Characterization of the perioperative changes of exosomal immune-related cytokines induced by prostatectomy in early-stage prostate cancer patients.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are relevant in prostate cancer microenvironment collaborating in tumor development. The main tumor marker used in this disease, prostate-specific antigen (PSA), does not provide information related to this tumor microenvironment. Cancer cells secrete exosomes carrying bioactive molecules contributing to MDSCs recruitment and induction. The aim of this study was to characterize the perioperative changes of exosomal cytokines relevant in MDSCs recruitment induced by prostatectomy in prostate cancer patients. METHODS: Blood was drawn from 26 early-stage prostate cancer patients before and after radical prostatectomy and from 16 healthy volunteers. Serum exosomes were separated by precipitation. Cytokines related with MDSC cell recruitment and activation CCL2, CXCL2, CXCL5, CXCL8, CXCL12, MIF, S100A9 and TGF-ß were measured in serum and serum-derived exosomes using immunometric assays. RESULTS: All cytokines were detected both in serum and exosomes, except for CXCL12, which was detected only in serum. Exosomes were enriched specially in MIF, TGF-ß and CXCL2. Presurgical MIF levels in exosomes correlated negatively with serum PSA. Also, presurgical TGF-ß decreased both in serum and exosomes as Gleason score rises. Patients presurgical exosomes had increased CCL2, CXCL5 and TGF-ß levels than exosomes from healthy controls. These differences were not observed when cytokines were analyzed in serum, except for TGF-ß. Cytokine levels of CCL2, CXCL5 decreased in patients' postsurgical exosomes, while TGF-ß further increased. On the contrary, S100A9 levels were lower in patients presurgical exosomes but increased after radical prostatectomy. CONCLUSIONS: Blood exosomal content in cytokines constitute an attractive source to evaluate MDSCs immunomodulators providing additional information related to tumor microenvironment in prostate cancer.

Performance comparison of two next-generation sequencing panels to detect actionable mutations in cell-free DNA in cancer patients.

Background Genomic alterations studies in cell-free DNA (cfDNA) have increasing clinical use in oncology. Next-generation sequencing (NGS) technology provides the most complete mutational analysis, but nowadays limited data are available related to the comparison of results reported by different platforms. Here we compare two NGS panels for cfDNA: Oncomine™ Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific), suitable for clinical laboratories, and Guardant360® (GuardantHealth), with more genes targeted but only available in an outsourcing laboratory. Methods Peripheral blood was obtained from 16 advanced cancer patients in which Guardant360® (G360) was requested as part of their clinical assistance. Blood samples were sent to be analyzed with G360 panel, and an additional blood sample was drawn to obtain and analyze cfDNA with Oncomine™ Pan-Cancer (OM) panel in an Ion GeneStudio S5™ System. Results cfDNA analysis globally rendered 101 mutations. Regarding the 55/101 mutations claimed to be included by manufacturers in both panels, 17 mutations were reported only by G360, 10 only by OM and 28 by both. In those coincident cases, there was a high correlation between the variant allele fractions (VAFs) calculated with each panel (r = 0.979, p < 0.01). Regarding the six actionable mutations with an FDA-approved therapy reported by G360, one was missed with OM. Also, 12 mutations with clinical trials available were reported by G360 but not by OM. Conclusions In summary, G360 and OM can produce different mutational profile in the same sample, even in genes included in both panels, which is especially important if these mutations are potentially druggable.

Identification of mutations associated with acquired resistance to sunitinib in renal cell cancer.

Sunitinib is one of the most widely used targeted therapeutics for renal cell carcinoma (RCC), but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in RCC, we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and after development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in silico prediction models, six predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1, and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function renders tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the six proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.

Combined clinical and genomic signatures for the prognosis of early stage non-small cell lung cancer based on gene copy number alterations.

