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Several therapies to improve the management of lymphoma are currently being investigated, necessitating the development of new biomarkers. However, this requires high-quality and clinically annotated biological material. Therefore, we established a lymphoma biobank including all available biological material (tissue specimens and matched biological resources) along with associated clinical data for lymphoma patients diagnosed, according to the WHO classification, between 2005 and 2022 in the Laboratory of Clinical and Experimental Pathology, Nice, France. We retrospectively included selected cases in a new collection at the Côte d’Azur Biobank, which contains 2150 samples from 363 cases (351 patients). The male/female ratio was 1.3, and the median age at diagnosis was 58 years. The most common lymphoma types were classical Hodgkin lymphoma, diffuse large B-cell lymphoma, and extra-nodal marginal zone lymphoma of MALT tissue. The main sites of lymphoma were the mediastinum, lymph node, Waldeyer’s ring, and lung. The Côte d’Azur Biobank is ISO 9001 and ISO 20387 certified and aims to provide high quality and diverse biological material to support translational research projects into lymphoma. The clinico-pathological data generated by this collection should aid the development of new biomarkers to enhance the survival of patients with lymphoid malignancies.

Supplementary Figures. Fig.S1: Additional confocal images showing that expression of EFA6Bvsvg in MCF7 cells restores normal apico-basal polarity and promotes lumen formation Fig.S2: Analysis of the specificity of EFA6B repression using siRNAs Fig.S3: Additional confocal images showing that repression of EFA6B in MCF7 cells abrogates the assembly of TJ and leads to unorganized aggregates Fig.S4: Exogenous expression of EFA6Bvsvg abrogates the TGFβ-induced EMT in MDCK cells Fig.S5: Analysis of EFA6B/PSD4 mutations and copy number alterations from the cBioPortal website

As new SARS-CoV-2 variants emerge, there is an urgent need to increase the efficiency and availability of viral genome sequencing, notably to detect the lineage in samples with a low viral load. SARS-CoV-2 genome next-generation sequencing (NGS) was performed retrospectively in a single center on 175 positive samples from individuals. An automated workflow used the Ion AmpliSeq SARS-CoV-2 Insight Research Assay on the Genexus Sequencer. All samples were collected in the metropolitan area of the city of Nice (France) over a period of 32 weeks (from 19 July 2021 to 11 February 2022). In total, 76% of cases were identified with a low viral load (Ct ≥ 32, and ≤200 copies/µL). The NGS analysis was successful in 91% of cases, among which 57% of cases harbored the Delta variant, and 34% the Omicron BA.1.1 variant. Only 9% of cases had unreadable sequences. There was no significant difference in the viral load in patients infected with the Omicron variant compared to the Delta variant (Ct values, p = 0.0507; copy number, p = 0.252). We show that the NGS analysis of the SARS-CoV-2 genome provides reliable detection of the Delta and Omicron SARS-CoV-2 variants in low viral load samples.

Introduction International guidelines recommend BRAF mutational status assessment in treatment-naive advanced non-squamous non-small cell lung carcinoma (NSCLC) patients since the presence of a BRAFV600 mutation enables specific BRAF inhibitor treatment. For this purpose, the mutational status needs to be obtained in 10 working days. Herein, we prospectively evaluated the feasibility of systematic assessment of the BRAF status using immunohistochemistry (IHC) in a single institution (LPCE, Nice) at baseline for NSCLC diagnosed. Methods 1317 NSCLC were evaluated using BRAF IHC from 2011 to 2019. Initially the BRAF status was prospectively assessed using NGS and/or pyrosequencing in 618 consecutively diagnosed NSCLC patients from 2012 to 2016; BRAFV600E and BRAF nonV600E mutated tumors detected in this cohort were retrospectively evaluated using BRAF IHC. Secondarily, 699 biopsies of NSCLC were prospectively analyzed between 2017 and 2019 using BRAF IHC. BRAF IHC positive tumors were tested using a rapid BRAF specific PCR based assay. Results Initially, 21/618 (3%) of tumors (15 early and 6 late stage tumors) were BRAFV600E mutated according to the results of NGS and/or pyrosequencing. BRAF IHC was positive in 21/21 of these cases and negative in 51/51 (100 %) BRAF non V600E mutated cases. In the prospective BRAF IHC tested cohort of patients, 24/699 (3%) tumors (13 early and 11 late stage tumors) were positive with VE1 IHC. The BRAF PCR assay was positive in 20/24 (83 %) of these cases. Conclusion BRAFV600E IHC screening of treatment-naive NSCLC patients is a rapid, specific and very sensitive method which can lead in advanced stage positive NSCLC tumors to a BRAF inhibitor treatment. This test can be routinely integrated into mandatory predictive biomarker ‘testing of NSCLC. According to the organization of patient care and the physician’s request, this practice can be proposed as an alternative to NGS-based tissue biopsy made at baseline.

The number of genomic alterations required for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has increased and become more complex these last few years. These molecular abnormalities lead to treatment that provides improvement in overall survival for certain patients. However, these treated tumors inexorably develop mechanisms of resistance, some of which can be targeted with new therapies. The characterization of the genomic alterations needs to be performed in a short turnaround time (TAT), as indicated by the international guidelines. The origin of the tissue biopsies used for the analyses is diverse, but their size is progressively decreasing due to the development of less invasive methods. In this respect, the pathologists are facing a number of different challenges requiring them to set up efficient molecular technologies while maintaining a strategy that allows rapid diagnosis. We report here our experience concerning the development of an optimal workflow for genomic alteration assessment as reflex testing in routine clinical practice at diagnosis for NS-NSCLC patients by using an ultra-fast-next generation sequencing approach (Ion Torrent Genexus Sequencer, Thermo Fisher Scientific). We show that the molecular targets currently available to personalized medicine in thoracic oncology can be identified using this system in an appropriate TAT, notably when only a small amount of nucleic acids is available. We discuss the new challenges and the perspectives of using such an ultra-fast NGS in daily practice.

