Characterization of the RofA regulon in the pandemic M1global and emergent M1UK lineages of Streptococcus pyogenes.

The standalone regulator RofA is a positive regulator of the pilus locus in Streptococcus pyogenes. Found in only certain emm genotypes, RofA has been reported to regulate other virulence factors, although its role in the globally dominant emm1 S. pyogenes is unclear. Given the recent emergence of a new emm1 (M1UK) toxigenic lineage that is distinguished by three non-synonymous SNPs in rofA, we characterized the rofA regulon in six emm1 strains that are representative of the two contemporary major emm1 lineages (M1global and M1UK) using RNAseq analysis, and then determined the specific role of the M1UK-specific rofA SNPs. Deletion of rofA in three M1global strains led to altered expression of 14 genes, including six non-pilus locus genes. In M1UK strains, deletion of rofA led to altered expression of 16 genes, including nine genes that were unique to M1UK. Only the pilus locus genes were common to the RofA regulons of both lineages, while transcriptomic changes varied between strains even within the same lineage. Although introduction of the three SNPs into rofA did not impact gene expression in an M1global strain, reversal of three SNPs in an M1UK strain led to an unexpected number of transcriptomic changes that in part recapitulated transcriptomic changes seen when deleting RofA in the same strain. Computational analysis predicted that interactions with a key histidine residue in the PRD domain of RofA would differ between M1UK and M1global. RofA is a positive regulator of the pilus locus in all emm1 strains but effects on other genes are strain- and lineage-specific, with no clear, common DNA binding motif. The SNPs in rofA that characterize M1UK may impact regulation of RofA; whether they alter phosphorylation of the RofA PRD domain requires further investigation.

Clinical Characteristics of Patients with Advanced ALK-Translocated Non-small Cell Lung Cancers and Long-Term Responses to Crizotinib (CRIZOLONG GFPC 05-19 Study).

BACKGROUND: Although ALK-translocated (ALK+) advanced non-small cell lung cancers (aNSCLCs) are currently treated with second- or third-generation ALK inhibitors (ALK-TKIs), some patients respond durably to the first-generation ALK-TKI crizotinib. OBJECTIVE: This study aimed to describe the clinical characteristics of these long-term responders. PATIENTS AND METHODS: This national, multicenter, retrospective, non-interventional study included patients with ALK+ aNSCLCs and long-term responses to first (L1)- or subsequent (≥ L2)-line crizotinib, defined, respectively, as treatments lasting > 18 and > 10 months. Median treatment duration (mDOT) was the primary endpoint. RESULTS: A total of 85 patients (32 L1 and 53 ≥ L2 responders) from 23 centers were included (receiving crizotinib between 10/24/2011-10/02/2018): median age of 59 years, 83.6% non-smokers or ex-smokers, 85.9% performance status (PS) 0/1, 94.1% with adenocarcinomas, median of one metastatic site, and 22.4% with brain metastases (BMs). After median follow-up of 73.4 [95% confidence interval, 67.5-79.9] months, respective L1 and ≥ L2 mDOTs were 43.3 [26.7-56.8] and 29.6 [22.6-35.8] months, with overall survival (OS) not reached (NR) and 116.2 [83.4-NR] months. BM presence or absence did not affect mDOT (31.4 versus 32.9 months) but significantly impacted median OS (70.6 versus 158.6 months; p = 0.0008). Progression on crizotinib was paucisymptomatic (74.1%) and oligometastatic (34.8%), especially BMs (42.4%). After crizotinib discontinuation, 65 (76.5%) patients received subsequent systemic therapy: 57 (67.1%) with second-generation ALK-TKIs. Respective mDOTs of first- and second-line post-crizotinib ALK-TKIs lasted 19.4 [14.9-25.6] and 11.1 [4.8-17.9] months, respectively. CONCLUSIONS: Most ALK+ aNSCLC patients with prolonged crizotinib efficacy had paucisymptomatic and oligometastatic disease without BMs. They subsequently benefited from a sequential strategy with other ALK-TKIs.

Three-Year Overall Survival of Patients With Advanced Non-Small-Cell Lung Cancers With ≥50% PD-L1 Expression Treated With First-Line Pembrolizumab Monotherapy in a Real-World Setting (ESCKEYP GFPC Study).

