RET Fusion Testing in Patients With NSCLC: The RETING Study.

Introduction: RET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. Methods: This situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion-positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. Results: The most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). Conclusions: In-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.

Comprehensive NGS profiling to enable detection of ALK gene rearrangements and MET amplifications in non-small cell lung cancer.

Introduction: Next-generation sequencing (NGS) is currently widely used for biomarker studies and molecular profiling to identify concurrent alterations that can lead to the better characterization of a tumor's molecular landscape. However, further evaluation of technical aspects related to the detection of gene rearrangements and copy number alterations is warranted. Methods: There were 12 ALK rearrangement-positive tumor specimens from patients with non-small cell lung cancer (NSCLC) previously detected via fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and an RNA-based NGS assay, and 26 MET high gene copy number (GCN) cases detected by FISH, selected for this retrospective study. All 38 pre-characterized cases were reassessed utilizing the PGDx™ elio™ tissue complete assay, a 505 gene targeted NGS panel, to evaluate concordance with these conventional diagnostic techniques. Results: The detection of ALK rearrangements using the DNA-based NGS assay demonstrated excellent sensitivity with the added benefit of characterizing gene fusion partners and genomic breakpoints. MET copy number alterations were also detected; however, some discordances were observed likely attributed to differences in algorithm, reporting thresholds and gene copy number state. TMB was also assessed by the assay and correlated to the presence of NSCLC driver alterations and was found to be significantly lower in cases with NGS-confirmed canonical driver mutations compared with those without (p=0.0019). Discussion: Overall, this study validates NGS as an accurate approach for detecting structural variants while also highlighting the need for further optimization to enable harmonization across methodologies for amplifications.

Pre-existing tumor host immunity characterization in resected non-small cell lung cancer.

INTRODUCTION: Neoadjuvant and adjuvant immune checkpoint blockade (ICB) have recently become standard of care in resectable non-small cell lung cancer (NSCLC). Yet, biomarkers that inform patients who benefit from this approach remain largely unknown. Here, we interrogated the tumor immune microenvironment (TIME) in early-stage NSCLC patients that underwent up-front surgery. METHODS: A total of 185 treatment-naïve patients with early-stage NSCLC, that underwent up-front surgical treatment between 2006 and 2018 at Hospital del Mar were included. 124 lung adenocarcinomas (LUADs), and 61 squamous cell carcinoma (LUSCs) were included in a tissue microarray. Immunohistochemistry for CD3, CD4, CD8, CD68, CD80, CD103, FOXP3, PD-1, PD-L1, PD-L2 and HLA class II were evaluated by digital image analysis (QuPath software). TIME was categorized into four groups using PD-L1 expression in tumor cells (<1 % or ≥1 %) and tumor resident memory (CD103+) immune cells (using the median as cut-off). We explored the association between different TIME dimensions and patient's clinicopathological features and outcomes. RESULTS: We found increased levels of T cell markers (CD3+, CD4+, CD8+ cells), functional immune markers (FOXP3+ cells) as well as, higher HLA-II tumor membrane expression in LUADs compared to LUSCs (p < 0.05 for all). In contrast, LUSCs displayed higher percentage of intratumor macrophages (CD68+ cells) as well as, higher PD-L1 and PD-L2 tumor membrane expression (p < 0.05 for all). Unsupervised analysis revealed three different tumor subsets characterized by membrane tumor expression of PD-L1, PD-L2 and HLA-class II. Enrichment of T cells (CD3+, CD8+ cells), regulatory T cells (FOXP3+ cells) and macrophages (CD68+ cells) was observed in the CD103+/PD-L1+ group (p < 0.05 for all). Multivariate analysis showed that infiltration by CD103+ immune cells was associated with improved OS (p = 0.009). CONCLUSIONS: TIME analysis in resected NSCLC highlighted differences by histology, PD-L1 expression and molecular subgroups. Biomarker studies using IHC might aid to individually tailor adjuvant treatment in early-stage NSCLC.

Multicenter Evaluation of the Idylla GeneFusion in Non-Small-Cell Lung Cancer.

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next-generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods.

