Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Here, we provide detailed procedures for a variety of multiparameter fluorescence microscopy imaging methods to explore the spatial organization of DC in tissues and to dissect how DC migrate, communicate, and mediate their multiple functional roles in immunity in a variety of tissue settings. The protocols presented here entail approaches to study DC dynamics and T cell cross-talk by intravital microscopy, large-scale visualization, identification, and quantitative analysis of DC subsets and their functions by multiparameter fluorescence microscopy of fixed tissue sections, and an approach to study DC interactions with tissue cells in a 3D cell culture model. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Detection of ALK fusion variants by RNA-based NGS and clinical outcome correlation in NSCLC patients treated with ALK-TKI sequences.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) fusions identify a limited subset of non-small cell lung cancer (NSCLC) patients, whose therapeutic approach have been radically changed in recent years. However, diagnostic procedures and clinical-radiological responses to specific targeted therapies remain heterogeneous and intrinsically resistant or poor responder patients exist. METHODS: A total of 290 patients with advanced NSCLC defined as ALK+ by immunohistochemistry (IHC) and/or fluorescent in situ hybridisation (FISH) test and treated with single or sequential multiple ALK inhibitors (ALKi) from 2011 to 2017 have been retrospectively retrieved from a multicentre Italian cancer network database. In 55 patients with enough leftover tumour tissue, specimens were analysed with both targeted and customised next generation sequencing panels. Identified fusion variants have been correlated with clinical outcomes. RESULTS: Of the 55 patients, 24 received crizotinib as first-line therapy, 1 received ceritinib, while 30 received chemotherapy. Most of the patients (64%) received ALKi in sequence. An ALK fusion variant was identified in 73% of the cases, being V3 variant (E6A20) the most frequent, followed by V1 (E13A20) and more rare ones (e.g. E6A19). In three specimens, four new EML4-ALK fusion breakpoints have been reported. Neither fusion variants nor brain metastases were significantly associated with overall survival (OS), while it was predictably longer in patients receiving a sequence of ALKi. The presence of V1 variant was associated with progression-free survival (PFS) improvement when crizotinib was used (p = 0.0073), while it did not affect cumulative PFS to multiple ALKi. CONCLUSION: Outcomes to sequential ALKi administration were not influenced by fusion variants. Nevertheless, in V1+ patients a prolonged clinical benefit was observed. Fusion variant identification by NGS technology may add relevant information about rare chromosomal events that could be potentially correlated to worse outcomes.

Immunotherapy in NSCLC Patients With Brain and Leptomeningeal Metastases.

Immunotherapy has now been integrated as a treatment strategy for most patients with non-small cell lung cancer (NSCLC). However, the pivotal clinical trials that demonstrated its impressive efficacy often did not include patients with active, untreated brain metastases or leptomeningeal carcinomatosis. Nevertheless, NSCLC is the most common tumor to metastasize to the brain, and patients develop brain and meningeal involvement in approximately 40 and 10% of cases, respectively. Consequently, the appropriate care of these patients is a recurrent clinical concern. Although there are many aspects that would merit further investigation to explain the mechanism of intracranial response to immune checkpoint inhibitors (ICPs), some data suggest that they are able to cross the blood-brain barrier, resulting in local tumor microenvironment modification. This results in a similar clinical benefit in patients with stable, previously treated brain metastases compared to the general population. Despite important limitations, some real-life studies have described that the ICPs' efficacy was maintained also in less selected patients with untreated or symptomatic brain metastases. In contrast, few data are available about patients with leptomeningeal carcinomatosis. Nevertheless, neurological complications due to ICP treatment in patients with brain metastases have to be evaluated and carefully monitored. Despite the fact that limited data are available in the literature, the purpose of this review is to show that the multimodal treatment of these patients with brain metastases and/or leptomeningeal disease should be discussed during tracing of the history of the disease, participating in the local and possibly systemic control of NSCLC.

Immune Checkpoint Inhibitor Rechallenge and Resumption: a Systematic Review.