Outside clinical trials, few data are available on the effect of long-term first-line pembrolizumab in patients with advanced non-small-cell lung cancers with ≥50% of tumor cells expressing programmed cell death ligand 1 (PD-L1). This French, multicenter study included consecutive advanced patients with non-small-cell lung cancer given first-line pembrolizumab alone between May 2017 (authorization date for this indication) and November 2019 (authorization date for pembrolizumab-chemotherapy combination). Information was collected from patients' medical files, with a local evaluation of the response and progression-free survival (PFS). Overall survival (OS) was calculated from pembrolizumab onset using the Kaplan-Meier method. The analysis concerned 845 patients, managed in 33 centers: median age: 65 (range: 59-72) years, 67.8% men, 78.1% Eastern Cooperative Oncology Group performance status 0/1, 38.9%/51.5%/6.6% active, ex or never-smokers, respectively, 10.9%/16.8% taking or recently took corticosteroids/antibiotics, 69.6% nonsquamous histology, 48.9% ≥75% PD-L1-positive, and 20.8% had brain metastases at diagnosis. After a median (95% CI) follow-up of 45 (44.1-45.9) months, respective median (95% CI) PFS and OS lasted 8.2 (6.9-9.2) and 22 (8.5-25.9) months; 3-year PFS and OS rates were 25.4% and 39.4%, respectively. Multivariate analysis retained never-smoker status, adenocarcinoma histology, Eastern Cooperative Oncology Group performance status ≥2, and neutrophil/lymphocyte ratio >4 as being significantly associated with shorter survival, but not brain metastases at diagnosis or <75% PD-L1 tumor-cell expression. These long-term results of pembrolizumab efficacy based on a nationwide "real-world" cohort reproduced those obtained in clinical trials.

Venous thrombotic events and impact on outcomes in patients treated with first-line single-agent pembrolizumab in PD-L1 ≥ 50% advanced non small cell lung cancer.

BACKGROUND: Few data are available on the impact of venous thrombotic events (VTE) in patients with metastatic non-small cell lung cancer (mNSCLC) treated with immunotherapy. METHODS: This is a secondary analysis of the ESKEYP study, a national, retrospective, multicenter study that consecutively included all PD-L1 ≥ 50% mNSCLC patients who initiated first-line treatment with pembrolizumab monotherapy. From May 2017 to November 2019, 845 patients were included (from availability of pembrolizumab in this indication in France to the authorization of the combination with chemotherapy). Impact of VTE and patient characteristics were analyzed. RESULTS: Of the 748 patients (88.5%) with available data, the incidence of VTE was 14.8% (111/748). At pembrolizumab initiation, Khorana score was ≥ 2 for 55.0% (61/111) of them. Recurrence of VTE was reported for 4 of the 111 patients and 5 had bleeding complications. Patients with VTE were significantly younger, had more frequently long-term corticosteroids treatment and more often liver metastases. Progression-free survival (PFS) was significantly shorter in patients with VTE compared to patients without VTE: 6.1 (95% CI 4.1-9.0) months vs. 8.3 (6.9-10.3) months (p = 0.03). VTE did not significantly impact overall survival (OS): 15.2 (10.0-24.7) months with VTE and 22.6 (18.4-29.8) months without VTE (p = 0.07). In multivariate analysis for PFS and OS, HRs for VTE were 1.3 (0.99-1.71), p = 0.06 and 1.32 (0.99-1.76), p = 0.05. CONCLUSION: The incidence of VTE appears to be as high with in first-line immunotherapy as with chemotherapy in patients with mNSCLC, with in patient with VTE, a no significant trend for lower PFS and OS in multivariate analysis. more marked impact on PFS than on OS.

Single-cell profiling of Anopheles gambiae spermatogenesis defines the onset of meiotic silencing and premeiotic overexpression of the X chromosome.

Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell-types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes.

Brief Report: Severe Sotorasib-Related Hepatotoxicity and Non-Liver Adverse Events Associated With Sequential Anti-Programmed Cell Death (Ligand)1 and Sotorasib Therapy in KRASG12C-Mutant Lung Cancer.