International audience; The histological distinction of lung neuroendocrine carcinoma, including small cell lung carcinoma (SCLC), large cell neuroendocrine carcinoma (LCNEC) and atypical carcinoid (AC), can be challenging in some cases, while bearing prognostic and therapeutic significance. To assist pathologists with the differentiation of histologic subtyping, we applied a deep learning classifier equipped with a convolutional neural network (CNN) to recognize lung neuroendocrine neoplasms. Slides of primary lung SCLC, LCNEC and AC were obtained from the Laboratory of Clinical and Experimental Pathology (University Hospital Nice, France). Three thoracic pathologists blindly established gold standard diagnoses. The HALO-AI module (Indica Labs, UK) trained with 18,752 image tiles extracted from 60 slides (SCLC = 20, LCNEC = 20, AC = 20 cases) was then tested on 90 slides (SCLC = 26, LCNEC = 22, AC = 13 and combined SCLC with LCNEC = 4 cases; NSCLC = 25 cases) by F1-score and accuracy. A HALO-AI correct area distribution (AD) cutoff of 50% or more was required to credit the CNN with the correct diagnosis. The tumor maps were false colored and displayed side by side to original hematoxylin and eosin slides with superimposed pathologist annotations. The trained HALO-AI yielded a mean F1-score of 0.99 (95% CI, 0.939–0.999) on the testing set. Our CNN model, providing further larger validation, has the potential to work side by side with the pathologist to accurately differentiate between the different lung neuroendocrine carcinoma in challenging cases.

The evaluation of an increasing number of diagnostic and predictive markers is playing a central role in precision thoracic oncology. Multiplex immunohistochemistry (mIHC), alongside next-generation sequencing, is ideally situated for this purpose and maximizes tumor tissue preservation for molecular analyses that use increasingly large panels. However, the standardization and validation of mIHC that supports routine clinical laboratory processes are mandatory. After a previous proof-of-concept study, we now (i) optimized two automated four-plex assays on a commercially available IHC autostainer for use in daily practices worldwide and (ii) evaluated the repeatability and concordance of the assessment of the cell density.Two four-plex mIHC assays [i) TTF1, p40, PD-L1, CD8; and, ii) ALK, ROS1, BRAFV600E, NTRK] were optimized on the BenchMark ULTRA autostainer (Ventana Medical Systems, Inc.), as determined in comparison to conventional IHC chromogenic assays. Intra-site repeatability was evaluated on serial tumor sections from non-small cell lung carcinomas (NSCLC). The concordance was assessed by linear fit to plots of the percentage staining evaluated on tumor sections from 89 NSCLC patients.Following optimization, an average concordance for a staining rate of 95.4% was achieved between conventional IHC and mIHC across all selected markers. Assessment of intra-site repeatability showed strong concordance for all these markers (average, ROur optimized mIHC assay gave a sensitive and repeatable assessment of two panels of eight diagnostic and predictive biomarkers for NSCLC. The availability of standardized protocols to determine these biomarkers on a widely available IHC platform will expand the number of pathology laboratories able to determine the eligibility of patients with NSCLC for targeted treatment or immunotherapy in a reliable and concordant manner, thus providing a unique sample-sparing tool to characterize limited tissue samples in thoracic oncology.

Due to increased demand for testing, as well as restricted supply chain resources, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to face many hurdles. Pooling several samples has been proposed as an alternative approach to address these issues. We investigated the feasibility of pooling nasopharyngeal swab (NPS) or saliva samples for SARS-CoV-2 testing with a commercial assay (Idylla SARS-CoV-2 test; Biocartis). We evaluated the 10-pool and 20-pool approaches for 149 subjects, with 30 positive samples and 119 negative samples. The 10-pool approach had sensitivity of 78.95% (95% confidence interval [CI], 54.43% to 93.95%) and specificity of 100% (95% CI, 71.51% to 100%), whereas the 20-pool approach had sensitivity of 55.56% (95% CI, 21.20% to 86.30%) and specificity of 100% (95% CI, 25% to 100%). No significant difference was observed between the results obtained with pooled NPS and saliva samples. Given the rapidity, full automation, and practical advantages of the Idylla SARS-CoV-2 assay, pooling of 10 samples has the potential to significantly increase testing capacity for both NPS and saliva samples, with good sensitivity. IMPORTANCE To control outbreaks of coronavirus disease 2019 (COVID-19) and to avoid reagent shortages, testing strategies must be adapted and maintained for the foreseeable future. We analyzed the feasibility of pooling NPS and saliva samples for SARS-CoV-2 testing with the Idylla SARS-CoV-2 test, and we found that sensitivity was dependent on the pool size. The SARS-CoV-2 testing capacity with both NPS and saliva samples could be significantly expanded by pooling 10 samples; however, pooling 20 samples resulted in lower sensitivity.

Resume L’evaluation des images histologiques peut etre parfois subjective et assez peu reproductible selon les cas. Par consequent, l’analyse computationnelle de l’imagerie histologique via des approches dites d’intelligence artificielle (IA) a recemment recu une attention considerable afin d’ameliorer cette precision diagnostique. Ainsi, l’analyse computationnelle de lames virtuelles a recemment ete evaluee pour l’optimisation du diagnostic histologique ou cytologique, la prediction du pronostic ou du profil genomique des patients atteints de cancer. L’utilisation de l’IA progresse de plus en plus dans le domaine de la recherche translationnelle en anatomopathologie, mais n’a pas encore ete mise en œuvre de la maniere la plus pratique pour le transfert en routine clinique. Malgre des resultats prometteurs, il subsiste plusieurs defis ou enjeux majeurs a relever afin de reussir le transfert reel de cette nouvelle approche en routine clinique. Il s'agit ici de faire le point sur les applications recentes de l’IA en pathologie cancereuse, mais aussi d’apporter des clarifications sur les avantages et les limites de cette approche, ainsi que les perspectives a mettre en œuvre pour un transfert potentiel dans la pratique quotidienne des pathologistes.