BACKGROUND: The development of a more refined prognostic methodology for early non-small cell lung cancer (NSCLC) is an unmet clinical need. An accurate prognostic tool might help to select patients at early stages for adjuvant therapies. RESULTS: A new integrated bioinformatics searching strategy, that combines gene copy number alterations and expression, together with clinical parameters was applied to derive two prognostic genomic signatures. The proposed methodology combines data from patients with and without clinical data with a priori information on the ability of a gene to be a prognostic marker. Two initial candidate sets of 513 and 150 genes for lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively, were generated by identifying genes which have both: a) significant correlation between copy number and gene expression, and b) significant prognostic value at the gene expression level in external databases. From these candidates, two panels of 7 (ADC) and 5 (SCC) genes were further identified via semi-supervised learning. These panels, together with clinical data (stage, age and sex), were used to construct the ADC and SCC hazard scores combining clinical and genomic data. The signatures were validated in two independent datasets (n = 73 for ADC, n = 97 for SCC), confirming that the prognostic value of both clinical-genomic models is robust, statistically significant (P = 0.008 for ADC and P = 0.019 for SCC) and outperforms both the clinical models (P = 0.060 for ADC and P = 0.121 for SCC) and the genomic models applied separately (P = 0.350 for ADC and P = 0.269 for SCC). CONCLUSION: The present work provides a methodology to generate a robust signature using copy number data that can be potentially used to any cancer. Using it, we found new prognostic scores based on tumor DNA that, jointly with clinical information, are able to predict overall survival (OS) in patients with early-stage ADC and SCC.

Role of [¹8F]FDG PET in prediction of KRAS and EGFR mutation status in patients with advanced non-small-cell lung cancer.

PURPOSE: The tumour molecular profile predicts the activity of epidermal growth factor receptor (EGFR) inhibitors in non-small-cell lung cancer (NSCLC). However, tissue availability and tumour heterogeneity limit its assessment. We evaluated whether [(18)F]FDG PET might help predict KRAS and EFGR mutation status in NSCLC. METHODS: Between January 2005 and October 2011, 340 NSCLC patients were tested for KRAS and EGFR mutation status. We identified patients with stage III and IV disease who had undergone [(18)F]FDG PET/CT scanning for initial staging. SUVpeak, SUVmax and SUVmean of the single hottest tumour lesions were calculated, and their association with KRAS and EGFR mutation status was assessed. A receiver operator characteristic (ROC) curve analysis and a multivariate analysis (including SUVmean, gender, age and AJCC stage) were performed to identify the potential value of [(18)F]FDG PET/CT for predicting KRAS mutation. RESULTS: From 102 patients staged using [(18)F]FDG PET/CT, 28 (27%) had KRAS mutation (KRAS+), 22 (22%) had EGFR mutation (EGFR+) and 52 (51%) had wild-type KRAS and EGFR profiles (WT). KRAS+ patients showed significantly higher [(18)F]FDG uptake than EGFR+ and WT patients (SUVmean 9.5, 5.7 and 6.6, respectively; p < 0.001). No significant differences were observed in [(18)F]FDG uptake between EGFR+ patients and WT patients. ROC curve analysis for KRAS mutation status discrimination yielded an area under the curve of 0.740 for SUVmean (p < 0.001). The multivariate analysis showed a sensitivity and specificity of 78.6% and 62.2%, respectively, and the AUC was 0.773. CONCLUSION: NSCLC patients with tumours harbouring KRAS mutations showed significantly higher [(18)F]FDG uptake than WT patients, as assessed in terms of SUVpeak, SUVmax and SUVmean. A multivariate model based on age, gender, AJCC stage and SUVmean might be used as a predictive marker of KRAS mutation status in patients with stage III or IV NSCLC.

First-line Management of Metastatic Urothelial Cancer: Current and Future Perspectives After the EV-302 and CheckMate-901 Studies.

The standard of care for the first-line management of metastatic urothelial carcinoma has been recently challenged, with the combination of pembrolizumab and enfortumab vedotin (P-EV) strongly arising as a practice-changing option from classical platinum-based chemotherapies. With this paradigm shift on the horizon new questions, including the most suitable second line of treatment for these patients, and the role that the molecular characterization of these tumours will have when selecting these therapies will inevitably arise. Furthermore, after the negative results of the Keynote 361 and IMvigor 130 trials, the combination of nivolumab with platinum-based chemotherapy followed by nivolumab maintenance (Nivo GC-Nivo) has also shown positive results when compared with chemotherapy alone. Translational studies at a molecular, cellular, and functional level will be key to better explain these discordant results. In this Current Perspective, we discuss the potential impact of these results in clinical practice and propose specific guidance for prospective translational research.