INTRODUCTION: Sequential anti-programmed cell death protein 1 (PD-1) or anti-programmed death-ligand 1 (PD-L1) followed by small targeted therapy use is associated with increased prevalence of adverse events (AEs) in NSCLC. KRASG12C inhibitor sotorasib may trigger severe immune-mediated hepatotoxicity when used in sequence or in combination with anti-PD-(L)1. This study was designed to address whether sequential anti-PD-(L)1 and sotorasib therapy increases the risk of hepatotoxicity and other AEs. METHODS: This is a multicenter, retrospective study of consecutive advanced KRASG12C-mutant NSCLC treated with sotorasib outside clinical trials in 16 French medical centers. Patient records were reviewed to identify sotorasib-related AEs (National Cancer Institute Common Classification Criteria for Adverse Events-Version 5.0). Grade 3 and higher AE was considered as severe. Sequence group was defined as patients who received an anti-PD-(L)1 as last line of treatment before sotorasib initiation and control group as patients who did not receive an anti-PD-(L)1 as last line of treatment before sotorasib initiation. RESULTS: We identified 102 patients who received sotorasib, including 48 (47%) in the sequence group and 54 (53%) in the control group. Patients in the control group received an anti-PD-(L)1 followed by at least one treatment regimen before sotorasib in 87% of the cases or did not receive an anti-PD-(L)1 at any time before sotorasib in 13% of the cases. Severe sotorasib-related AEs were significantly more frequent in the sequence group compared with those in the control group (50% versus 13%, p < 0.001). Severe sotorasib-related AEs occurred in 24 patients (24 of 48, 50%) in the sequence group, and among them 16 (67%) experienced a severe sotorasib-related hepatotoxicity. Severe sotorasib-related hepatotoxicity was threefold more frequent in the sequence group compared with that in the control group (33% versus 11%, p = 0.006). No fatal sotorasib-related hepatotoxicity was reported. Non-liver severe sotorasib-related AEs were significantly more frequent in the sequence group (27% versus 4%, p < 0.001). Severe sotorasib-related AEs typically occurred in patients who received last anti-PD-(L)1 infusion within 30 days before sotorasib initiation. CONCLUSIONS: Sequential anti-PD-(L)1 and sotorasib therapy are associated with a significantly increased risk of severe sotorasib-related hepatotoxicity and severe non-liver AEs. We suggest avoiding starting sotorasib within 30 days from the last anti-PD-(L)1 infusion.

Characterization of emergent toxigenic M1UK Streptococcus pyogenes and associated sublineages.

Streptococcus pyogenes genotype emm1 is a successful, globally distributed epidemic clone that is regarded as inherently virulent. An emm1 sublineage, M1UK, that produces increased levels of SpeA toxin was associated with increased scarlet fever and invasive infections in England in 2015/2016. Defined by 27 SNPs in the core genome, M1UK is now dominant in England. To more fully characterize M1UK, we undertook comparative transcriptomic and proteomic analyses of M1UK and contemporary non-M1UK emm1 strains (M1global). Just seven genes were differentially expressed by M1UK compared with contemporary M1global strains. In addition to speA, five genes in the operon that includes glycerol dehydrogenase were upregulated in M1UK (gldA, mipB/talC, pflD, and phosphotransferase system IIC and IIB components), while aquaporin (glpF2) was downregulated. M1UK strains have a stop codon in gldA. Deletion of gldA in M1global abrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes gldA, consistent with a feedback effect. Phylogenetic analysis identified two intermediate emm1 sublineages in England comprising 13/27 (M113SNPs) and 23/27 SNPs (M123SNPs), respectively, that had failed to expand in the population. Proteomic analysis of invasive strains from the four phylogenetic emm1 groups highlighted sublineage-specific changes in carbohydrate metabolism, protein synthesis and protein processing; upregulation of SpeA was not observed in chemically defined medium. In rich broth, however, expression of SpeA was upregulated ~10-fold in both M123SNPs and M1UK sublineages, compared with M113SNPs and M1global. We conclude that stepwise accumulation of SNPs led to the emergence of M1UK. While increased expression of SpeA is a key indicator of M1UK and undoubtedly important, M1UK strains have outcompeted M123SNPs and other emm types that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1UK on an inherently successful emm1 streptococcal background.