Background Recent advances obtained with immune checkpoint inhibitors (ICIs) targeting the programmed cell death-1 (PD-1) protein have significantly improved the outcome of patients with metastatic melanoma. The PD-L1 expression in tumour cells as detected by immunohistochemistry is a predictive biomarker in some solid tumours, but appears insufficient as prognostic or predictive factor of response to ICIs in metastatic melanomas. Objectives We investigated whether the presence and the features of pretreatment CD8+ tumour-infiltrating T lymphocytes (TILs) could be a complementary prognostic or predictive biomarker in patients with metastatic melanoma. Methods In this retrospective study, we evaluated the association of PD-L1 expression ≥5% of tumour cells combined with TIL features (CD8, CD28, Ki67) with the overall survival (OS) among 51 patients treated with ICIs and 54 patients treated with other treatment options (non-ICIs). Results PD-L1 positivity was observed in 33% and 39% of primary melanomas and matched metastases, respectively, with, however, poor concordance between the primary and the matched metastatic site (κ = 0.283). No significant association was noted between PD-L1 expression and CD8+ TIL profile analysed as single markers and OS or response to immunotherapy. Instead, their combined analysis in primary melanoma samples showed that the PD-L1-/CD8+ status was significantly associated with prolonged OS in the whole population (P = 0.04) and in the subgroup treated with non-ICIs (P = 0.009). Conversely, the PD-L1+/CD8+ status was a good prognostic factor in patients treated with ICIs (P = 0.022), whereas was significantly associated with poor prognosis in patients treated with non-ICIs (P = 0.014). While the expression of CD28 was not related to outcome, the Ki67 expression was significantly associated with poor OS in the subgroup CD8+ TIL+/PD-L1- (P = 0.02). Conclusions The pretreatment combination of PD-L1 expression with the level of CD8+ TILs could better assess OS and predict therapeutic response of patients with metastatic melanoma treated by either immunotherapy or other treatment regimens.

The outcome of patients with cutaneous melanoma has been strongly modified by recent advances obtained with Immune Checkpoint Inhibitors (ICIs). However, despite this breakthrough, durable response to ICIs is limited to a subset of patients. We investigated whether the expression of TRF2, which preserves telomere integrity, and have an effect on tumor immunosurveillance notably by directly recruiting and activating myeloid-derived suppressor cells (MDSCs), could be a prognostic biomarker in patients with relapsed or metastatic melanoma based on different treatment regimens. We evaluated retrospectively the association of TRF2 expressed in melanoma cells in combination with intratumoral CD33+ CD15+ CD14- MDSCs, as detected by immunohistochemistry and quantified by digital analysis, to clinicopathological features and overall survival (OS) among 48 patients treated with ICIs and 77 patients treated with other treatment options. The densities/mm2 of TRF2+ cells (P=.003) and CD33+ cells (P=.004) were individually significantly related to poor OS. In addition, only the combined expression of CD33+/CD15+/CD14- cells/mm2 was significantly correlated to poor OS (P=.017) in the whole study population as well as in patients treated by ICIs (P=.023). There was no significant difference in OS when analyzing the other markers individually or in combination according to the treatment regimen. The pre-treatment assessment of TRF2 expression and CD33+ cells/mm2 along with the density of CD33+/CD15+/CD14- cells/mm2 could assess OS and better predict clinical response of patients with melanoma treated by ICIs.

In non-small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with programmed cell death ligand 1 expression that is induced by interferon-γ-produced by tumor-infiltrating CD8+ T cells. However, not all tumors with a PD-L1 expression and/or CD8+ T cell infiltrate respond to ICB, and some tumors without any PD-L1 expression respond to ICB. Moreover, little is known about all the mechanisms governing ICB resistance in NSCLC. The objective of the study was to investigate a circulating immunological signature (cytokines, chemokines and immune checkpoints) which could be predictive of resistance to ICB in patients with advanced NSCLC. We performed a multiplexed analysis on 23 TruCulture® (in vitro T cells activation system) and 41 plasma samples using the Luminex® platform (Bio-Techne, MN USA). We investigated the relationship between the levels at baseline of 30 circulating analytes and the response to ICB of advanced NSCLC patients. Through the TruCulture® samples analysis, we identified two types of responders depending on T cell functionality. The responders with a functional T cell activation had lower levels of neutrophil associated analytes (CXCL5/6; p-value Citation Format: Wassila Khatir, Olivier Humbert, Jaap Neels, Léa Berland, Jonathan Benzaquen, Fabian Andrés Gallardo Rivera, Maryline Allegra, Myriam Salah, Virginie Tanga, Olivier Bordone, Julien Fayada, Virginie Lespinet-Fabre, Elodie Long-Mira, Sandra Lassalle, Patrick Brest, Valérie Vouret, Charlotte Maniel, Jacques Boutros, Simon Heeke, Véronique Hofman, Charles-Hugo Marquette, Paul Hofman, Marius Ilie. Identification of a predictive circulating immunological signature of response to immune checkpoint inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1265.

In only a few months after its inception, the COVID-19 pandemic lead to the death of hundreds of thousands of patients and to the infection of millions of people on most continents, mostly in the United States and in Europe. During this crisis, it was demonstrated that a better understanding of the pathogenicity, virulence, and contagiousness of SARS-CoV-2, all of which were initially underestimated, was urgently needed. The development of diagnostic tests to identify SARS-CoV-2 or to detect anti-SARS-CoV2 antibodies in blood, of vaccines, and of preventive and curative treatments has been relying on intense activity of scientists in academia and industry. It is noteworthy that these scientists depend on the use of high-quality biological samples taken from positive COVID-19 patients in a manner that preserves their integrity. Given this unique and emergent situation, it was necessary to urgently establish biological collections clinically annotated for immediate development of clinical and translational research projects focusing on COVID-19 biological aspects. It is in this very specific context that biobanks must rapidly adapt their infrastructure and/or operational capacity to fulfill new critical needs. We report the establishment of a biobank dedicated to the collection of blood-derived products (plasma, serum, and leukocytes) from COVID-19 patients hospitalized in the Nice Pasteur Hospital (Nice, France).