PURPOSE OF THE REVIEW: The reintroduction of immune checkpoint inhibitors (ICIs) after disease progression (rechallenge) or immune-related adverse events (irAEs) recovering (resumption) raises questions in terms of efficacy and safety. RECENT FINDINGS: Here, we reviewed literature data about ICIs rechallenge/resumption in cancer patients along with their clinical characteristics to explore those factors associated with better outcomes. Heterogenous results were pointed out across rechallenge studies with an overall response rate between 0 and 54%, and a progression free survival ranged from 1.5 to 12.9 months and an overall survival between 6.5 and 23.8 months. Better outcomes have been recorded in patients with good ECOG PS, longer duration of initial ICI, discontinuation reason of initial ICI other than progression, and those who received ICI sequence other than the switch between anti-PD1 and anti-PDL1. Studies about ICI resumption highlighted that certain types of irAEs were more likely to relapse at retreatment. These results suggest that ICI rechallenge/resumption can be an interesting strategy for selected patients.

Immune checkpoint inhibitors and hospitalization at home in France.

CONTEXT: The administration of immune checkpoints inhibitors (ICIs) within hospitalization at home (HaH) organizations is an interesting alternative to conventional care. Three surveys were carried out to describe the different organizational models of French HaHs and criteria used by physicians in patient selection. METHODS: Three surveys were conducted between April 1 and August 31, 2020. The first one was addressed to all French HaHs, and the two others to public HaHs and oncologists treating patients with solid cancer in the Auvergne-Rhone-Alpes region. RESULTS: Overall, 54 French HaHs and 23 oncologists participated to the study. The health professionals involved in the patients' care were very heterogeneous, although in 92% of cases, the treatment prescription was made by the oncologist. HaH physicians were more involved in clinical assessment the day before treatment (19% vs. 0%), treatment validation (56% vs. 15%), and treatment prescription (19% vs. 0%), while nurses were better equipped (emergency kit available in 81% versus 50% of cases) when HaHs did carry out ICIs compared to when they did not. Most oncologists agreed that age, neuropsychiatric disorders, home environment, as well as treatment duration and good tolerance should be considered in patient selection. ECOG PS status and treatment response were less consensually considered. CONCLUSION: These results highlight the variability in French HaH organizations and patient selection criteria for employing ICIs at home. This study resulted in recommendations for administrating ICIs in HaH settings, which will likely be instrumental in further promoting this activity across France.

Dpb4 promotes resection of DNA double-strand breaks and checkpoint activation by acting in two different protein complexes.

Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase ε (Pol ε) holoenzyme. In Saccharomyces cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol ε complex. Here, we show that Dpb4 plays two functions in sensing and processing DNA double-strand breaks (DSBs). Dpb4 promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. Persistence of both Isw2 and Rad9 at DSBs is enhanced by the A62S mutation that is located in the Dpb4 histone fold domain and increases Dpb4 association at DSBs. Thus, Dpb4 exerts two distinct functions at DSBs depending on its interactors.

Epidemiology of oligometastatic non-small cell lung cancer: results from a systematic review and pooled analysis.

BACKGROUND: To describe the incidence and the clinical characteristics of oligometastatic non-small cell lung cancer (NSCLC) patients. Oligometastatic NSCLC is gaining recognition as a clinical condition with a different prognosis compared to multi metastatic disease. Usually, four different scenarios of oligometastatic disease can be described but not epidemiological data are available. To date, it is difficult to delineate an exhaustive epidemiological scenario because no uniform or shared definition of oligometastatic status exists, even though a recent consensus defined synchronous oligometastatic disease as having a maximum of 5 metastases in 3 different organs. METHODS: A systematic review and a pooled analysis of literature were performed. Article selection was based on the following characteristics: focus on lung cancers; dealing with oligometastatic settings and providing a definition of oligometastatic disease; number of metastatic lesions with or without the number of organs involved; providing some incidence or clinical characteristics of oligometastatic NSCLC patients. Series focusing on a specific single metastatic organ were excluded. The research was launched in MEDLINE (OvidSP) in March 2020. Full articles were individually and collectively read by the authors according to the previous criteria. Each author inspected the reference list included in the eligible articles. If the selection criteria were recognized, the article was reviewed by all authors and then included. Data on patient clinical features were pooled together from 31 articles selected. RESULTS: A total number of 31 articles have been selected for the analysis. The following variables were extracted from the publications: (I) number of metastases, (II) number of organs involved, (III) number of patients, (IV) number and percentage of males and females, (V) number and percentage of squamous and non-squamous histology, (VI) T and N status and/or stage of primary disease for oligometastatic setting. The data collected have been analyzed according to the oligometastatic setting. CONCLUSIONS: Oligometastatic status is globally identified as a different clinical condition from multi metastatic NSCLC, although the clinical characteristics were consistent in the general metastatic population, even with a lower-than-expected TN status. The brain and bones were the most frequent organs involved. Lacking consensus definition, these results must be interpreted cautiously and a prospective evaluation is urgently needed.

Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners.

The Mre11-Rad50-Xrs2 (MRX) complex detects and processes DNA double-strand breaks (DSBs). Its DNA binding and processing activities are regulated by transitions between an ATP-bound state and a post-hydrolysis cutting state that is nucleolytically active. Mre11 endonuclease activity is stimulated by Sae2, whose lack increases MRX persistence at DSBs and checkpoint activation. Here we show that the Rif2 protein inhibits Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identify a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2 inhibition of Mre11 nuclease. This residue is located near a Rad50 surface that binds Sae2 and is important in stabilizing the Mre11-Rad50 (MR) interaction in the cutting state. We propose that Sae2 stimulates Mre11 endonuclease activity by stabilizing a post-hydrolysis MR conformation that is competent for DNA cleavage, whereas Rif2 antagonizes this Sae2 function and stabilizes an endonuclease inactive MR conformation.

Resection of a DNA Double-Strand Break by Alkaline Gel Electrophoresis and Southern Blotting.

Generation of 3' single-stranded DNA (ssDNA) at the ends of a double-strand break (DSB) is essential to initiate repair by homology-directed mechanisms. Here we describe a Southern blot-based method to visualize the generation of ssDNA at the ends of site-specific DSBs generated in the Saccharomyces cerevisiae genome.

Advances and Therapeutic Perspectives in Extended-Stage Small-Cell Lung Cancer.

Extended small cell lung cancer (ED-SCLC) is a very aggressive disease, characterized by rapid growth and an early tendency to relapse. In contrast to non-small cell lung cancer, no therapeutic innovation has improved survival in patients with ED-SCLC over the past 20 years. Recently, immunotherapy has shown an important role in the management of these patients, emerging as the treatment of first choice in combination with chemotherapy and completely changing the therapeutic paradigm. However, patients' selection for this strategy is still challenging due to a lack of reliable predictive biomarkers. Conversely, the immunotherapy efficacy beyond the first line is pretty disappointing and innovative chemotherapies or target agents seem to be more promising in this setting. Some of them are also under evaluation as an upfront strategy and they will probably change the treatment algorithm in the next future. This proposal provides a comprehensive overview of available treatment strategies for ED-SCLC patients, highlighting their strengths and weaknesses.

Implementing ctDNA Analysis in the Clinic: Challenges and Opportunities in Non-Small Cell Lung Cancer.

Tumor genomic profiling has a dramatic impact on the selection of targeted treatment and for the identification of resistance mechanisms at the time of progression. Solid tissue biopsies are sometimes challenging, and liquid biopsies are used as a non-invasive alternative when tissue is limiting. The clinical relevance of tumor genotyping through analysis of ctDNA is now widely recognized at all steps of the clinical evaluation process in metastatic non-small cell lung cancer (NSCLC) patients. ctDNA analysis through liquid biopsy has recently gained increasing attention as well in the management of early and locally advanced, not oncogene-addicted, NSCLC. Its potential applications in early disease detection and the response evaluation to radical treatments are promising. The aim of this review is to summarize the landscape of liquid biopsies in clinical practice and also to provide an overview of the potential perspectives of development focusing on early detection and screening, the assessment of minimal residual disease, and its potential role in predicting response to immunotherapy. In addition to available studies demonstrating the clinical relevance of liquid biopsies, there is a need for standardization and well-designed clinical trials to demonstrate its clinical utility.