Plasmatic KRAS Kinetics for the Prediction of Treatment Response and Progression in Patients With KRAS-mutant Lung Adenocarcinoma / Estudio de la cinética del KRAS plasmático para predecir la respuesta al tratamiento y la progresión en pacientes con adenocarcinoma de pulmón con mutación de KRAS

Introduction: KRAS is the most common driver mutation in lung cancer. ctDNA-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Monitoring KRAS mutational load in ctDNA may be useful in the management of the patients.Methods: Consecutive patients diagnosed with KRAS mutant lung adenocarcinoma in the tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. KRAS mutations in plasma were quantified using digital PCR and correlated with mutations in tumor and with radiological response and progression.Results: Two hundred and forty-five plasma samples from 56 patients were analyzed. The rate of detection of KRAS mutations in plasma in our previously characterized KRAS-mutant cases was 82% overall, reaching 96% in cases with more than 1 metastatic location. The dynamics of KRAS mutational load predicted response in 93% and progression in 63% of cases, 33 and 50 days respectively in advance of radiological evaluation. Progression-free survival for patients in whom ctDNA was not detectable in plasma after treatment initiation was significantly longer than for those in whom ctDNA remained detectable (7.7 versus 3.2 months; HR: 0.44, p=0.004).Conclusions: The detection of KRAS mutations in ctDNA showed a good correlation with that in tumor biopsy and, in most cases, predicted tumor response and progression to chemotherapy in advance of radiographic evaluation. The liquid biopsies for ctDNA-based molecular analyses are a reliable tool for KRAS testing in clinical practice. (AU)

Introducción: La mutación en KRAS es la mutación iniciadora más común en el cáncer de pulmón. La valoración basada en el ctDNA ofrece ventajas frente a la tumoral, al ser un método mínimamente invasivo capaz de capturar la heterogeneidad del tumor. La monitorización de la carga de KRAS mutado en el ctDNA puede ser útil en el manejo de los pacientes.Métodos: En este estudio se incluyó, mediante selección consecutiva, a pacientes diagnosticados con adenocarcinoma de pulmón con mutación en KRAS en la biopsia tumoral. Se obtuvieron muestras de plasma en diferentes momentos durante el curso de la enfermedad. Las mutaciones de KRAS en plasma se cuantificaron mediante PCR digital y se correlacionaron con las mutaciones en el tumor y con la respuesta radiológica y la progresión.Resultados: Se analizaron 245 muestras de plasma de 56 pacientes. La tasa de detección de mutaciones KRAS en plasma en aquellos casos previamente definidos con dicha mutación fue del 82% globalmente, porcentaje que alcanzó el 96% en aquellos casos con más de una ubicación metastásica. La dinámica de la carga de KRAS mutado predijo la respuesta en el 93% de los casos y la progresión en el 63%, a los 33 y 50 días, respectivamente, anteriores a la evaluación radiológica. La supervivencia libre de progresión para pacientes en los que el ctDNA no era detectable en plasma después del inicio del tratamiento fue significativamente más larga que para aquellos en los que el ctDNA permaneció detectable (7,7 frente a 3,2 meses; HR: 0,44; p = 0,004).Conclusiones: La detección de mutaciones KRAS en el ctDNA mostró una buena correlación con la de la biopsia tumoral y, en la mayoría de los casos, predijo la respuesta tumoral a la quimioterapia y la progresión antes de la evaluación radiológica. Las biopsias líquidas para análisis moleculares basados en ctDNA son una herramienta fiable para la valoración de KRAS en la práctica clínica. (AU)

Plasmatic KRAS Kinetics for the Prediction of Treatment Response and Progression in Patients With KRAS-mutant Lung Adenocarcinoma.