Atezolizumab with or without bevacizumab and platinum-pemetrexed in patients with stage IIIB/IV non-squamous non-small cell lung cancer with EGFR mutation, ALK rearrangement or ROS1 fusion progressing after targeted therapies: A multicentre phase II open-label non-randomised study GFPC 06-2018.

BACKGROUND: Previous reports showed limited efficacy of immune checkpoint inhibitors as single-agent treatment for non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutation or ALK/ROS1 fusion. We aimed at evaluating the efficacy and safety of immune checkpoint inhibitor combined with chemotherapy and bevacizumab (when eligible) in this patient subgroup. METHODS: We conducted a French national open-label multicentre non-randomised non-comparative phase II study in patients with stage IIIB/IV NSCLC, oncogenic addiction (EGFR mutation or ALK/ROS1 fusion), with disease progression after tyrosine kinase inhibitor and no prior chemotherapy. Patients received platinum, pemetrexed, atezolizumab, bevacizumab (PPAB cohort) or, if not eligible to bevacizumab, platinum-pemetrexed-atezolizumab (PPA cohort). The primary end-point was the objective response rate (RECIST v1.1) after 12 weeks, evaluated by blind independent central review. RESULTS: 71 patients were included in PPAB cohort and 78 in PPA cohort (mean age, 60.4/66.1 years; women 69.0%/51.3%; EGFR mutation, 87.3%/89.7%; ALK rearrangement, 12.7%/5.1%; ROS1 fusion, 0%/6.4%, respectively). After 12 weeks, objective response rate was 58.2% (90% confidence interval [CI], 47.4-68.4) in PPAB cohort and 46.5% (90% CI, 36.3-56.9) in PPA cohort. Median progression-free survival and overall survival were 7.3 (95% CI 6.9-9.0) months and 17.2 (95% CI 13.7-NA) months in PPAB cohort and 7.2 (95% CI 5.7-9.2) months and 16.8 (95% CI 13.5-NA) months in PPA cohort, respectively. Grade 3-4 adverse events occurred in 69.1% of patients in PPAB cohort and 51.4% in PPA cohort; Grade 3-4 atezolizumab-related adverse events occurred in 27.9% and 15.3%, respectively. CONCLUSION: Combination approach with atezolizumab with or without bevacizumab and platinum-pemetrexed achieved promising activity in metastatic EGFR-mutated or ALK/ROS1-rearranged NSCLC after tyrosine kinase inhibitor failure, with acceptable safety profile.

Injury primes mutation-bearing astrocytes for dedifferentiation in later life.

Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis.

First-line single-agent pembrolizumab for PD-L1-positive (tumor proportion score ≥ 50%) advanced non-small cell lung cancer in the real world: impact in brain metastasis: a national French multicentric cohort (ESCKEYP GFPC study).

BACKGROUND: Few real-world data are available in patients with advanced metastatic non-small cell lung cancer (NSCLC) treated with first-line immunotherapy, particularly in those with brain metastases at treatment initiation. METHODS: This was a national, retrospective, multicenter study that consecutively included all patients with PD-L1-positive (tumor proportion score ≥ 50%) advanced NSCLC who initiated first-line treatment with pembrolizumab as a single agent between May 2017 (date of availability of pembrolizumab in this indication in France) to November 22, 2019 (approval of the pembrolizumab-chemotherapy combination). Data were collected from medical records with local response assessment. RESULTS: The cohort included 845 patients and 176 (20.8%) had brain metastases at diagnosis. There were no significant differences in outcomes for patients with and without brain metastases: 9.2 (95% CI 5.6-15) and 8 (95% CI 6.7-9.2, p = 0.3) months for median progression-free survival (PFS) and, 29.5 (95% CI 17.2-NA) and 22 (95% CI 17.8-27.1, p = 0.3) months for median overall survival (OS), respectively. Overall response rates were 47% and 45% in patients with and without cerebral metastases. In multivariate analysis, performance status 2-4 vs. 0-1 and neutrophil-to-lymphocyte ratio ≥ 4 vs. < 4 were the main independent negative factors for OS; brain metastasis was not an independent factor for OS. CONCLUSION: In this large multicenter cohort, nearly 20% of patients initiating pembrolizumab therapy for advanced NSCLC had cerebral metastases. There was no significant difference in response rates, PFS and OS between patients with and without brain metastases.