Whole exome sequencing and machine learning germline analysis of individuals presenting with extreme phenotypes of high and low risk of developing tobacco-associated lung adenocarcinoma.

BACKGROUND: Tobacco is the main risk factor for developing lung cancer. Yet, while some heavy smokers develop lung cancer at a young age, other heavy smokers never develop it, even at an advanced age, suggesting a remarkable variability in the individual susceptibility to the carcinogenic effects of tobacco. We characterized the germline profile of subjects presenting these extreme phenotypes with Whole Exome Sequencing (WES) and Machine Learning (ML). METHODS: We sequenced germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age (extreme cases) or who did not develop lung cancer at an advanced age (extreme controls), selected from databases including over 6600 subjects. We selected individual coding genetic variants and variant-rich genes showing a significantly different distribution between extreme cases and controls. We validated the results from our discovery cohort, in which we analysed by WES extreme cases and controls presenting similar phenotypes. We developed ML models using both cohorts. FINDINGS: Mean age for extreme cases and controls was 50.7 and 79.1 years respectively, and mean tobacco consumption was 34.6 and 62.3 pack-years. We validated 16 individual variants and 33 variant-rich genes. The gene harbouring the most validated variants was HLA-A in extreme controls (4 variants in the discovery cohort, p = 3.46E-07; and 4 in the validation cohort, p = 1.67E-06). We trained ML models using as input the 16 individual variants in the discovery cohort and tested them on the validation cohort, obtaining an accuracy of 76.5% and an AUC-ROC of 83.6%. Functions of validated genes included candidate oncogenes, tumour-suppressors, DNA repair, HLA-mediated antigen presentation and regulation of proliferation, apoptosis, inflammation and immune response. INTERPRETATION: Individuals presenting extreme phenotypes of high and low risk of developing tobacco-associated lung adenocarcinoma show different germline profiles. Our strategy may allow the identification of high-risk subjects and the development of new therapeutic approaches. FUNDING: See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript.

PSA reactivity in extracellular microvesicles to commercial immunoassays.

AIMS: Characterization of PSA in extracellular microvesicles (EVs) and its reactivity to commercial methods. MATERIALS AND METHODS: EVs derived from serum of 47 prostate cancer (PCa) patients, 27 benign prostatic hyperplasia (BPH) patients and 42 healthy controls were analyzed. EVs isolation and quantification of PSA immunoreactive to total (ev-T-PSA) or free (ev-F-PSA) PSA immunoassays, were performed using commercial assays. PSA in CD81+ or CD63+ EVs was determined directly in serum by an immunocapture-ELISA (IC-ELISA). RESULTS: Ev-T-PSA immunoreactive to Elecsys assay was detected in all samples. Median T-PSA ev/srm ratio was 2.20 % (Q1-Q3: 0.80-4.00 %), although in some samples this ratio reached 59 %. T-PSA ev/srm ratio was higher in those samples with serum T-PSA below 4 µg/L than in those exceeding that cut-off (p < 0.001). T-PSA ev/srm ratio was lower in PCa patients compared to healthy controls and BPH patients (p < 0.001). Elecsys immunoassays detected higher concentrations of ev-T-PSA and ev-F-PSA than Immulite (p < 0.001). PSA was detected by IC-ELISA more intensely in CD81+ EVs than in CD63+ EVs, and ev-T-PSA correlated with PSA+ CD63+ (p < 0.001) but not with PSA+ CD81+. CONCLUSION: EVs-bound PSA is another form of circulating PSA whose measurement could be easily performed in clinical laboratories by automated immunoassays.

Long-Term Outcome of Critically Ill Advanced Cancer Patients Managed in an Intermediate Care Unit.