INTRODUCTION: KRAS is the most common driver mutation in lung cancer. ctDNA-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Monitoring KRAS mutational load in ctDNA may be useful in the management of the patients. METHODS: Consecutive patients diagnosed with KRAS mutant lung adenocarcinoma in the tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. KRAS mutations in plasma were quantified using digital PCR and correlated with mutations in tumor and with radiological response and progression. RESULTS: Two hundred and forty-five plasma samples from 56 patients were analyzed. The rate of detection of KRAS mutations in plasma in our previously characterized KRAS-mutant cases was 82% overall, reaching 96% in cases with more than 1 metastatic location. The dynamics of KRAS mutational load predicted response in 93% and progression in 63% of cases, 33 and 50 days respectively in advance of radiological evaluation. Progression-free survival for patients in whom ctDNA was not detectable in plasma after treatment initiation was significantly longer than for those in whom ctDNA remained detectable (7.7 versus 3.2 months; HR: 0.44, p=0.004). CONCLUSIONS: The detection of KRAS mutations in ctDNA showed a good correlation with that in tumor biopsy and, in most cases, predicted tumor response and progression to chemotherapy in advance of radiographic evaluation. The liquid biopsies for ctDNA-based molecular analyses are a reliable tool for KRAS testing in clinical practice.

Concomitant genomic alterations in KRAS mutant advanced lung adenocarcinoma.

OBJECTIVES: KRAS mutations are one of the most prevalent alterations in non-small cell lung cancer. However, patients with this driver alteration present heterogeneous clinical outcomes. In this study, we have explored the potential clinical impact of coexisting alterations in this subset of patients. MATERIALS AND METHODS: Samples from a cohort of 69 lung adenocarcinoma patients homogenously treated with platinum doublet as first-line therapy were evaluated using targeted next generation sequencing (NGS). Mutations and copy number alterations were assessed in 37 advanced KRAS-mutant (KRASm) and in 32 KRAS wild-type (KRASwt). RESULTS: TP53 was the most frequent additional alteration found in both cohorts. Interestingly, TP53 mutations were more frequent in KRASwt than in KRASm patients (80 % vs. 34 %; p <  0.05) as well as STK11 mutations (17 % vs 8 %, p=NS). FGFR3 mutations were only found concomitantly with KRASm (11 %). No genomic co-alteration had an impact on overall survival within the KRASm patients treated with chemotherapy. CONCLUSIONS: KRAS mutated lung adenocarcinoma is a heterogeneous entity and comprehensive characterization of co-alterations using NGS may lead to more accurate patient stratification.

Complementary value of electron microscopy and immunohistochemistry in the diagnosis of non-small cell lung cancer: A potential role for electron microscopy in the era of targeted therapy.

With the identification of therapeutic targets for lung adenocarcinoma, it has become mandatory to distinguish it from other entities. Some cases remain classified as non-small cell lung carcinoma, not otherwise specified (NSCLC-NOS) with immunohistochemistry. Electron microscopy (EM) can be useful, allowing the identification of glandular differentiation. The aim of this study was to determine the complementary value of immunohistochemistry and EM.Forty-eight NSCLC-NOS cases were selected (PSMAR-Biobank, Barcelona, Spain). Immunohistochemistry (TTF-1, p40) was performed. Tissue was retrieved from paraffin blocks. Results were compared to the final diagnosis, derived from combination of light microscopy, immunohistochemistry, EM, molecular studies and resection specimen.Immunohistochemistry concurred with final diagnosis in 36 cases (75%, Kappa = 0.517). EM agreed with final diagnosis in 35 (72.9%, Kappa = 0.471). Immunohistochemistry had a sensitivity = 73%, specificity = 100%, positive predictive value (PPV) = 100% and negative predictive value (NPV) = 52.4% for adenocarcinoma. All adenocarcinoma cases not solved by immunohistochemistry (n = 10) were classified by EM, and vice versa. Data from EM were identical to those of immunohistochemistry: sensitivity = 73%, specificity = 100%, PPV = 100% and NPV = 52.4%. Combining both techniques, 47 cases were coincident with final diagnosis (97.9%, Kappa = 0.943).EM can provide valuable information in subtyping NSCLC-NOS, being particularly useful when immunohistochemistry is inconclusive. EM could be considered as a complementary tool for decision-making in NSCLC-NOS.

HER-Family Ligands Promote Acquired Resistance to Trastuzumab in Gastric Cancer.

