-generation EGFR TKIs in EGFR-mutant lung cancer. However, efficacy is marked by interindividual heterogeneity. We present the molecular profiles of pretreatment and post-treatment samples from patients treated with third-generation EGFR TKIs and their impact on treatment outcomes. METHODS Using the databases of two lung cancer networks and two lung cancer centers, we molecularly characterized 124 patients with EGFR p.T790M-positive AR to early-generation EGFR TKIs. In 56 patients, correlative analyses of third-generation EGFR TKI treatment outcomes and molecular characteristics were feasible. In addition, matched post-treatment biopsy samples were collected for 29 patients with progression to third-generation EGFR TKIs. RESULTS Co-occurring genetic aberrations were found in 74.4% of EGFR p.T790-positive samples (n = 124). Mutations in TP53 were the most frequent aberrations detected (44.5%; n = 53) and had no significant impact on third-generation EGFR TKI treatment. Mesenchymal-epithelial transition factor ( MET) amplifications were found in 5% of samples (n = 6) and reduced efficacy of third-generation EGFR TKIs significantly (eg, median progression-free survival, 1.0 months; 95% CI, 0.37 to 1.72 v 8.2 months; 95% CI, 1.69 to 14.77 months; P ≤ .001). Genetic changes in the 29 samples with AR to third-generation EGFR TKIs were found in EGFR (eg, p.T790M loss, acquisition of p.C797S or p.G724S) or in other genes (eg, MET amplification, KRAS mutations). CONCLUSION Additional genetic aberrations are frequent in EGFR-mutant lung cancer and may mediate innate and AR to third-generation EGFR TKIs. MET amplification was strongly associated with primary treatment failure and was a common mechanism of AR to third-generation EGFR TKIs. Thus, combining EGFR inhibitors with TKIs targeting common mechanisms of resistance may delay AR.

Zusammenfassung Einführung Nicht kleinzellige Lungenkarzinome (engl. non-small cell lung cancer = NSCLC) im Stadium III und ihre mögliche multimodale Therapie stellen eine Herausforderung für die behandelnden Onkologen, Thoraxchirurgen und Strahlentherapeuten dar. Ziel dieser retrospektiven Studie war die Analyse und Evaluation des Therapiealgorithmus unserer Klinik bei Patienten mit NSCLC im Stadium III (intention to treat). Es sollte ermittelt werden, ob sich ein aggressives Therapieregime mit primärer trimodaler Therapie (Hochdosisradiochemotherapie und Resektion) unabhängig von einem „multilevel“-N2- oder „single-level“-N3-Status: Diese Resultate wurden anschließend mit einem historischen Kollektiv unserer Patienten verglichen, die eine alleinige simultane Radio-/Chemotherapie erhielten (= bimodale Therapie). Material und Methoden 156 Patienten wurden in dem Erhebungszeitraum mit einem NSCLC Stadium III diagnostiziert und einer trimodalen Therapie zugeführt. Das mediane Alter war 71 Jahre, 103 Patienten (60%) waren männlich, 53 (34%) waren weiblich. In der Gruppe mit bimodaler Therapie konnten 102 Patienten ausgewertet werden. Ergebnisse Nach radiologischem Restaging und Überprüfung der funktionellen Resektabilität konnten 90 Patienten (57,7%) aus der trimodalen Therapiegruppe einer sekundären Resektion zugeführt werden. Hierbei wurden 37 (41,1%) Lobektomien/Bilobektomien, 37 (41,1%) Sleeve-Lobektomien, 13 (14,4%) Pneumonektomien und 3 (3,3%) Segmentektomien (bei schwerer lungenfunktioneller Einschränkung) durchgeführt. Das mediane Überleben in der trimodalen Therapiegruppe betrug 535 Tage, in der bimodalen Therapiegruppe hingegen 388 Tage, der Unterschied war statistisch nicht signifikant (p = 0,1377). Abschließend wurde eine Analyse der 5-Jahres-Überlebensrate nach tatsächlicher Therapie durchgeführt („as-treated trimodally“ vs. „as-treated bimodally“). Hier betrug das mediane Überleben 807 Tage für Patienten, die eine trimodale Therapie erhielten, 427 Tage für Patienten die eine bimodale Therapie erhielten. Schlussfolgerung Eine hochdosierte neoadjuvante Radiochemotherapie gefolgt von einer sekundären Resektion stellt weiterhin eine wertvolle Option für selektionierte Patienten in der Behandlung des NSCLC im Stadium III dar. Diese retrospektive Analyse konnte jedoch einen statistisch signifikanten Überlebensvorteil für die „intention-to-treat-trimodally“-Patienten nicht nachweisen.