Management of patients with synchronous head-and-neck and lung cancers: SYNCHRON GFPC 15-01 study.

Purpose: Few data have been published on the management of patients with synchronous head-and-neck cancer (HNC) and lung cancer (LC). This observational study was undertaken to describe the management of these patients in multiple centers. Materials and Methods: All patients consecutively diagnosed with synchronous HNC and LC in 26 French centers were included. Information was collected on patients' clinical characteristics, management, and outcomes. Those characteristics and treatments were analyzed descriptively. Kaplan-Meier progression-free and overall survival probabilities were estimated. Results: The study included 132 patients: 83% male; median age: 63.7 (range: 62.1-65.4) years; all current or former smokers; Eastern Cooperative Oncology Group performance status: 0 or 1 for 21.9% or 65.9% of the patients, respectively; cardiovascular comorbidities: 63%; chronic obstructive pulmonary disease: 33%; and previous cancer: 11%. HNC histology was 98% squamous: 23.5% oral cavity, 26.5% oropharyngeal, 22.0% hypopharyngeal, and 28.0% laryngeal. LCs were mainly localized (47.7% Stage I and 9.9% Stage II): 38% squamous, 49% adenocarcinomas, and 13% others. LC diagnosis impacted HNC management for 38% of the patients, with a median time from HNC diagnosis to first HNC treatment of 40 days. HNC impacted LC management for 48% of the patients, with a median time from LC diagnosis-to-LC treatment interval of 41 days. Conclusions: Synchronous LC at HNC diagnosis impacted management and outcomes of both cancers. Specific recommendations should be elaborated to improve the management of these patients.

Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization.

Cellular barcoding techniques are powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been broadly applied to protozoan parasites, which have unique genomic structures and virulence strategies compared with viral and bacterial pathogens. Here, we present a CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. Using libraries of barcoded T. gondii, we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes representing a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. These data establish a cellular barcoding approach for protozoa and evidence that the blood-brain barrier is not a major bottleneck to colonization by T. gondii.

PECATI: A Multicentric, Open-Label, Single-Arm Phase II Study to Evaluate the Efficacy and Safety of Pembrolizumab and Lenvatinib in Pretreated B3-Thymoma and Thymic Carcinoma Patients.

Thymic epithelial tumors are rare neoplastic proliferations of thymic epithelial cells. The aggressiveness of these malignancies increases as higher is the histologic subtype, being thymic carcinoma the most aggressive subtype, with a greater tendency to metastatic spread. In metastatic setting, there is no standard treatment after progression on platinum-based chemotherapy. In this scenario, monotherapy treatment either with lenvatinib, a multi-tyrosine kinase inhibitor with antiangiogenic properties, or pembrolizumab, an immune-checkpoint inhibitor, has reported clinical activity. Potential combination of both agents may have synergistic activity as reported in other cancer types. PECATI trial is a single-arm, investigator-initiated phase II study aiming to assess the activity and safety of the combination of lenvatinib and pembrolizumab in 43 patients with advanced B3-thymoma or thymic carcinoma who progressed on or after at least one previous line of platinum-based chemotherapy. The primary endpoint of the trial is 5-month progression-free survival rate and the secondary endpoints include overall response rate, duration of response, and overall survival.

Treatment strategies for thymic carcinoma in a real-life setting. Insights from the RYTHMIC network.