Introduction Tumor mutational burden (TMB) has been proposed as a novel predictive biomarker for the stratification of patients with NSCLC undergoing immune checkpoint inhibitor (ICI) treatment. The assessment of TMB has recently been established using large targeted sequencing panels, and numerous studies are ongoing to harmonize TMB assessment. "Correlation" or the coefficient of determination has generally been used to evaluate the association between different panels. We hypothesized that these metrics might overestimate the comparability, especially for lower TMB values. Methods A total of 30 samples from patients with NSCLC undergoing ICI treatment were consecutively sequenced using the following three large, targeted sequencing panels: FoundationOne, Oncomine TML, and QiaSeq TMB. The TMB values were compared in the whole patient population and in a subset of patients in which the TMB assessed by FoundationOne was between 5 and 25 mutations/Mb. Prediction of durable clinical benefit (>6 mo with no progression) was assessed using receiver operator characteristics, and optimal cutoff values were calculated using the Youden J statistic. Results Correlation between the three targeted sequencing panels was strong in the whole patient population (R2 > 0.79) but was dramatically reduced in the subset of patients with TMB of 5 to 25 mutations/Mb. The agreement assessed using the Bland-Altman method was also very low. All panels were able to predict durable clinical benefit in the TMB-high population. Conclusions Assessment of TMB using the three targeted sequencing panels was possible and predictive of response to ICI treatment, but correlation was an inappropriate measurement to assess the association between the respective panels.

Background: Assessment of actionable EGFR mutations is mandatory for treatment-naïve advanced or metastatic non-squamous lung carcinoma (NSLC), but the results need to be obtained in less than 10 working days. For rapid EGFR testing, an EGFR-specific polymerase chain reaction (PCR) assay is an alternative and simple approach compared to next generation sequencing (NGS). Here, we describe how a rapid EGFR-specific PCR assay can be implemented in a single laboratory center (LPCE, Nice, France) as reflex testing in treatment-naïve NSLC. Methods: A total of 901 biopsies from NSLC with more than 10% of tumor cells were prospectively and consecutively evaluated for EGFR mutation status between November 2017 and December 2019 using the Idylla system (Biocartis NV, Mechelen, Belgium). NGS was performed for nonsmokers with NSLC wild type for EGFR, ALK, ROS1, and BRAF and with less than 50% PD-L1 positive cells using the Hotspot panel (Thermo Fisher Scientific, Waltham, MA, USA). Results: Results were obtained from 889/901 (97%) biopsies with detection of EGFR mutations in 114/889 (13%) cases using the Idylla system. Among the 562 EGFR wild type tumors identified with Idylla, NGS detected one actionable and one nonactionable EGFR mutation. Conclusions: Rapid and targeted assessment of EGFR mutations in treatment-naïve NSLC can be implemented in routine clinical practice. However, it is mandatory to integrate this approach into a molecular algorithm that allows evaluation of potentially actionable genomic alterations other than EGFR mutations.

The mutation status of the BRAF and NRAS genes in tumor tissue is used to select patients with metastatic melanoma for targeted therapy. Cell-free circulating DNA (cfDNA) represents an accessible, non-invasive surrogate sample that could provide a snapshot of the BRAF and NRAS genotype in these patients. We investigated the feasibility of the Idylla™ assay for detection of BRAF and NRAS mutations in cfDNA of 19 patients with metastatic melanoma at baseline and during the course of treatment. The cfDNA genotype obtained with Idylla was compared to the results obtained with matched-tumor tissue and to clinical outcome. At baseline, 47% of patients harbored a BRAFV600 mutation in their cfDNA. Two months after targeted treatment the BRAFV600 mutant cfDNA was undetectable in all patients and 3 were disease-free. Moreover, 15% of patients harbored a NRAS mutation that was detected with plasma before treatment. The sensitivity and specificity were 80% and 89% for the BRAF status, and 79% and 100% for the NRAS status in pretreatment cfDNA compared to results obtained with a tissue test. Due to the small size of the population, no significant correlation was observed between the presence of BRAF or NRAS mutations in cfDNA and the metastatic tumor load or overall survival. In conclusion, this study demonstrated that evaluation with the Idylla system of the BRAF and NRAS mutation status in cfDNA may be a surrogate for determination of the BRAF and NRAS status in tumor tissue.

// Marius Ilie 1, 2, 3 , Catherine Butori 1, 3 , Sandra Lassalle 1, 2, 3 , Simon Heeke 2 , Nicolas Piton 4 , Jean-Christophe Sabourin 4 , Virginie Tanga 3 , Kevin Washetine 1, 3 , Elodie Long-Mira 1, 2, 3 , Priscilla Maitre 3 , Nathalie Yazbeck 2 , Olivier Bordone 3 , Virginie Lespinet 3 , Sylvie Leroy 5 , Charlotte Cohen 6 , Jerome Mouroux 2, 6 , Charles Hugo Marquette 2, 5 , Veronique Hofman 1, 2, 3 and Paul Hofman 1, 2, 3 1 Laboratory of Clinical and Experimental Pathology, Pasteur Hospital, FHU OncoAge, University Cote d’Azur, Nice, France 2 IRCAN Inserm U1081/CNRS 7284, FHU OncoAge, University Cote d’Azur, Nice, France 3 Hospital-related Biobank (BB-0033-00025), Pasteur Hospital, FHU OncoAge, University Cote d’Azur, Nice, France 4 Department of Pathology, Rouen University Hospital, Rouen, France 5 Department of Pulmonary Medicine and Thoracic Oncology, Pasteur Hospital, FHU OncoAge, University Cote d’Azur, Nice, France 6 Department of Thoracic Surgery, Pasteur Hospital, FHU OncoAge, University Cote d’Azur, Nice, France Correspondence to: Paul Hofman, email: hofman.p@chu-nice.fr Keywords: NSCLC, EGFR, targeted therapy, optimization, PCR Received: March 21, 2017 Accepted: September 19, 2017 Published: October 04, 2017 ABSTRACT Treatment with EGFR inhibitors is limited to patients with advanced/metastatic non-small cell lung cancer who have known EGFR mutations. Currently, patient care has to respond to several imperatives to make these inhibitors broadly available to all patients; fast and accurate detection of EGFR mutations by a sensitive and specific standardized cost-effective method, easy-to-implement in settings with limited expertise in molecular diagnostics. We evaluated the Idylla™ EGFR Mutation Assay (Biocartis) for the detection of EGFR mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples from a series of 55 patients with lung adenocarcinoma and compared these results with those obtained by a pyrosequencing ISO-15189 accredited laboratory method. The comparison was made on both whole surgical tumor sections and on three artificially constructed small biopsies (~1 mm) from the same FFPE blocks. Cost-effectiveness and turnaround time comparison between the two methods was performed. On both whole tissue sections and on biopsy cores, the Idylla™ and pyrosequencing had an agreement of 95% (52/55). The Idylla™ EGFR Assay produced results faster and more cost-effective than pyrosequencing. The Idylla™ system showed a good sensitivity and was cost-saving in our setting. Because of the easy workflow, the Idylla™ system has the potential to expand EGFR testing to more pathology laboratories in a reliable and fast manner.