Background Large-cell neuroendocrine carcinoma of the lung (LCNEC) is a rare disease with poor prognosis and limited treatment options. Neuroendocrine tumors frequently show overactivation of the mTOR pathway. Based on the good activity of the mTOR inhibitor everolimus in different types of neuroendocrine tumors and the results of a previous phase I trial, we evaluated the efficacy and safety of everolimus in combination with carboplatin and paclitaxel as upfront treatment for patients with advanced LCNEC. Patients and methods In this prospective, multicenter phase II trial chemotherapy-naive patients with stage IV LCNEC received 5 mg everolimus daily combined with paclitaxel 175 mg/m2 and carboplatin AUC 5 every 3 weeks for a maximum of four cycles followed by maintenance everolimus 5 mg daily until progression. Efficacy parameters were determined based on central radiologic assessment. Results Forty-nine patients with a mean age of 62 ±9 years and a predominance of male (71%) smokers (98%) were enrolled in 10 German centers. The overall response rate was 45% (95% confidence interval [CI] 31%-60%), the disease control rate 74% (CI 59%-85%), the median progression-free survival 4.4 (CI 3.2-6) months and the median overall survival 9.9 (CI 6.9-11.7) months. The progression-free survival rate at 3 months (primary end point) was 76% (CI 64%-88%) according to Kaplan-Meier. Grade-3/4 toxicities occurred in 51% of patients and mainly consisted of general physical health deterioration (8%), cytopenias (24%), infections (10%) and gastrointestinal problems (8%). Typical everolimus-related adverse events, like stomatitis, rash and ocular problems occurred only in a minority of patients (

Concurrent chemoradiotherapy (CRT) is considered standard for inoperable stage III non-small cell lung cancer (NSCLC). Consolidation chemotherapy (CC) following CRT is intended to further improve outcomes, yet studies have shown discordant results. This phase III study assessed CRT followed by best supportive care (BSC) or consolidation with oral vinorelbine and cisplatin.Patients received two cycles of oral vinorelbine (50 mg/m(2) days 1, 8 and 15) + cisplatin (20 mg/m(2) days 1-4) q4w + radiotherapy (RT; 66 Gy). Patients with at least stable disease (SD) were randomised to either two cycles oral vinorelbine (60-80 mg/m(2) days 1 and 8) + cisplatin (80 mg/m(2) day 1) q3w + BSC or BSC alone. Primary endpoint was progression-free survival (PFS).A total of 279 patients were enrolled for CRT and 201 patients were randomised to CC or BSC. Both CRT and CC were well tolerated, with limited radiation-mediated grade 3/4 toxicities (CRT/CC/BSC: oesophagitis-related events 12.9 %/3.1 %/0 %; grade 3 pneumonitis 0 %/0 %/2 %) and chemotherapy-mediated grade 3/4 toxicities (CRT/CC: neutropenia 11.2 %/22.1 %; leukopenia 18.3 %/26.7 %; grade 3 nausea 5.0 %/2.3 %, grade 3 vomiting 3.2 %/3.5 %). Median PFS from randomisation was 6.4 (5.0-8.7) and 5.5 (3.8-7.4) months in the CC and BSC arms (hazard ratio, HR = 0.93 [0.69-1.26]; p = 0.63), respectively; median overall survival (OS) 20.8 (13.5-25.3) and 18.5 (13.6-24.7) months, respectively.Consolidation chemotherapy after concurrent CRT did not prolong PFS or OS. Concurrent RT with oral vinorelbine and cisplatin demonstrated a favourable safety profile and represents a suitable treatment regimen for inoperable stage III NSCLC.