BACKGROUND: Thymic carcinomas are aggressive and difficult to treat a subset of thymic epithelial tumours that represent a heterogeneous group of rare intrathoracic malignancies. The treatment strategy of thymic carcinomas is based on whether surgical resection may be achieved, which represents the most significant favourable prognostic factor on survival. For this study, we took advantage of the unique prospective Réseau tumeurs THYMiques et Cancer (RYTHMIC) database to describe baseline characteristics, analyse treatment strategies in light of existing guidelines and provide landmark patient outcomes data with regards to response and survival of patients in a real-life clinical practice setting. METHODS: Inclusion criteria for this analysis were the following: (1) histologically-confirmed thymic carcinomas - excluding neuroendocrine tumours-after pathological review by the RYTHMIC pathology panel, (2) discussion of the case at the RYTHMIC multidisciplinary tumour board, (3) at least one active treatment modality. RESULTS: A total of 213 patients were analysed. Overall, 60 (28%) patients were considered as surgical candidates upfront, 91 (43%) patients received primary chemotherapy, and 62 (29%) patients received exclusive chemotherapy. Median overall survival (OS) was 49.2 months (IC95%: 34.8-63.6); OS was significantly longer in patients with a lower stage at diagnosis (p < 0.001), who were operated on upfront, as opposed to patients who received primary or exclusive chemotherapy (p < 0.001). Surgery, conducted upfront or after primary chemotherapy, was significantly associated with more prolonged OS (p < 0.001); complete resection and postoperative radiotherapy were also predictors of better outcome (p = 0.018 and p = 0.051, respectively). CONCLUSIONS: Our cohort is the first to analyse in-depth outcomes and treatment strategies in a prospective cohort of consecutive patients with thymic carcinoma. While we confirm the major prognostic impact of surgery, our data highlight the need for optimised multidisciplinary management and innovative therapies as the survival of patients remains limited.

LUMI-PCR: an Illumina platform ligation-mediated PCR protocol for integration site cloning, provides molecular quantitation of integration sites.

BACKGROUND: Ligation-mediated PCR protocols have diverse uses including the identification of integration sites of insertional mutagens, integrating vectors and naturally occurring mobile genetic elements. For approaches that employ NGS sequencing, the relative abundance of integrations within a complex mixture is typically determined through the use of read counts or unique fragment lengths from a ligation of sheared DNA; however, these estimates may be skewed by PCR amplification biases and saturation of sequencing coverage. RESULTS: Here we describe a modification of our previous splinkerette based ligation-mediated PCR using a novel Illumina-compatible adapter design that prevents amplification of non-target DNA and incorporates unique molecular identifiers. This design reduces the number of PCR cycles required and improves relative quantitation of integration abundance for saturating sequencing coverage. By inverting the forked adapter strands from a standard orientation, the integration-genome junction can be sequenced without affecting the sequence diversity required for cluster generation on the flow cell. Replicate libraries of murine leukemia virus-infected spleen samples yielded highly reproducible quantitation of clonal integrations as well as a deep coverage of subclonal integrations. A dilution series of DNAs bearing integrations of MuLV or piggyBac transposon shows linearity of the quantitation over a range of concentrations. CONCLUSIONS: Merging ligation and library generation steps can reduce total PCR amplification cycles without sacrificing coverage or fidelity. The protocol is robust enough for use in a 96 well format using an automated liquid handler and we include programs for use of a Beckman Biomek liquid handling workstation. We also include an informatics pipeline that maps reads, builds integration contigs and quantitates integration abundance using both fragment lengths and unique molecular identifiers. Suggestions for optimizing the protocol to other target DNA sequences are included. The reproducible distinction of clonal and subclonal integration sites from each other allows for analysis of populations of cells undergoing selection, such as those found in insertional mutagenesis screens.

Role of the YAP-1 Transcriptional Target cIAP2 in the Differential Susceptibility to Chemotherapy of Non-Small-Cell Lung Cancer (NSCLC) Patients with Tumor RASSF1A Gene Methylation from the Phase 3 IFCT-0002 Trial.

RASSF1 gene methylation predicts longer disease-free survival (DFS) and overall survival (OS) in patients with early-stage non-small-cell lung cancer treated using paclitaxel-based neo-adjuvant chemotherapy compared to patients receiving a gemcitabine-based regimen, according to the randomized Phase 3 IFCT (Intergroupe Francophone de Cancérologie Thoracique)-0002 trial. To better understand these results, this study used four human bronchial epithelial cell (HBEC) models (HBEC-3, HBEC-3-RasV12, A549, and H1299) and modulated the expression of RASSF1A or YAP-1. Wound-healing, invasion, proliferation and apoptosis assays were then carried out and the expression of YAP-1 transcriptional targets was quantified using a quantitative polymerase chain reaction. This study reports herein that gemcitabine synergizes with RASSF1A, silencing to increase the IAP-2 expression, which in turn not only interferes with cell proliferation but also promotes cell migration. This contributes to the aggressive behavior of RASSF1A-depleted cells, as confirmed by a combined knockdown of IAP-2 and RASSF1A. Conversely, paclitaxel does not increase the IAP-2 expression but limits the invasiveness of RASSF1A-depleted cells, presumably by rescuing microtubule stabilization. Overall, these data provide a functional insight that supports the prognostic value of RASSF1 gene methylation on survival of early-stage lung cancer patients receiving perioperative paclitaxel-based treatment compared to gemcitabine-based treatment, identifying IAP-2 as a novel biomarker indicative of YAP-1-mediated modulation of chemo-sensitivity in lung cancer.