Resume Les tests d’evaluation du statut PD-L1 realises en immunohistochimie sont proposes dans les essais cliniques en oncologie thoracique afin de predire la reponse aux immunotherapies ciblant l’axe PD-1/PD-L1. Avec l’arrivee de cette nouvelle classe de molecules en deuxieme ligne et tres rapidement en premiere ligne dans le traitement des patients atteints d’un cancer pulmonaire non a petites cellules de stade avance ou metastatique, ces tests devront certainement etre realises en routine des l’obtention de l’autorisation de mise sur le marche de ces traitements. La mise en place tres rapide de tests « compagnons » ou « complementaires » semble indispensable pour selectionner les patients devant beneficier de ces therapeutiques efficaces mais aussi couteuses et parfois toxiques. Bien que contestee (en particulier du fait que certains patients n’exprimant pas PD-L1 puissent parfois repondre aux therapeutiques ciblant l’axe PD1/PD-L1), l’approche immunohistochimique anti-PD-L1 parait incontournable en 2017. Cette nouvelle activite deployee dans les laboratoires d’ACP souleve desormais plusieurs questions : quel clone anti-PD-L1 utiliser ? Sur quel automate ? Quel seuil de positivite doit-on considerer ? Faut-il evaluer le marquage PD-L1 sur les cellules tumorales mais aussi sur les cellules immunitaires ? Quels controles doit-on utiliser ? Des etudes comparatives sont en cours ou ont ete deja realisees afin de pouvoir repondre a certaines de ces questions. Cette revue aborde les differents criteres d’evaluation immunohistochimique des principaux anticorps anti-PD-L1 utilises a ce jour et les travaux realises recemment utilisant ces anticorps en oncologie thoracique.

International audience; Background - The introduction of liquid biopsy using PCR-based assays into routine practice has had a strong impact on the treatment of EGFR-mutated lung adenocarcinoma and is now commonly used for routine testing of EGFR mutations in certain clinical settings. To assess whether the claimed benefits of PCR-based assays hold true in daily practice at a multicenter clinical institution, we assessed how treatment decisions are affected by PCR-based assays for the analysis of EGFR mutations from plasma samples in a centralized laboratory (LPCE, Nice, France). Patients and methods - A total of 345 samples were analyzed using the US Food and Drug Administration-approved Cobas EGFR Mutation Test v2 and 103 using the Therascreen EGFR Plasma RGQ PCR Kit over 3 years (395 samples from 324 patients). Eleven plasma samples were validated independently using Cobas at 3 institutions, and 130 samples were analyzed using Stilla digital PCR. Clinical data were collected for 175 (54%) of 324 patients. Results - Cobas was superior to the Therascreen assay and demonstrated 100% reproducibility. Digital PCR showed only 48%, 83%, and 58% concordance with Cobas for exon 19 deletions, L858R mutations, and T790M mutations, respectively. Liquid biopsies helped inform and change treatment when resistance occurred and enabled the detection of EGFR mutations in patients when biopsy tissue results were unavailable. Conclusion - PCR-based assays are a fast and convenient test, allowing the detection of primary and secondary EGFR mutations from plasma. Cobas proved to be a reliable test, whereas digital PCR produced too many inconclusive results to be currently recommended as a principal testing device.

Introduction The detection of a ROS1 rearrangement in advanced and metastatic lung adenocarcinoma (LUAD) led to a targeted treatment with tyrosine kinase inhibitors with favorable progression-free survival and overall survival of the patients. Thus, it is mandatory to screen for the ROS1 rearrangement in all these patients. ROS1 rearrangements can be detected using break-apart fluorescence in situ hybridization (FISH), which is the gold standard; however, ROS1 immunohistochemistry (IHC) can be used as a screening test because it is widely available, easy and rapid to perform, and cost-effective. Methods We evaluated the diagnostic accuracy and interpathologist agreement of two anti-ROS1 IHC clones, SP384 (Ventana, Tucson, Arizona) and D4D6 (Cell Signaling, Danvers, Massachusetts), in a training cohort of 51 positive ROS1 FISH LUAD cases, and then in a large validation cohort of 714 consecutive cases of LUAD from six routine molecular pathology platforms. Results In the two cohorts, the SP384 and D4D6 clones show variable sensitivity and specificity rates on the basis of two cutoff points greater than or equal to 1+ (all % tumor cells) and greater than or equal to 2+ (>30% stained tumor cells). In the validation cohort, the D4D6 yielded the best accuracy for the presence of a ROS1 rearrangement by FISH. Interpathologist agreement was moderate to good (interclass correlation 0.722–0.874) for the D4D6 clone and good to excellent (interclass correlation: 0.830–0.956) for the SP384 clone. Conclusions ROS1 IHC is an effective screening tool for the presence of ROS1 rearrangements. However, users must be acutely aware of the variable diagnostic performance of different anti-ROS1 antibodies before implementation into routine clinical practice.