Despite the clinical benefit of trastuzumab, eventually all HER2-amplified gastric cancer tumors develop drug resistance. We aimed to identify molecular mechanisms of acquired resistance to trastuzumab in gastric cancer by using well-established cell line-based preclinical models, as well as samples from patients with HER2-positive gastric cancer treated with trastuzumab. We studied trastuzumab resistance in NCI-N87 and OE19, two gastric cancer cell lines that overexpress HER2 receptor and are trastuzumab sensitive. Differences at protein, DNA, and RNA levels between the parental and resistant cells were characterized and functional studies were performed. Paired pre- and post-trastuzumab blood and tissue samples from patients with gastric cancer treated with trastuzumab were analyzed. We found that resistant cells were associated with increased activation of MAPK/ERK and PI3K/mTOR pathways driven by SRC activation. Upstream, resistant cells showed increased coexpression of multiple HER-family ligands that allowed for compensatory activation of alternative HER receptors upon HER2 blockade. Simultaneous inhibition of EGFR, HER2, and HER3 by the novel antibody mixture, Pan-HER, effectively reverted trastuzumab resistance in vitro and in vivo Similarly, an increase in HER-family ligands was observed in serum and tumor from patients with gastric cancer after trastuzumab therapy. We propose that trastuzumab resistance in gastric cancer is mediated by HER-family ligand upregulation that allows a compensatory activation of HER receptors and maintains downstream signaling activation despite trastuzumab therapy. Resistance is reverted by simultaneous inhibition of EGFR, HER2, and HER3, thereby revealing a potential therapeutic strategy to overcome trastuzumab resistance in patients with gastric cancer.

Accurate Identification of Predictive Biomarkers of Response to Targeted Therapies in Lung Cancer With Next Generation Sequencing / Identificación precisa de biomarcadores predictivos de respuesta a terapias dirigidas en cáncer de pulmón con secuenciación de nueva generación

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Next-generation Sequencing for ALK and ROS1 Rearrangement Detection in Patients With Non-small-cell Lung Cancer: Implications of FISH-positive Patterns.

BACKGROUND: Detection of ALK and ROS1 gene rearrangements in non-small-cell lung cancer is required for directing patient care. Although fluorescence in situ hybridization (FISH) and immunohistochemistry have been established as gold standard methods, next-generation sequencing (NGS) platforms are called to be at least equally successful. Comparison of these methods for translation into daily use is currently under investigation. PATIENTS AND METHODS: Forty non-small-cell lung cancer paraffin-embedded samples with previous ALK (n = 33) and ROS1 (n = 7) FISH results were examined with the Oncomine Focus Assay and tested for ALK and ROS1 immunoreactivity. Clinical implications of concurrent molecular alterations and concordance between methods were evaluated. RESULTS: NGS was successful in 32 (80%) cases: 25 ALK and 7 ROS1. Few concomitant alterations were detected: 1 ALK rearranged case had an ALK p.L1196M-resistant mutation, 4 had CDK4, MYC, and/or ALK amplifications, and 1 ROS1 rearranged case showed a FGFR4 amplification. Comparison between techniques revealed 5 (16%) discordant cases that had lower progression-free survival than concordant cases: 7.6 (95% confidence interval, 2.2-13) versus 19.4 (95% confidence interval, 10.1-28.6). Remarkably, 4 of these cases had isolated 3' signal FISH pattern (P = .026). CONCLUSION: Our data support that the identification of 3' isolated signal FISH pattern in ALK and ROS1 cases might suggest a false-positive result. NGS seems a reliable technique to assess ALK and ROS1 rearrangements, offering the advantage over immunohistochemistry of detecting other molecular alterations with potential therapeutic implications.

Dynamics of EGFR Mutation Load in Plasma for Prediction of Treatment Response and Disease Progression in Patients With EGFR-Mutant Lung Adenocarcinoma.