The emergence of acquired resistance against targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. In a subgroup of these patients we identified an association between selection of EGFRT790M-negative but EGFRG724S-positive subclones and osimertinib resistance. We demonstrate that EGFRG724S limits the activity of third-generation EGFR inhibitors both in vitro and in vivo. Structural analyses and computational modeling indicate that EGFRG724S mutations may induce a conformation of the glycine-rich loop, which is incompatible with the binding of third-generation TKIs. Systematic inhibitor screening and in-depth kinetic profiling validate these findings and show that second-generation EGFR inhibitors retain kinase affinity and overcome EGFRG724S-mediated resistance. In the case of afatinib this profile translates into a robust reduction of colony formation and tumor growth of EGFRG724S-driven cells. Our data provide a mechanistic basis for the osimertinib-induced selection of EGFRG724S-mutant clones and a rationale to treat these patients with clinically approved second-generation EGFR inhibitors., Acquired resistance to targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. Here, the authors show how the acquired EGFRG724S mutation induces resistance to third-generation EGFR inhibitors and why the mutant kinase remains susceptible to second-generation inhibitors.

Purpose: MET is a potential therapeutic target in lung cancer and both MET tyrosine kinase inhibitors and monoclonal antibodies have entered clinical trials. MET signaling can be activated by various mechanisms, including gene amplification. In this study, we aimed to investigate MET amplification status in adeno- and squamous cell carcinomas of the lung. We propose clearly defined amplification scores and provide epidemiologic data on MET amplification in lung cancer. Experimental Design: We evaluated the prevalence of increased MET gene copy numbers in 693 treatment-naïve cancers by FISH, defined clear cutoff criteria, and correlated FISH results to MET IHC. Results: Two thirds (67%) of lung cancers do not have gains in MET gene copy numbers, whereas 3% show a clear-cut high-level amplification (MET/centromer7 ratio ≥2.0 or average gene copy number per nucleus ≥6.0 or ≥10% of tumor cells containing ≥15 MET copies). The remaining cases can be subdivided into intermediate- (6%) and low-level gains (24%). Importantly, MET amplifications occur at equal frequencies in squamous and adenocarcinomas without or with EGFR or KRAS mutations. Conclusion: MET amplification is not a mutually exclusive genetic event in therapy-naïve non–small cell lung cancer. Our data suggest that it might be useful to determine MET amplification (i) before EGFR inhibitor treatment to identify possible primary resistance to anti-EGFR treatment, and (ii) to select cases that harbor KRAS mutations additionally to MET amplification and, thus, may not benefit from MET inhibition. Furthermore, our study provides comprehensive epidemiologic data for upcoming trials with various MET inhibitors. Clin Cancer Res; 21(4); 907–15. ©2014 AACR.

This study was performed to evaluate for a potentially important role of T cells in the pathophysiology and treatment sensitivity of large cell neuroendocrine lung carcinoma (LCNEC), an orphan disease with poor prognosis and scarce data to guide novel therapeutic strategies.We performed T-cell receptor (TCR) β-chain spectratyping on blood samples of patients treated within the CRAD001KDE37 trial (n = 35) using age-matched current or former (n = 11) and never smokers (n = 10) as controls. The data were analyzed in conjunction with the complete blood counts of the probands as well as the data about response to treatment and overall survival in the clinical trial.Untreated stage IV LCNEC patients had significant T-cell repertoire alterations (p 0.001) compared to age-matched smokers. These changes correlated positively with blood lymphocyte counts (r = 0.49, p 0.01), suggesting antigen-induced T-cell proliferation as the causative mechanism. At the same time, LCNEC patients showed mild lymphopenia (1.54 vs. 2.51/nl in median, p 0.01), which reveals a second, antigen-independent mechanism of systemic immune dysregulation. More pronounced T-cell repertoire alterations and higher blood lymphocyte counts at diagnosis were associated with a better treatment response by RECIST and with a longer overall survival (441 vs. 157 days in median, p = 0.019). A higher degree of T-cell repertoire normalization after 3 months of therapy also distinguished a patient group with more favourable prognosis (median overall survival 617 vs. 316 days, p = 0.036) independent of radiological response. Thus, LCNEC induces clinically relevant changes of the T-cell repertoire, which are measurable in the blood and could be exploited for prognostic, predictive and therapeutic purposes. Their pathogenesis appears to involve antigen-induced oligoclonal T-cell expansions superimposed on TCR-independent lymphopenia.