Histopathology is the fundamental tool of pathology used for more than a century to establish the final diagnosis of lung cancer. In addition, the phenotypic data contained in the histological images reflects the overall effect of molecular alterations on the behavior of cancer cells and provides a practical visual reading of the aggressiveness of the disease. However, the human evaluation of the histological images is sometimes subjective and may lack reproducibility. Therefore, computational analysis of histological imaging using so-called “artificial intelligence” (AI) approaches has recently received considerable attention to improve this diagnostic accuracy. Thus, computational analysis of lung cancer images has recently been evaluated for the optimization of histological or cytological classification, prognostic prediction or genomic profile of patients with lung cancer. This rapidly growing field constantly demonstrates great power in the field of computing medical imaging by producing highly accurate detection, segmentation or recognition tasks. However, there are still several challenges or issues to be addressed in order to successfully succeed the actual transfer into clinical routine. The objective of this review is to emphasize recent applications of AI in pulmonary cancer pathology, but also to clarify the advantages and limitations of this approach, as well as the perspectives to be implemented for a potential transfer into clinical routine., L’histopathologie est l’outil fondamental utilisé en anatomopathologie depuis plus d’un siècle pour établir le diagnostic final d’un carcinome bronchopulmonaire. L’information phénotypique présente sur les images histologiques reflète l’effet global des altérations moléculaires sur le comportement des cellules cancéreuses et fournit une lecture visuelle pratique de l’agressivité de la maladie. Cependant, l’évaluation humaine de l’image histologique peut être parfois subjective et assez peu reproductible selon les cas. Par conséquent, l’analyse computationnelle de l’imagerie histologique via des approches dites « d’intelligence artificielle » (IA) a récemment reçu une attention considérable afin d’améliorer cette précision diagnostique. Ainsi, l’analyse computationnelle d’images de cancer du poumon a récemment été évaluée pour l’optimisation de la classification histologique ou cytologique, la prédiction du pronostic ou du profil génomique des patients atteints d’un cancer pulmonaire. Ce domaine, en pleine croissance, fait constamment preuve d’une grande puissance dans le domaine de l’informatique d’imagerie médicale en produisant des tâches de détection, de segmentation ou de reconnaissance d’une très grande précision. Cependant, il subsiste plusieurs défis ou enjeux majeurs à relever afin de réussir le transfert réel de cette nouvelle approche en routine clinique. L’objectif de cette revue est de faire le point sur les applications récentes de l’IA en pathologie cancéreuse pulmonaire, mais aussi d’apporter des clarifications sur les avantages et les limites de cette approche, ainsi que les perspectives à mettre en œuvre pour un transfert potentiel dans la pratique quotidienne des pathologistes.

As targeted molecular therapies and immuno-oncology have become pivotal in the management of patients with lung cancer, the essential requirement for high throughput analyses and clinical validation of biomarkers has become even more intense, with response rates maintained in the 20%–30% range. Moreover, the list of treatment alternatives, including combination therapies, is rapidly evolving. The molecular profiling and specific tumor-associated immune contexture may be predictive of response or resistance to these therapeutic strategies. Multiplexed immunohistochemistry is an effective and proficient approach to simultaneously identify specific proteins or molecular abnormalities, to determine the spatial distribution and activation state of immune cells, as well as the presence of immunoactive molecular expression. This method is highly advantageous for investigating immune evasion mechanisms and discovering potential biomarkers to assess mechanisms of action and to predict response to a given treatment. This review provides views on the current technological status and evidence for clinical applications of multiplexing and how it could be applied to optimize clinical management of patients with lung cancer.

Resume L’activite des laboratoires d’anatomo-cyto-pathologie (ACP) s’est considerablement modifiee ces dernieres annees en raison de l’evolution des pratiques cliniques et des progres therapeutiques. Ainsi, par exemple, le developpement des therapies ciblees et de l’immunotherapie a recemment bouleverse la prise en charge des echantillons tissulaires et cytologiques, mais aussi des echantillons sanguins a visee diagnostique, pronostique et predictive de reponse aux nouvelles therapies. Dans ce contexte, il est devenu essentiel d’optimiser les bonnes pratiques dans les laboratoires d’ACP, afin d’assurer une offre de soins de qualite en collaboration etroite avec les cliniciens. Si la plupart des pays industrialises ont mis en place depuis plusieurs annees un systeme d’accreditation dans les laboratoires d’ACP, ceci n’est pas, ou peu, le cas en France. La norme d’accreditation applicable en France pour les laboratoires d’analyse medicale est la norme ISO 15189. Cette accreditation est delivree par un organisme, le COFRAC (comite francais d’accreditation), apres audit de la structure. Si cette norme est bien adaptee aux pratiques de la biologie medicale, elle est souvent discutable pour les pathologistes. Il est neanmoins incontournable de s’engager rapidement dans une demarche d’accreditation pour les laboratoires d’ACP en France afin de repondre aux nouvelles exigences de la pathologie clinique et moleculaire et notamment pour rester competitif en Europe. Cette revue aborde les enjeux actuels et futurs de l’accreditation des laboratoires d’ACP en France mais aussi les principales contraintes et potentielles faiblesses de la norme ISO 15189 dans le cadre des activites de l’ACP.

Resume Les biobanques ou centres de ressources biologiques (CRB) sont les maillons essentiels de la recherche translationnelle. Ces structures beneficient de financements publics recurrents afin d’etablir des collections d’echantillons biologiques utilisees dans le cadre de projets de recherche. Plus de 100 biobanques ont ete developpees sur le territoire national au sein des hopitaux universitaires et des centres de lutte contre le cancer. Le developpement de nouvelles techniques et de nouvelles methodes d’analyses dans la recherche medicale bouleversent les pratiques et soulevent de nouveaux defis scientifiques et organisationnels dans la collecte, la transformation et la conservation des echantillons. Relever ces defis devient un enjeu primordial pour les biobanques. La mise en place d’une strategie dynamique pour anticiper les besoins des utilisateurs est necessaire et repose sur de nouveaux modes de fonctionnement. Cet article decrit les enjeux a court et a moyen terme pour les biobanques francaises et propose des elements d’organisation pour y repondre.