BACKGROUND: The assessment of epidermal growth factor receptor (EGFR) mutations is crucial for the management of patients with lung adenocarcinoma. Circulating tumor DNA (ctDNA)-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. PATIENTS AND METHODS: Consecutive patients diagnosed with EGFR-mutant lung adenocarcinoma in tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. EGFR mutations in plasma were quantified using BEAMing (beads, emulsions, amplification, and magnetics) or digital PCR and were correlated with mutations in tumor and with radiologic response and progression. RESULTS: Two hundred twenty-one plasma samples from 33 patients were analyzed. EGFR mutations in plasma were detected in 83% of all patients and 100% of those with extrathoracic metastases. The dynamics of the EGFR mutation load predicted response in 93% and progression in 89% of cases well in advance of radiologic evaluation. Progression-free survival for patients in whom ctDNA was not detected in plasma during treatment was significantly longer than for those in whom ctDNA remained detectable (295 vs. 55 days; hazard ratio, 17.1; P < .001). CONCLUSION: The detection of EGFR mutations in ctDNA showed good correlation with that in tumor biopsy and predicted tumor response and progression in most patients. The liquid biopsy for ctDNA-based assessment of EGFR mutations is a reliable technique for diagnosis and follow-up in patients with EGFR-mutant lung adenocarcinoma in routine clinical practice.

CD274 (PDL1) and JAK2 genomic amplifications in pulmonary squamous-cell and adenocarcinoma patients.

AIMS: CD274 (PDL1) and JAK2 (9p24.1) gene amplifications have been recently described in pulmonary carcinomas in association with programmed death-ligand 1 (PD-L1) expression. Furthermore, PTEN loss has been explored preclinically in relation to PD-L1 expression. Our aim was to determine whether these genomic alterations affect PD-L1 expression levels in non-small-cell lung cancer. METHODS AND RESULTS: PD-L1 and PTEN expression determined by immunohistochemistry (IHC), and CD274, JAK2 and PTEN copy number alterations (CNAs) determined by fluorescence in-situ hybridisation, were studied in 171 pulmonary carcinoma specimens. PD-L1 expression was positive in 40 cases (23.3%), and CD274 amplification was present in 14 tumours (8.8%). Concordance between both events was found in 12 of 14 amplified cases (P = 0.0001). We found nine JAK2-amplified cases (5.7%), seven with PD-L1 expression (P = 0.0006). Moreover, six of the seven cases had JAK2 and CD274 coamplification (9p24.1 genomic amplification). Remarkably, the average PD-L1 IHC score was higher in these amplified cases (230 versus 80; P = 0.001). Non-statistical associations were observed between PD-L1 expression and PTEN loss and PTEN deletions. CONCLUSIONS: We describe a subset of patients (8.2%) who had 9p24.1 amplifications resulting in high expression of PD-L1. Our results provide evidence for genomic up-regulation of PD-L1 expression in non-small-cell lung cancer.

Heterogeneity of Tumor and Immune Cell PD-L1 Expression and Lymphocyte Counts in Surgical NSCLC Samples.

BACKGROUND: Immune-checkpoint inhibitors against programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) have shown remarkable therapeutic activity in non-small-cell lung cancer (NSCLC). However, biomarker-based patient selection remains a challenge. Our aim was to assess the heterogeneity of various immune markers between different tumor areas of surgically resected NSCLC specimens. MATERIALS AND METHODS: We included 94 adenocarcinoma (ADC) and 50 squamous cell carcinoma (SCC) specimens. Two distinct tumor areas of each tumor sample were selected and incorporated into tissue microarrays. PD-L1 expression in tumor cells (TCs) and immune cells (ICs) was assessed using clone SP142 (Ventana). PDL1 gene amplification was assessed using fluorescence in situ hybridization. CD3 and CD8 densities were quantified using digital image-based analysis. Heterogeneity was assessed using kappa agreement index (KI) and intraclass correlation coefficient. RESULTS: Prevalence of PD-L1 expression was 16.8% in TCs and 27.8% in ICs. Eleven tumors (7.6%) showed PDL1 amplification. In ADC, KI of PD-L1 TC and IC expression between cores was 0.465 and 0.260, compared with 0.274 and 0.124 in SCC, respectively. Higher concordance was observed for PDL1 amplification (KI, 0.647 in ADC and KI, 1 in SCC). Eleven (61.1%) of 18 amplified cores showed PD-L1 staining in < 5% of TCs. Intraclass correlation coefficients for CD3 and CD8 were 0.293 and 0.186 in ADC and 0.489 and 0.610 in SCC samples, respectively. CONCLUSIONS: We found significant heterogeneity of PD-L1 expression in both ADC and SCC samples, especially in the IC compartment. Heterogeneous expression of PD-L1 could misclassify patients for PD-1/PD-L1-directed therapies.