Wolfgang Schuette,1 Claus-Peter Schneider,2,3 Walburga Engel-Riedel,4 Christian Schumann,5,6 Martin Kohlhaeufl,7 Monika Heidi Ursel Serke,8 Gert Hoeffken,9 Cornelius Kortsik,10 Martin Reck11 1Department of Internal MedicineII, Hospital Martha-Maria Halle-Doelau, Halle, 2Department of Pneumonology, Central Hospital, Bad Berka, 3Department of Internal Medicine, DRK Manniske Hospital, Bad Frankenhausen, 4Cologne Clinics, Merheim Lung Hospital, Cologne, 5Pneumonology, Department of Internal MedicineII, University Hospital Ulm, 6Clinic for Pneumology, Thoracic Oncology, Sleep, and Respiratory Critical Care, Kempten-Oberallgäu, 7Center of Pneumonology and Chest Surgery, Hospital Schillerhoehe, Gerlingen, 8Pneumonology, Lung Clinics, Hemer, 9Center of Pneumonology, Chest and Vascular Surgery, Specialty Hospital Coswig, Coswig, 10Department of Pneumonology, Catholic Hospital, Mainz, 11Department of Thoracic Oncology, Lung Clinic, Grosshansdorf, Germany Background: The aim of the study was to investigate in terms of noninferiority the efficacy and safety of a monochemotherapy regimen of pemetrexed plus bevacizumab (BevPem) versus carboplatin/pemetrexed plus bevacizumab (BevCPem) in elderly patients as first-line treatment for advanced metastatic or recurrent nonsquamous non-small-cell lung cancer (NSCLC).Materials and methods: 65Plus was a Phase III, randomized, open-label study. In total, 253 patients received BevPem (n=119) or BevCPem (n=134). The primary outcome measure was progression-free survival. Secondary end points were overall survival, tumor response, and safety outcomes. Evaluations were performed for the whole study population and stratified according to Eastern Cooperative Oncology Group (ECOG) performance status (PS).Results: Noninferiority of BevPem in comparison to BevCPem could not be demonstrated for the overall population (P=0.7864). Significant superiority of the combined treatment BevCPem was seen in patients of ECOG PS 0–1 (median PFS 5.1 vs 6.9 months, HR 1.353, 95% CI 1.03–1.777), while the opposite tendency was observed in patients with ECOG PS 2 (median PFS 2.9 vs 1.5 months, HR 0.628, 95% CI 0.195–2.025). Overall, better tolerability was found for the BevPem group, irrespective of ECOG PS.Conclusion: Results from the 65plus study give evidence that BevPem and BevCPem treatments may exert differential effects on PFS, depending on the patients ECOG PS. It appears that patients with better ECOG PS (0–1) benefited more from the combined treatment with carboplatin, while the group comprising more severely impaired patients (ECOG PS 2) benefited more from the monochemotherapy. Keywords: bevacizumab, pemetrexed, carboplatin, NSCLC, elderly patients

Oncotarget 6(2), 1315-1326 (2015). doi:10.18632/oncotarget.2834, Published by Impact Journals LLC, [S.l.]

e20550 Background: Patient selection, dosing regimens and resistance mechanisms for immune checkpoint inhibitor combination therapy remain unmet medical needs in lung cancer. Combining blockade of PD-1 and CTLA-4 can be more effective than monotherapy but is accompanied by an increase in toxicity. Thus, to circumvent unnecessary toxicity it is of great interest to identify patients who will benefit from PD-1/PD-L1 blockade alone and to add ipilimumab only in case of primary or secondary progression. We present interim data from the non-small-cell lung cancer (NSCLC) cohort of the ongoing BIOLUMA trial which evaluates efficacy and safety of nivolumab and ipilimumab in lung cancer with a broad translational program to identify potential biomarkers predictive of response and/or resistance including whole exome sequencing (WES) of serial biopsies, functional analysis of peripheral T-cells and gut microbiome analyses. Methods: BIOLUMA is a multicentre non-randomised phase II trial in 2nd line patients with non-squamous NSCLC. Patients are treated with nivolumab 240 mg until disease progression and subsequently with a combination therapy of nivolumab 3 mg/kg q2w and ipilimumab 1mg/kg q6w. Primary endpoint is overall response rate (ORR) after addition of ipilimumab to nivolumab treatment. Analysis of sequential tumor biopsies, blood and gut microbiome is performed at different timepoints. Results: To date, 26 patients have been enrolled and 9 patients were transferred to the combination therapy after progression on nivolumab monotherapy. Drop out rate between the treatment arms is high, mainly due to rapid disease progression and adverse events which don’t allow addition of ipilimumab. ORR is available for 8 of these patients: 6 patients (75%) had PD as best response, and 1 (12.5%) each had a stable disease and partial response, respectively. The patient who achieved a PR had experienced primary tumor progression on nivolumab monotherapy before. Toxicity rate was similar to what has been reported from other trials. Conclusions: In NSCLC, addition of ipilimumab to nivolumab in nivolumab refractory patients seems to be safe, but the response rate is low and the drop out between the treatment parts high. Given these data, early termination of this cohort is currently discussed. Clinical trial information: NCT03083691.