PD-1/PD-L1 inhibitors demonstrated durable clinical responses in patients with lung squamous cell carcinoma. However, the expression pattern of PD-L1 and the presence of CD8+ and PD-1+ tumor-infiltrating T cells in the basaloid variant of squamous cell carcinoma remain unknown. immunohistochemistry analysis of PD-L1 expression, with three recently validated monoclonal antibodies used in clinical trials (clones SP142, SP263, and 28-8), and detection of CD8+ and PD-1+ tumor-infiltrating T cells was performed on whole-tissue sections from 56 patients following surgery for basaloid squamous cell carcinoma. Data were correlated to clinicopathological parameters and outcome. Fair to poor concordance was observed between the SP142 vs SP263 clones, and SP142 vs 28-8 (κ range, 0.018-0.412), while the 28-8 and SP263 demonstrated a strong correlation in both the tumor cell and immune cell compartments (κ=0.883, and κ=0.721). Expression of PD-L1 correlated with a high content of CD8+ and PD-1+ tumor-infiltrating T cells when using SP142 (P=0.012; P=0.022), but not with SP263 or 28-8 (P=0.314; P=0.611). In the multivariate analysis, we found significantly better disease-free and overall survival rates for high PD-L1 expression with SP142, CD8+ and PD-1+ tumor-infiltrating T cells (P=0.003; P=0.007). No significant prognosis value was observed for SP263 and 28-8 clones, except a correlation between improved overall survival and SP263 in the univariate analysis (P=0.039), not confirmed in the multivariate model. In conclusion, we report that the expression of PD-L1 and the content of CD8+ and PD-1+ tumor-infiltrating T cells is an independent indicator of better outcome in basaloid squamous cell carcinoma patients, although the observed effect is dependent on the PD-L1 immunohistochemistry assay.

Resume L’immunotherapie ciblant l’axe PD-L1/PD-1 a recemment montre des resultats prometteurs chez les patients atteints d’un cancer du poumon en phase metastatique. Cette approche semble d’autant plus efficace qu’il existe une forte expression immunohistochimique de PD-L1 sur les biopsies bronchiques. Un tel traitement sera donc tres prochainement propose chez ces patients atteints d’un cancer du poumon en phase metastatique. L’indication d’une immunotherapie pourrait etre conditionnee a l’evaluation immunohistochimique de PD-L1, celle-ci devenant ainsi un test diagnostique « compagnon ». Cette perspective a court terme souleve plusieurs interrogations et en particulier le seuil de positivite du marquage PD-L1 a definir pour considerer que le patient est eligible a une immunotherapie. D’autres parametres devront etre pris en compte, comme l’evaluation du compartiment cellulaire positif (cellules immunitaires et/ou cellules tumorales), le nombre de cellules positives et le clone utilise. Un certain nombre de patients peut repondre au traitement ciblant l’axe PD-L1/PD-1 malgre la faible ou l’absence d’expression immunohistochimique de PD-L1. Malgre cela, l’approche immunohistochimique pourrait etre la seule conseillee en oncologie thoracique avant de decider un traitement ciblant PD-L1/PD1. Cet article discute les principaux challenges de l’approche immunohistochimique ciblant PD-L1 en tant que test diagnostique « compagnon » associe a l’immunotherapie des cancers du poumon.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib. Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.

In the last few years, the treatment of patients with non-small cell lung cancer (NSCLC) has impressively benefitted from immunotherapy, in particular from the inhibition of immune checkpoints such as programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1). However, despite the significant survival benefit for some patients with advanced NSCLC, the objective response rates (ORRs) remain relatively low no more than 20-30% with a large proportion of patients demonstrating primary resistance. Although the selection of NSCLC patients for the first-line treatment is currently guided by the expression of PD-L1 in tumor cells as detected by immunohistochemistry, this is not the case for the second-line setting. Moreover, the sensitivity and specificity of PD-L1 expression is modest which has prompted the search for additional predictive biomarkers. In this context, the assessment of the tumor mutational burden (TMB), defined as the total number of nonsynonymous mutations in the coding regions of genes, has recently emerged as an additional powerful biomarker to select patients for immunotherapy. The purpose of our review is to highlight the recent advances as well as the challenges and perspectives in the field of TMB and immunotherapy for patients with NSCLC.

Tumor mutational burden (TMB) has emerged as an important potential biomarker for prediction of response to immune-checkpoint inhibitors (ICIs), notably in non-small cell lung cancer (NSCLC). However, its in-house assessment in routine clinical practice is currently challenging and validation is urgently needed. We have analyzed sixty NSCLC and thirty-six melanoma patients with ICI treatment, using the FoundationOne test (FO) in addition to in-house testing using the Oncomine TML (OTML) panel and evaluated the durable clinical benefit (DCB), defined by &gt, 6 months without progressive disease. Comparison of TMB values obtained by both tests demonstrated a high correlation in NSCLC (R2 = 0.73) and melanoma (R2 = 0.94). The association of TMB with DCB was comparable between OTML (area-under the curve (AUC) = 0.67) and FO (AUC = 0.71) in NSCLC. Median TMB was higher in the DCB cohort and progression-free survival (PFS) was prolonged in patients with high TMB (OTML HR = 0.35, FO HR = 0.45). In contrast, we detected no differences in PFS and median TMB in our melanoma cohort. Combining TMB with PD-L1 and CD8-expression by immunohistochemistry improved the predictive value. We conclude that in our cohort both approaches are equally able to assess TMB and to predict DCB in NSCLC.