Background: To analyze the long-term outcomes for advanced cancer patients admitted to an intermediate care unit (ImCU), an analysis of a do not resuscitate orders (DNR) subgroup was made. Methods: A retrospective observational study was conducted from 2006 to January 2019 in a single academic medical center of cancer patients with stage IV disease who suffered acute severe complications. The Simplified Acute Physiology Score 3 (SAPS 3) was used as a prognostic and severity score. In-hospital mortality, 30-day mortality and survival after hospital discharge were calculated. Results: Two hundred and forty patients with stage IV cancer who attended at an ImCU were included. In total, 47.5% of the cohort had DNR orders. The two most frequent reasons for admission were sepsis (32.1%) and acute respiratory failure (excluding sepsis) (38.7%). Mortality in the ImCU was 10.8%. The mean predicted in-hospital mortality according to SAPS 3 was 51.9%. The observed in-hospital mortality was 37.5% (standard mortality ratio of 0.72). Patients discharged from hospital had a median survival of 81 (30.75−391.25) days (patients with DNR orders 46 days (19.5−92.25), patients without DNR orders 162 days (39.5−632)). The observed mortality was higher in patients with DNR orders: 52.6% vs. 23.8%, p 0 < 0.001. By multivariate logistic regression, a worse ECOG performance status (3−4 vs. 0−2), a higher SAPS 3 Score and DNR orders were associated with a higher in-hospital mortality. By multivariate analysis, non-invasive mechanical ventilation, higher bilirubin levels and DNR orders were significantly associated with 30-day mortality. Conclusion: For patients with advanced cancer disease, even those with DNR orders, who suffer from acute complications or require continuous monitoring, an ImCU-centered multidisciplinary management shows encouraging results in terms of observed-to-expected mortality ratios.

YES1 Is a Druggable Oncogenic Target in SCLC.

INTRODUCTION: SCLC is an extremely aggressive subtype of lung cancer without approved targeted therapies. Here we identified YES1 as a novel targetable oncogene driving SCLC maintenance and metastasis. METHODS: Association between YES1 levels and prognosis was evaluated in SCLC clinical samples. In vitro functional experiments for proliferation, apoptosis, cell cycle, and cytotoxicity were performed. Genetic and pharmacologic inhibition of YES1 was evaluated in vivo in cell- and patient-derived xenografts and metastasis. YES1 levels were evaluated in mouse and patient plasma-derived exosomes. RESULTS: Overexpression or gain/amplification of YES1 was identified in 31% and 26% of cases, respectively, across molecular subgroups, and was found as an independent predictor of poor prognosis. Genetic depletion of YES1 dramatically reduced cell proliferation, three-dimensional organoid formation, tumor growth, and distant metastasis, leading to extensive apoptosis and tumor regressions. Mechanistically, YES1-inhibited cells revealed alterations in the replisome and DNA repair processes, that conferred sensitivity to irradiation. Pharmacologic blockade with the novel YES1 inhibitor CH6953755 or dasatinib induced marked antitumor activity in organoid models and cell- and patient-derived xenografts. YES1 protein was detected in plasma exosomes from patients and mouse models, with levels matching those of tumors, suggesting that circulating YES1 could represent a biomarker for patient selection/monitoring. CONCLUSIONS: Our results provide evidence that YES1 is a new druggable oncogenic target and biomarker to advance the clinical management of a subpopulation of patients with SCLC.

Assessment of epidermal growth factor receptor and K-ras mutation status in cytological stained smears of non-small cell lung cancer patients: correlation with clinical outcomes.

OBJECTIVE: Epidermal growth factor receptor (EGFR) and K-ras mutations guide treatment selection in non-small cell lung cancer (NSCLC) patients. Although mutation status is routinely assessed in biopsies, cytological specimens are frequently the only samples available. We determined EGFR and K-ras mutations in cytological samples. METHODS: DNA was extracted from 150 consecutive samples, including 120 Papanicolau smears (80%), 10 cell blocks (7%), nine fresh samples (6%), six ThinPrep® tests (4%), and five body cavity fluids (3.3%). Papanicolau smears were analyzed when they had >50% malignant cells. Polymerase chain reaction and direct sequencing of exons 18-21 of EGFR and exon 2 of K-ras were performed. EGFR mutations were simultaneously determined in biopsies and cytological samples from 20 patients. Activity of EGFR tyrosine kinase inhibitors (TKIs) was assessed. RESULTS: The cytological diagnosis was adenocarcinoma in 110 samples (73%) and nonadenocarcinoma in 40 (27%) samples. EGFR mutations were identified in 26 samples (17%) and K-ras mutations were identified in 18 (12%) samples. EGFR and K-ras mutations were mutually exclusive. In EGFR-mutated cases, DNA was obtained from stained smears in 24 cases (92%), pleural fluid in one case (4%), and cell block in one case (4%). The response rate to EGFR TKIs in patients harboring mutations was 75%. The mutation status was identical in patients who had both biopsies and cytological samples analyzed. CONCLUSION: Assessment of EGFR and K-ras mutations in cytological samples is feasible and comparable with biopsy results, making individualized treatment selection possible for NSCLC patients from whom tumor biopsies are not available.