ROS1 copy number alterations are frequent in non-small cell lung cancer.

OBJECTIVES: We aimed to determine the prevalence and partners of ROS1 rearrangements, to explore the correlation between FISH and IHC assays, and to investigate clinical implications of ROS1 copy number alterations (CNAs). METHODS: A total of 314 NSCLC patients were screened using ROS1 FISH break-apart probes. Of these, 47 surgical tumors were included in TMAs to analyze ROS1 heterogeneity assessed either by FISH and IHC, and chromosome 6 aneusomy. To characterize ROS1 partners, probes for CD74, EZR, SLC34A2 and SDC3 genes were developed. ROS1 positive FISH cases were screened also by IHC. RESULTS: Five patients were ROS1 positive (1.8%). We identified two known fusion partners in three patients: CD74 and SLC34A2. Four out of five ROS1 rearranged patients were female, never smokers and with adenocarcinoma histology. Rearranged cases were also positive by IHC as well. According to ROS1 CNAs, we found a prevalence of 37.8% gains/amplifications and 25.1% deletions. CONCLUSIONS: This study point out the high prevalence of ROS1 CNAs in a large series of NSCLC. ROS1 gains, amplifications and deletions, most of them due to chromosome 6 polysomy or monosomy, were heterogeneous within a tumor and had no impact on overall survival.

MET expression and copy number heterogeneity in nonsquamous non-small cell lung cancer (nsNSCLC).

OBJECTIVE: We aimed to assess MET intratumoral heterogeneity and its potential impact on biomarker-based patient selection as well as potential surrogate biomarkers of MET activation. METHODS: Our study included 120 patients with non-squamous Non-small-cell Lung Cancer (nsNSCLC), of which 47 were incorporated in tissue microarrays (TMA). Four morphologically distinct tumor areas were selected to assess MET heterogeneity. MET positivity by immunohistochemistry (IHC) was defined as an above-median H-score and by +2/+3 staining intensity in >50% of tumor cells (Metmab criteria). MET FISH positivity was defined by MET/CEP7 ratio ≥ 2.0 and/or MET ≥ 5.0. MET staining pattern (cytoplasmic vs. membranous) and mesenchymal markers were investigated as surrogates of MET activation. RESULTS: Median MET H-score was 140 (range 0-400) and 47.8% of patients were MET positive by Metmab criteria. Eight cases (6.8%) were MET FISH positive and showed higher H-scores (p = 0.021). MET positivity by IHC changed in up to 40% of cases among different tumor areas, and MET amplification in 25-50%. Cytoplasmic MET staining and positivity for vimentin predicted poor survival (p = 0.042 and 0.047, respectively). CONCLUSIONS: MET status is highly heterogeneous among different nsNSCLC tumor areas, hindering adequate patient selection for MET-targeted therapies. MET cytoplasmic staining and vimentin might represent surrogate markers for MET activation.

Assessment of ALK status by FISH on 1000 Spanish non-small cell lung cancer patients.

INTRODUCTION: Patients with non-small cell lung cancer (NSCLC) harboring anaplastic lymphoma kinase (ALK) rearrangement selectively respond to ALK inhibitors. Thus, identification of ALK rearrangements has become a standard diagnostic test in advanced NSCLC patients. Our institution has been a referral center in Spain for ALK determination by Fluorescent in situ hybridization (FISH). The aim of our study was to assess the feasibility and the FISH patterns of the ALK gene and to evaluate the clinical and pathological features of patients with ALK alterations. METHODS: Between 2010 and 2014, 1092 samples were evaluated for ALK using FISH technique (927 histological samples, 165 cytological samples). Correlation with available clinical-pathological information was assessed. RESULTS: ALK rearrangement was found in 35 patients (3.2%). Cytological samples (using either direct smears or cell blocks), were more frequently non-assessable than histological samples (69% versus 89%, respectively) (p < 0.001). Within the ALK-rearranged cases the majority were female, non-smokers, and stage IV. CONCLUSIONS: Although assessable in cytological samples, biopsies are preferred when available for ALK evaluation by FISH. The ALK translocation prevalence and the associated clinico-pathological features in Spanish NSCLC patients are similar to those previously reported.