Incidence of thyroid dysfunctions during treatment with nivolumab for non-small cell lung cancer: Retrospective study of 105 patients.

INTRODUCTION: Immunotherapy is a standard not only in second line but also in first line treatment in patients with non-small cell lung cancer (NSCLC) and other tumors. Thyroid dysfunctions are the most common endocrine toxicities. OBJECTIVE: To determine the incidence of thyroid dysfunctions during treatment with a PD-1 monoclonal antibody (nivolumab) in patients with NSCLC. METHODS: Retrospective study of patients treated with nivolumab for NSCLC between May 2015 and December 2016; euthyroidism within the 3 months preceding immunotherapy; monitoring of thyroid function tests until stopping nivolumab, death or February 2017. Patients treated with levothyroxine, amiodarone or another immunotherapy were excluded. RESULTS: Among 183 patients treated, 105 fullfilled the inclusion criteria (72 males, median age: 61 years [range: 41-80]). Fifteen patients (14.3%) experienced a thyroid dysfunction; among them, compared to the "control" group (n=90), we found more females (53.3% vs. 27.8%; P=0.07), and younger patients (median age: 56 years vs. 62 years; P=0.02). Thirteen patients had thyrotoxicosis (median onset: 8 weeks), and then hypothyroidism was observed in 5 patients. Isolated hypothyroidism was rare (n=2) and late (median: 30 weeks). Three patients had anti-TPO antibodies. Three patients discontinued immunotherapy transiently due to thyroid dysfunctions. After a median follow-up of 9 months [95% CI, 7.5-10.3], one patient (6.7%) in the "thyroid dysfunctions" group and 30 patients (33.3%) in the "control" group died, with a trend toward a higher overall survival in the "thyroid dysfunctions" group (HR: 0.16 [95% CI, 0.02-1.15]; P=0.07). CONCLUSION: Thyroid dysfunctions (isolated thyrotoxicosis, biphasic thyroiditis and hypothyroidism) were common, and required patients with NSCLC to be screened during nivolumab therapy.

Lung cancer survivors and employment: A systematic review.

BACKGROUND: The aim of this systematic review is to identify, in a comprehensive manner, the impact of lung cancer on the employment status of survivors. METHODS: The Preferred Reported Items for Systematic Reviews and Meta-analyses (PRISMA) statement was used as a formal guideline. The systematic review includes scientific papers published between January 2000 and October 2018. The search strategy queried the database MEDLINE. Inclusion criteria comprised: (1) inclusion of patients diagnosed with lung cancer (LC) (2); assessment of employment status or employment outcomes or work adjustments or return to work (3); inclusion of scientific papers published in peer-reviewed journals (4); inclusion of articles written either in English or in French. Literature reviews were not included. RESULTS: A total of 642 scientific papers were retrieved. Twenty-three articles were included in the systematic review: 5 longitudinal studies and 18 cross-sectional studies. LC survivors are 2-3 times more likely to be unemployed as compared with control groups. Previous studies highlight a median duration of sickness absence increased for LC survivors compared to control groups. The strongest decline in earnings was observed among LC survivors as compared to other cancer types. CONCLUSIONS: LC is associated with a significant impact on employment of patients. The promising results of recent therapeutic strategies could lead to a better social and professional prognosis. A reduction of indirect costs is to be expected.

Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance.

This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue.

Author Correction: Subclonal mutation selection in mouse lymphomagenesis identifies known cancer loci and suggests novel candidates.

The original version of this Article contained an error in the hyperlink for the online repository http://mulvdb.org which was incorrectly given as http://mulv.lms.mrc.ac.uk. This has been corrected in both the PDF and HTML versions of the Article.