Whole exome sequencing characterization of individuals presenting extreme phenotypes of high and low risk of developing tobacco-induced lung adenocarcinoma.

BACKGROUND: Tobacco is the main risk factor for developing lung cancer. Yet, some heavy smokers do not develop lung cancer at advanced ages while others develop it at young ages. Here, we assess for the first time the genetic background of these clinically relevant extreme phenotypes using whole exome sequencing (WES). METHODS: We performed WES of germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age (extreme cases, n=50) or did not present lung adenocarcinoma or other tumors at an advanced age (extreme controls, n=50). We selected non-synonymous variants located in exonic regions and consensus splice sites of the genes that showed significantly different allelic frequencies between both cohorts. We validated our results in all the additional extreme cases (i.e., heavy smokers who developed lung adenocarcinoma at an early age) available from The Cancer Genome Atlas (TCGA). RESULTS: The mean age for the extreme cases and controls was respectively 49.7 and 77.5 years. Mean tobacco consumption was 43.6 and 56.8 pack-years. We identified 619 significantly different variants between both cohorts, and we validated 108 of these in extreme cases selected from TCGA. Nine validated variants, located in relevant cancer related genes, such as PARP4, HLA-A or NQO1, among others, achieved statistical significance in the False Discovery Rate test. The most significant validated variant (P=4.48×10-5) was located in the tumor-suppressor gene ALPK2. CONCLUSIONS: We describe genetic variants associated with extreme phenotypes of high and low risk for the development of tobacco-induced lung adenocarcinoma. Our results and our strategy may help to identify high-risk subjects and to develop new therapeutic approaches.

Complement activation mediates cetuximab inhibition of non-small cell lung cancer tumor growth in vivo.

BACKGROUND: Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), increases survival in patients with advanced EGFR-positive non-small cell lung cancer when administrated in combination with chemotherapy. In this study, we investigated the role of complement activation in the antitumor mechanism of this therapeutic drug. RESULTS: EGFR-expressing lung cancer cell lines were able to bind cetuximab and initiate complement activation by the classical pathway, irrespective of the mutational status of EGFR. This activation led to deposition of complement components and increase in complement-mediated cell death. The influence of complement activation on the activity of cetuximab in vivo was evaluated in xenografts of A549 lung cancer cells on nude mice. A549 cells express wild-type EGFR and have a KRAS mutation. Cetuximab activity against A549 xenografts was highly dependent on complement activation, since complement depletion completely abrogated the antitumor efficacy of cetuximab. Moreover, cetuximab activity was significantly higher on A549 cells in which a complement inhibitor, factor H, was genetically downregulated. CONCLUSIONS: We demonstrate for the first time that the in vivo antitumor activity of cetuximab can be associated with a complement-mediated immune response. These results may have important implications for the development of new cetuximab-based therapeutic strategies and for the identification of markers that predict clinical response.

The Dynamic Use of EGFR Mutation Analysis in Cell-Free DNA as a Follow-Up Biomarker during Different Treatment Lines in Non-Small-Cell Lung Cancer Patients.

Epidermal growth factor receptor (EGFR) mutational testing in advanced non-small-cell lung cancer (NSCLC) is usually performed in tumor tissue, although cfDNA (cell-free DNA) could be an alternative. We evaluated EGFR mutations in cfDNA as a complementary tool in patients, who had already known EGFR mutations in tumor tissue and were treated with either EGFR-tyrosine kinase inhibitors (TKIs) or chemotherapy. We obtained plasma samples from 21 advanced NSCLC patients with known EGFR tumor mutations, before and during therapy with EGFR-TKIs and/or chemotherapy. cfDNA was isolated and EGFR mutations were analyzed with the multiple targeted cobas EGFR Mutation Test v2. EGFR mutations were detected at baseline in cfDNA from 57% of patients. The semiquantitative index (SQI) significantly decreased from the baseline (median = 11, IQR = 9.5-13) to the best response (median = 0, IQR = 0-0, p < 0.01), followed by a significant increase at progression (median = 11, IQR = 11-15, p < 0.01) in patients treated with either EGFR-TKIs or chemotherapy. The SQI obtained with the cobas EGFR Mutation Test v2 did not correlate with the concentration in copies/mL determined by droplet digital PCR. Resistance mutation p.T790M was observed at progression in patients with either type of treatment. In conclusion, cfDNA multiple targeted EGFR mutation analysis is useful for treatment monitoring in tissue of EGFR-positive NSCLC patients independently of the drug received.