Background:EGFR sequencing is crucial for therapeutic decisions in treatment regimens of lung cancer patients. Several mutations have been identified in the past, of which some were confirmed as activating or resistance mutations by in vitro phenotyping. In this study, novel mutations of so far unknown relevance were identified by a combination of Sanger and Pyrosequencing®. Materials & methods: Formalin-fixed paraffin-embedded lung biopsies from 20 randomly selected patients suffering from non-small-cell lung cancer with previous external EGFR mutation analyses were reanalyzed by Sanger sequencing according to a previous study, and Pyrosequencing with the EGFR Pyro Kit (Qiagen, Hilden, Germany). Sensitivity and specificity were determined in comparison with the results of an external supplier for all relevant mutations in exons 18, 19, 20 and 21. Results: Our analyses revealed that Pyrosequencing is faster and more sensitive for the common mutations compared with Sanger sequencing and the results of the external supplier. A new mutation, c.2160delC, in exon 18 in 40% of patients leading to a frameshift was identified. Another two frameshift mutations were detected in exon 18 in 10% of patients; c.2168delT in combination with c.2160delC, and c.2163insG alone. Conclusion: Divergences regarding the detection of the common mutations could be traced back to inhomogeneous or insufficient tumor material. Surprisingly, none of the newly identified mutations have been previously described, although they occurred in total in up to 40% of randomly selected cases. A possible explanation may be that commercial assays did not cover these deletions that are located nearby but not in the known mutation hotspot of exon 18, or that Sanger sequencing produced serious artifacts.

Introduction: Phospatidylinositol-3-kinases (PI3K) play an important role in various cell processes. Oncogenic mutations in the PIK3CA gene, which codes for the catalytic subunit, have been identified in various malignancies and activate the PI3K/AKT/mTOR pathway, which is a critical driver of tumorigenesis. Methods: We tested 41 tumor samples with known KRAS, BRAF, and EGFR mutation status for the occurrence of mutations in the PIK3CA gene, using a pyrosequencing assay. Results: Pyrosequencing revealed 2 mutations (4.9%) in the PIK3CA gene, one in exon 9 and the other in exon 20. Both mutations have not been identified yet in lung tumor tissue. Discussion: The screening of our small patient cohort by pyrosequencing identified 2 mutations (4.9%) in PIK3CA, one in exon 9 (Q546H) and the other in exon 20 (M1043T). Both mutations have not been described in lung tumors yet and seem to be rather uncommon mutations. Future screening of large patient cohorts with pyrosequencing may contribute to the detection of more mutations in lung cancer because of the low limit of detections of this method and may contribute to a better understanding of the interaction of mutations and tumorigenesis.

Companion diagnostics (CDx) have become a major tool in molecular pathology and assist in therapy decisions in an increasing number of various cancers. Particularly, the developments in lung cancer have been most impressing in the last decade and consequently lung cancer mutation testing and molecular profiling has become a major business of diagnostic laboratories. However, it has become difficult to decide which biomarkers are currently relevant for therapy decisions, as many of the new biomarkers are not yet approved as therapy targets, remain in the status of clinical studies, or still have not left the experimental phase. The current review is focussed on those markers that do have current therapy implications, practical implications arising from the respective companion diagnostics, and thus is focused on daily practice.

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis(1-3). We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 +/- 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice(4). Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