Histopathology is the fundamental tool of pathology used for more than a century to establish the final diagnosis of lung cancer. In addition, the phenotypic data contained in the histological images reflects the overall effect of molecular alterations on the behavior of cancer cells and provides a practical visual reading of the aggressiveness of the disease. However, the human evaluation of the histological images is sometimes subjective and may lack reproducibility. Therefore, computational analysis of histological imaging using so-called "artificial intelligence" (AI) approaches has recently received considerable attention to improve this diagnostic accuracy. Thus, computational analysis of lung cancer images has recently been evaluated for the optimization of histological or cytological classification, prognostic prediction or genomic profile of patients with lung cancer. This rapidly growing field constantly demonstrates great power in the field of computing medical imaging by producing highly accurate detection, segmentation or recognition tasks. However, there are still several challenges or issues to be addressed in order to successfully succeed the actual transfer into clinical routine. The objective of this review is to emphasize recent applications of AI in pulmonary cancer pathology, but also to clarify the advantages and limitations of this approach, as well as the perspectives to be implemented for a potential transfer into clinical routine.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (≥1%, ≥50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

With underrepresentation of elderly patients with lung adenocarcinoma (LADC) in anti-PD-1/PD-L1 clinical trials, better understanding of the interplay of PD-L1 and tumor-associated immune cells (TAICs) could assist clinicians in stratifying these patients for immunotherapy. One hundred and one patients with LADCs, stratified by age, were included for analysis of PD-L1 expression and density of TAICs expressing CD4, CD8, and CD33, by using multiplex chromogenic immunohistochemistry (IHC) assays and automated digital quantification. The CD4+/CD8+ ratio was significantly higher in elderly patients. In patients &lt, 75 years, the density of CD4+, CD8+, and PD-L1 in TAICs showed a positive significant correlation with PD-L1 expression in tumor cells (TCs), while a lower correlation was observed in the elderly population. In the latter, a high CD4+/CD8+ ratio, and combined PD-L1 expression &ge, 1% TCs with a low CD8+ density, low CD33+ density, and a high CD4+ density correlated to worse overall survival. We identified differences according to age in the CD4+/CD8+ ratio and in correlation between PD-L1 expression and the density of TAICs in LADC patients. Distinct groups of tumor microenvironments had an impact on the OS of elderly patients with LADC.

The identification of certain genomic alterations (EGFR, ALK, ROS1, BRAF) or immunological markers (PD-L1) in tissues or cells has led to targeted treatment for patients presenting with late stage or metastatic lung cancer. These biomarkers can be detected by immunohistochemistry (IHC) and/or by molecular biology (MB) techniques. These approaches are often complementary but depending on, the quantity and quality of the biological material, the urgency to get the results, the access to technological platforms, the financial resources and the expertise of the team, the choice of the approach can be questioned. The possibility of detecting simultaneously several molecular targets, and of analyzing the degree of tumor mutation burden and of the micro-satellite instability, as well as the recent requirement to quantify the expression of PD-L1 in tumor cells, has led to case by case development of algorithms and international recommendations, which depend on the quality and quantity of biological samples. This review will highlight the different predictive biomarkers detected by IHC for treatment of lung cancer as well as the present advantages and limitations of this approach. A number of perspectives will be considered.

Background High expression of programmed death ligand-1 (PD-L1) on tumor cells (TC) and/or on tumor-infiltrating immune cells (IC) is associated with a high response rate in patients with advanced nonsmall-cell lung cancer (NSCLC) treated with PD-L1 inhibitors. The use of a PD-L1 immunohistochemical (IHC) test in determining the responsiveness to immunotherapy has raised the question of the reliability and reproducibility of its evaluation in lung biopsies compared with corresponding resected surgical specimens. Patients and methods PD-L1 expression in TC and IC was assessed in 160 patients with operable NSCLC on both whole surgical tissue sections and matched lung biopsies, by using a highly sensitive SP142 IHC assay. The specimens were scored as TC 0–3 and IC 0–3 based on increasing PD-L1 expression. Results PD-L1 expression was frequently discordant between surgical resected and matched biopsy specimens (the overall discordance rate=48%; 95% confidence interval 4.64–13.24) and κ value was equal to 0.218 (poor agreement). In all cases, the biopsy specimens underestimated the PD-L1 status observed on the whole tissue sample. PD-L1-positive IC tumors were more common than PD-L1-positive TC tumors on resected specimens. The discrepancies were mainly related to the lack of a PD-L1-positive IC component in matched biopsies. Conclusions Our results indicate relatively poor association of the PD-L1 expression in TC and IC between lung biopsies and corresponding resected tumors. Although these results need to be further validated in larger cohorts, they indicate that the daily routine evaluation of the PD-L1 expression in diagnostic biopsies can be misleading in defining the sensitivity to treatment with PD-L1 targeted therapy.

Background Mortality in early stage, resectable lung cancer is sufficiently high to warrant consideration of post-surgical treatment. Novel markers to stratify resectable lung cancer patients may help with the selection of treatment to improve outcome. Methods Primary tumour tissue from 485 patients, surgically treated for stage I–II lung adenocarcinoma, was analysed for the RNA expression of 31 cell cycle progression (CCP) genes by quantitative polymerase chain reaction (PCR). The expression average, the CCP score, was combined with pathological stage into a prognostic score (PS). Cox proportional hazards regression assessed prediction of 5-year lung cancer mortality above clinical variables. The PS threshold was tested for risk discrimination by the Mantel–Cox log-rank test. Results The CCP score added significant information above clinical markers (all patients, P = 0.0029; stage I patients, P = 0.013). The prognostic score was a superior predictor of outcome compared to pathological stage alone (PS, P = 0.00084; stage, P = 0.24). Five-year lung cancer mortality was significantly different between the low-risk (90%, 95% confidence interval (CI) 81–95%), and high-risk groups (65%, 95% CI 57–72%), P = 4.2 × 10–6). Conclusions The CCP score is an independent prognostic marker in early stage lung adenocarcinoma. The prognostic score provides superior risk estimates than stage alone. The threefold higher risk in the high-risk group defines a subset of patients that should consider therapeutic choices to improve outcome.