Programmed death-ligand 1 expression on direct Pap-stained cytology smears from non-small cell lung cancer: Comparison with cell blocks and surgical resection specimens.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression, as assessed by immunohistochemistry (IHC), is used to select patients with non-small cell lung cancer (NSCLC) for anti-programmed cell death protein 1 (PD-1)/PD-L1 therapy. The current study evaluated the feasibility and efficacy of PD-L1 immunostaining and quantitation on direct Papanicolaou-stained cytological smears compared with formalin-fixed paraffin-embedded samples (cytological cell blocks and surgical resection specimens) in NSCLC cases using 2 commercially available assays: the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies/Dako, Carpinteria, CA, USA) and the Ventana SP263 Assay (Ventana Medical Systems Inc, Tucson, Arizona). METHODS: PD-L1 immunostaining using either both or one of the assays was tested in 117 sets of paired samples obtained from 62 NSCLC cases. The tumor proportion score was reported in every case following the recommendations of the International Association for the Study of Lung Cancer (IASLC). RESULTS: In 57 sets of samples, both PD-L1 assays were used. Due to the availability of samples, only 1 assay was performed in 3 sets of samples and in 2 cases, only cytology smears were used and tested for both assays. A total of 113 sets of paired samples finally were evaluated; 4 cases could not be studied due to intense nonspecific background staining. A significant concordance between the 2 assays on cytological smears was found. Concordance between paired cytological smears and formalin-fixed paraffin-embedded samples was observed in 97.3% of the cases. CONCLUSIONS: The quantification of PD-L1 expression on direct Papanicolaou-stained cytology smears is feasible and reliable for both PD-L1 assays.

Neoadjuvant nivolumab modifies the tumor immune microenvironment in resectable glioblastoma.

Glioblastoma is the most common primary central nervous system malignancy and has a poor prognosis. Standard first-line treatment, which includes surgery followed by adjuvant radio-chemotherapy, produces only modest benefits to survival1,2. Here, to explore the feasibility, safety and immunobiological effects of PD-1 blockade in patients undergoing surgery for glioblastoma, we conducted a single-arm phase II clinical trial (NCT02550249) in which we tested a presurgical dose of nivolumab followed by postsurgical nivolumab until disease progression or unacceptable toxicity in 30 patients (27 salvage surgeries for recurrent cases and 3 cases of primary surgery for newly diagnosed patients). Availability of tumor tissue pre- and post-nivolumab dosing and from additional patients who did not receive nivolumab allowed the evaluation of changes in the tumor immune microenvironment using multiple molecular and cellular analyses. Neoadjuvant nivolumab resulted in enhanced expression of chemokine transcripts, higher immune cell infiltration and augmented TCR clonal diversity among tumor-infiltrating T lymphocytes, supporting a local immunomodulatory effect of treatment. Although no obvious clinical benefit was substantiated following salvage surgery, two of the three patients treated with nivolumab before and after primary surgery remain alive 33 and 28 months later.

Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non-Small Cell Lung Carcinoma: the ROSING Study.

INTRODUCTION: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. METHODS: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). RESULTS: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). CONCLUSIONS: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm.

Dosimetric analysis of the patterns of local failure observed in patients with locally advanced non-small cell lung cancer treated with neoadjuvant chemotherapy and concurrent conformal (3D-CRT) chemoradiation.