Purpose: Blood-based surrogate markers would be attractive biomarkers for early detection, diagnosis, prognosis, and prediction of therapeutic outcome in cancer. Disease-associated gene expression signatures in peripheral blood mononuclear cells (PBMC) have been described for several cancer types. However, RNA-stabilized whole blood–based technologies would be clinically more applicable and robust. We evaluated the applicability of whole blood–based gene expression profiling for the detection of non–small cell lung cancer (NSCLC). Experimental Design: Expression profiles were generated from PAXgene-stabilized blood samples from three independent groups consisting of NSCLC cases and controls (n = 77, 54, and 102), using the Illumina WG6-VS2 system. Results: Several genes are consistently differentially expressed in whole blood of NSCLC patients and controls. These expression profiles were used to build a diagnostic classifier for NSCLC, which was validated in an independent validation set of NSCLC patients (stages I–IV) and hospital-based controls. The area under the receiver operator curve was calculated to be 0.824 (P < 0.001). In a further independent dataset of stage I NSCLC patients and healthy controls the AUC was 0.977 (P < 0.001). Specificity of the classifier was validated by permutation analysis in both validation cohorts. Genes within the classifier are enriched in immune-associated genes and show specificity for NSCLC. Conclusions: Our results show that gene expression profiles of whole blood allow for detection of manifest NSCLC. These results prompt further development of gene expression–based biomarker tests in peripheral blood for the diagnosis and early detection of NSCLC. Clin Cancer Res; 17(10); 3360–7. ©2011 AACR.

Darbepoetin alfa, an erythropoiesis-stimulating agent (ESA), is used in cancer patients as a supportive care for anemia. For small-cell lung cancer (SCLC), several studies have shown that the administration of ESAs does not affect survival but decreases the need for blood transfusions and improves the quality of life (QOL) of patients receiving chemotherapy. The present randomized phase II study assessed the feasibility, efficacy, and safety of the administration of darbepoetin alfa to patients with SCLC receiving dose-dense (every 2 weeks) standard chemotherapy consisting of carboplatin plus etoposide, pegfilgrastim prophylactically. Seventy-four chemotherapy-naive patients with limited or extensive SCLC received combination chemotherapy for 6 cycles, and half of the patients additionally received darbepoetin to achieve a target hemoglobin concentration of 12-13 g/dL. The primary study outcome, progression-free survival, showed no difference between the 2 arms of the study. Among the secondary endpoints, objective response was similar in the presence and absence of darbepoetin (best response rates = 75.0% vs. 77.8%). Likewise, 1-year survival rates were not different between the 2 treatment arms (40.1% vs. 45.9%). There were no significant differences in grade 3/4 toxicities. As expected, the need for blood transfusions differed significantly: 19.4% of patients in the darbepoetin arm received transfusions versus 38.9% in the control arm. Analysis of European Organization for Research and Treatment of Cancer quality of life questionnaire (EORTC QLQ-C30) scales at different time points showed that the darbepoetin group's QOL was significantly better for certain readouts and never significantly worse than that of the control group. Thus, the combination of darbepoetin alfa with dose-dense carboplatin plus etoposide was feasible and well tolerated. Addition of darbepoetin alfa to chemotherapy lowered the need for blood transfusions and did not affect measures of survival and objective response.

Introduction Rearrangements of RET are rare oncogenic events in patients with non–small cell lung cancer (NSCLC). While the characterization of Asian patients suggests a predominance of nonsmokers of young age in this genetically defined lung cancer subgroup, little is known about the characteristics of non-Asian patients. We present the results of an analysis of a European cohort of patients with RET rearranged NSCLC. Methods Nine hundred ninety-seven patients with KRAS/EGFR/ALK wildtype lung adenocarcinomas were analyzed using fluorescence in situ hybridization for RET fusions. Tumor specimens were molecularly profiled and clinicopathological characteristics of the patients were collected. Results Rearrangements of RET were identified in 22 patients, with a prevalence of 2.2% in the KRAS/EGFR/ALK wildtype subgroup. Co-occurring genetic aberrations were detected in 10 patients, and the majority had mutations in TP53 . The median age at diagnosis was 62 years (range, 39–80 years; mean ± SD, 61 ± 11.7 years) with a higher proportion of men (59% versus 41%). There was only a slight predominance of nonsmokers (54.5%) compared to current or former smokers (45.5%). Conclusions Patients with RET rearranged adenocarcinomas represent a rare and heterogeneous NSCLC subgroup. In some contrast to published data, we see a high prevalence of current and former smokers in our white RET cohort. The significance of co-occurring aberrations, so far, is unclear.