PURPOSE: Three-dimensional conformal radiation therapy (3D-CRT) represents an advance in the better delineation of the target contours and more accurate dose distributions. The purpose of this study was to identify local control prognostic factors in patients with locally advanced non-small cell lung cancer (LA-NSCLC) treated with 3D-CRT. MATERIAL AND METHODS: Between April 1995 and March 2002, 65 patients (NSCLC stage IIIA: 20%, IIIB: 77%) were treated with cisplatin-based induction and concurrent chemotherapy chemotherapy and hyperfractioned 3D-CRT (1.2Gy b.i.d.; median dose: 72.8 (range: 67-85.9). Clinical parameters (sex, age, performance status, stage, histology, tumor location), therapeutic factors (chemotherapy schedule, 3D-CRT dose, treatment response) and dosimetric factors (volume and dose of GTV, PTV-2, CTV and PTV-1) were evaluated. Local recurrences were divided into three dosimetric categories: those with more than 80% of their volume within high dose region (95% of prescription dose) were considered "central"; those between 20% and 80% were considered "marginal", and those with less than 20% of their volume within high dose region were considered "out-of-field". Local-failure free survival (LFFS) was obtained using the Kaplan-Meier method. Univariate and multivariate analyses were performed. RESULTS: There were 18 local failures (nine central, eight marginal and one out-of-field). The 2 and 5 year LFFS were 73% and 53%, respectively. In multivariate analysis, PTV-1>1146cc (HR=2.9, CI 95%: 1.1-7.5, p=0.026) was the only factor associated with worse LFFS. CONCLUSIONS: This study shows that local control was independently related to PTV-1 size. The great majority of local recurrences were located in the high-dose region. Dosimetric parameters may contribute to improving radiotherapy results in multidisciplinary treatment for LA-NSCLC.

[The role of chemotherapy in the treatment of hormone sensitive metastatic prostate cancer.] / Papel de la quimioterapia en el tratamiento del cáncer de próstata metastásico hormonosensible.

OBJECTIVE: Several studies have assessed the role of adding chemotherapy to hormonal treatment for metastatic hormone-sensitive prostate cancer (MHSPC). The objective of this manuscript is to review these studies and to provide recommendations for the management of these patients. METHODS: We identified published clinical trials comparing hormone blockade (HB) with HB plus docetaxel as first-line treatment of HSMPC and we analyzed their results in terms of efficacy and toxicity. RESULTS: Of the 3 trials published, two demonstrated increased overall survival by adding docetaxel to the first-line treatment of MHSPC (CHAARTED and Stampede-Docetaxel studies) and the third one did not show such an advantage (GETUG-AFU15). In the CHAARTED study, the survival advantage was limited to patients presenting high tumor volume. Toxicity was increased in patients who received docetaxel. CONCLUSIONS: The addition of docetaxel to treatment with HB should be considered in patients with MHSPC, especially in those with high tumor volume. However, the toxicity and recent results of trials performed with abiraterone in MHSPC should also be taken in consideration.

Genomic characterization of individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced lung cancer.

Single nucleotide polymorphisms (SNPs) may modulate individual susceptibility to carcinogens. We designed a genome-wide association study to characterize individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced non-small cell lung cancer (NSCLC), and we validated our results. We hypothesized that this strategy would enrich the frequencies of the alleles that contribute to the observed traits. We genotyped 2.37 million SNPs in 95 extreme phenotype individuals, that is: heavy smokers that either developed NSCLC at an early age (extreme cases); or did not present NSCLC at an advanced age (extreme controls), selected from a discovery set (n = 3631). We validated significant SNPs in 133 additional subjects with extreme phenotypes selected from databases including >39,000 individuals. Two SNPs were validated: rs12660420 (pcombined  = 5.66 × 10-5 ; ORcombined  = 2.80), mapping to a noncoding transcript exon of PDE10A; and rs6835978 (pcombined  = 1.02 × 10-4 ; ORcombined  = 2.57), an intronic variant in ATP10D. We assessed the relevance of both proteins in early-stage NSCLC. PDE10A and ATP10DmRNA expressions correlated with survival in 821 stage I-II NSCLC patients (p = 0.01 and p < 0.0001). PDE10A protein expression correlated with survival in 149 patients with stage I-II NSCLC (p = 0.002). In conclusion, we validated two variants associated with extreme phenotypes of high and low risk of developing tobacco-induced NSCLC. Our findings may allow to identify individuals presenting high and low risk to develop tobacco-induced NSCLC and to characterize molecular mechanisms of carcinogenesis and resistance to develop NSCLC.