We characterized genome alterations in 1255 clinically annotated lung tumors of all histological subgroups to identify genetically defined and clinically relevant subtypes. More than 55% of all cases had at least one oncogenic genome alteration potentially amenable to specific therapeutic intervention, including several personalized treatment approaches that are already in clinical evaluation. Marked differences in the pattern of genomic alterations existed between and within histological subtypes, thus challenging the original histomorphological diagnosis. Immunohistochemical studies confirmed many of these reassigned subtypes. The reassignment eliminated almost all cases of large cell carcinomas, some of which had therapeutically relevant alterations. Prospective testing of our genomics-based diagnostic algorithm in 5145 lung cancer patients enabled a genome-based diagnosis in 3863 (75%) patients, confirmed the feasibility of rational reassignments of large cell lung cancer, and led to improvement in overall survival in patients with EGFR-mutant or ALK-rearranged cancers. Thus, our findings provide support for broad implementation of genome-based diagnosis of lung cancer.

Objectives We investigated the feasibility of cisplatin or carboplatin combined with pemetrexed as adjuvant treatment in patients with completely resected Stage IB/II Non-Small-Cell Lung Cancer (NSCLC). Materials and methods Patients in this multicenter, open-label, parallel-group, non-comparative Phase 2 study were randomized (1:1) to pemetrexed (500 mg/m2) with either cisplatin (75 mg/m2) or carboplatin (AUC5) for 4 cycles of 21 days. The primary endpoint was treatment feasibility (defined as 4 cycles completed with no cycle delay >42 days and ≤2 dose reductions, with a median relative dose intensity (RDI) ≥95% [overall]; and no Grade ≥3 toxicities at the follow-up visit 30 days after last drug administration). Secondary objectives included overall survival (OS) and safety. Results We randomized 122 patients and treated 118. 71.9% (46/64) of patients in pemetrexed + cisplatin and 88.9% (48/54) in pemetrexed + carboplatin completed 4 cycles (median RDI >97% for all compounds). Neither treatment met the pre-defined feasibility level >60% of patients: 59.4% (95% confidence interval [CI]: 46.4;71.5) pemetrexed + cisplatin; 50.0% (95%CI: 36.1;63.9) in pemetrexed + carboplatin. In a post-hoc analysis considering only safety, both regimens were feasible with 81.3% (95%CI: 69.5;89.9) in pemetrexed + cisplatin and 90.7% (95%CI: 79.7;96.9) in pemetrexed + carboplatin. OS rates for both groups were 82–83% after 3 years and 80–83% after 5 years. Treatment-related Grade ≥3 adverse events (mostly hematological) were experienced by approximately 30% of patients in each group. Conclusion Although the study did not meet the primary objective, both treatment groups demonstrated good safety-related feasibility and tolerability as adjuvant treatment in patients with completely resected Stage IB/II NSCLC.

Targeted therapies are discussed as an approach to improve outcome in neuroendocrine tumors of the lung. With current treatment options like platinum derivates and etoposide, large cell neuroendocrine carcinoma of the lung (LCNEC) patients have a poor prognosis. Everolimus (RAD001) is an inhibitor of mTOR, a component of the PI3K/AKT/mTOR pathway known to be dysregulated in neuroendocrine tumors (NETs). In the presented trial for advanced LCNEC patients, daily RAD001 is combined in first-line with carboplatin and paclitaxel, a standard chemotherapy for this population. Enrolled patients receive 4 cycles of combined treatment followed by RAD001 maintenance therapy. The primary endpoint is the proportion of progression-free patients after 3 months. Main inclusion criteria are histologically confirmed stage IV LCNEC and at least one positive neuroendocrine marker. Main exclusion criteria are symptomatic CNS metastases and prior treatment for advanced LCNEC. Here a case report of a 56-year old patient with stage IV LCNEC enrolled in this trial is presented. The tumor histologically revealed NSCLC morphology of the large-cell type and a considerable CD56 and synaptophysin staining as well as a proliferation rate of more than 60% in Ki67 staining. The patient received in first-line 4 cycles of carboplatin and paclitaxel combined with RAD001 and subsequently RAD001 monotherapy for further 9 months. The tumor response revealed as per RECIST a partial remission during the first 3 months and an ongoing stabilization of the tumor for further 9 months before a disease progression was detected 12 months after initial diagnosis. At the fifth cycle of a second-line treatment with carboplatin and etoposide, the patient has still a stable disease 16 months after initial diagnosis of LCNEC. These data show that a combined therapy of carboplatin and paclitaxel with mTOR-inhibitor RAD001 in first-line might be a promising approach for more efficient treatment of LCNEC patients.

Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1(V561M)), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

ABSTRACT: A total of three cases with novel constellations regarding mutation patterns in non-small-cell lung cancer (NSCLC) are reported. The mutation patterns that are observed are novel and unexpected. First, a combined simultaneous KRAS mutation and EML4–ALK translocation, both in the main tumor and a bone metastasis, were observed, these mutations are assumed to mutually exclude each other. A further two cases include a father and a daughter, both of whom are suffering from NSCLC with different EGFR mutation patterns. A common cause was assumed; however, could not be deduced to mutations in the KRAS, BRAF and EGFR genes. The aforementioned cases are important, as it must be taken into account that mutations previously assumed to be exclusive can occur in combination, may influence the clinical outcome and may require different therapy compared with single mutated tumors. It has to be discussed whether diagnostic algorithms need to be adapted. The cases of father and daughter show that further unknown factors can influence development of NSCLC.

Zielsetzung: Nach Resektion eines NSCLC in den Stadien IB-IIIA ist eine adjuvante Chemotherapie indiziert. Bisherige Studien untersuchten meist eine platinbasierte Kombinationschemotherapie mit intravenösem Vinorelbin (Vrb). Carboplatin (Carbo) wurde hierbei bei Patienten eingesetzt, welche für[for full text, please go to the a.m. URL], Gemeinsame Jahrestagung der Deutschen, Österreichischen und Schweizerischen Gesellschaft für Thoraxchirurgie

We characterized genome alterations in 1255 clinically annotated lung tumors of all histological subgroups to identify genetically defined and clinically relevant subtypes. More than 55% of all cases had at least one oncogenic genome alteration potentially amenable to specific therapeutic intervention, including several personalized treatment approaches that are already in clinical evaluation. Marked differences in the pattern of genomic alterations existed between and within histological subtypes, thus challenging the original histomorphological diagnosis. Immunohistochemical studies confirmed many of these reassigned subtypes. The reassignment eliminated almost all cases of large cell carcinomas, some of which had therapeutically relevant alterations. Prospective testing of our genomics-based diagnostic algorithm in 5145 lung cancer patients enabled a genome-based diagnosis in 3863 (75%) patients, confirmed the feasibility of rational reassignments of large cell lung cancer, and led to improvement in overall survival in patients with EGFR-mutant or ALK-rearranged cancers. Thus, our findings provide support for broad implementation of genome-based diagnosis of lung cancer.

UNLABELLED 3'-deoxy-3'-[(18)F]fluoro-L-thymidine (FLT) and 2'-deoxy-2'-[(18)F]fluoro-D-glucose (FDG) are used to visualize proliferative and metabolic activity of tumors. In this study we aimed at evaluating the prognostic value of FLT and FDG uptake measured by positron emission tomography (PET) in patients with metastatic non-small cell lung cancer (NSCLC) prior to systemic therapy with erlotinib. FLT and FDG maximum standardized uptake (SUVmax) values per patient were analyzed in 40 chemotherapy naive patients with advanced NSCLC (stage IV) before treatment with erlotinib. Prior therapy median SUVmax was 6.6 for FDG and 3.0 for FLT, respectively. In univariate analysis, patients with an FDG SUVmax

BackgroundAdjuvant chemotherapy is beneficial in non-small-cell lung cancer (NSCLC). However, balancing toxicity and efficacy mandates improvement.Patients and methodsPatients with completely resected stages IB-pT3N1 NSCLC were randomly assigned to either four cycles cisplatin (C: 50 mg/m(2) day (d)1 + 8) and vinorelbine (V: 25 mg/m(2) d1, 8, 15, 22) q4 weeks or four cycles cisplatin (75 mg/m(2) d1) and pemetrexed (Px: 500 mg/m(2) d1) q3 weeks. Primary objective was the clinical feasibility rate (no grade (G)4 neutropenia/thrombocytopenia or thrombocytopenia with bleeding, no G3/4 febrile neutropenia or non-hematological toxicity; no premature withdrawal/death). Secondary objectives were drug delivery and efficacy.ResultsOne hundred and thirty two patients were randomized (stages: 38% IB, 10% IIA, 47% IIB, 5% pT3pN1; histology: 43% squamous, 57% non-squamous). The feasibility rates were 95.5% (cisplatin and pemetrexed, CPx) and 75.4% (cisplatin and vinorelbine, CVb) (P = 0.001); hematological G3/4 toxic effects were 10% (CPx) and 74% (CVb) (P

We recently reported fibroblast growth factor receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) was detected at a frequency of 16% and low-level amplification (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials.

SummaryLung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.