The 9-1-1 Complex Controls Mre11 Nuclease and Checkpoint Activation during Short-Range Resection of DNA Double-Strand Breaks.

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. Here, we show that abrogation of long-range resection induces a checkpoint response that decreases DNA damage resistance. This checkpoint depends on the 9-1-1 complex, which recruits Dpb11 and Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Dpb11 and Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, which is loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated.

Literature meta-analysis about the efficacy of re-challenge with PD-1 and PD-L1 inhibitors in cancer patients.

INTRODUCTION: Immune checkpoint inhibitor (ICPis) re-challenge could be an attractive therapeutic option considering its good safety profile. However, little data is available regarding anti-PD-1/anti-PD-L1 retreatment. We conducted a meta-analysis focusing on outcomes of solid cancer patients performing this strategy. METHODS: Fourteen full papers involving 74 patients were included. Individual data about best response or progression-free survival (PFS) upon the first and second course of anti-PD-1/anti-PD-L1 were collected. RESULTS: Non-small-cell lung cancer (53%) and melanoma (34%) were the most represented cancers. Higher objective response (46% versus 24%, P=4.10-4) and disease control rates (73% versus 52%, P=7.10-3) were obtained upon the first ICPi course compared to re-challenge. No association between responses obtained with the two ICPis courses was found (P=3.10-1). The PFS upon the first ICPi (PFS1) was longer than after re-challenge (PFSR) (6.6 versus 2.8 months, hazard ratio (HR) 0.57, P=2.10-3). A longer PFSR was obtained in patients with a longer PFS1 (P=6.10-3), in those who discontinued the first ICPi due to toxicity or per protocol (8.8 versus 2.1 months if disease progression occurs, P=2.10-3), and in those not receiving intercalated treatment between the two ICPis (6.6 versus 2.1 months for the treated ones, P=1.10-3). DISCUSSION: Anti-PD-1/anti-PD-L1 re-challenge showed interesting clinical activity in selected patients, mainly in those achieving a long-term response upon the first ICPi course, that do not discontinue therapy because of disease progression, or that are able to keep a treatment-free period.

Immune Checkpoint Inhibitors Rechallenge Efficacy in Non-Small-Cell Lung Cancer Patients.

BACKGROUND: Immune checkpoint inhibitor (ICPi) rechallenge could represent an attractive option in non-small-cell lung cancer (NSCLC), yet no sufficient data supporting this strategy are available. This retrospective observational multicenter national study explored the efficacy of anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) rechallenge in advanced NSCLC patients, looking for potential clinical features associated with greater outcomes. PATIENTS AND METHODS: We retrospectively collected data from 144 advanced NSCLC patients whose disease was rechallenged with ICPis after ≥ 12 weeks of discontinuation. The progression-free survival (PFS) and overall survival (OS) were calculated from first or second ICPi initiation to disease progression (PFS1 and PFSR, respectively), death, or last follow-up (OS1, OSR), respectively. RESULTS: The median (interquartile range) age was 63 (58-70) years. Most patients were male (67%) and smokers (87%). Most had adenocarcinomas (62%) and/or stage IV disease at diagnosis (66%). The best response at rechallenge was not associated with that under the first ICPi (P = 1.10-1). The median (95% confidence interval) PFS1 and PFSR were 13 (10-16.5) and 4.4 (3-6.5) months, respectively. The median (95% confidence interval) OS1 and OSR were 3.3 (2.9-3.9) and 1.5 (1.0-2.1) years, respectively. Longer PFSR and OSR were found in patients discontinuing first ICPi because of toxicity or clinical decision, those not receiving systemic treatment between the two ICPis, and those with good Eastern Cooperative Oncology Group performance status at rechallenge. Only performance status proved to affect outcomes at multivariate analysis. CONCLUSION: Patients discontinuing first ICPi because of toxicity or clinical decision, those able to maintain a treatment-free period, and those with good performance status may be potential candidates for rechallenge.

IFN-III is selectively produced by cDC1 and predicts good clinical outcome in breast cancer.

Dendritic cells play a key role in the orchestration of antitumor immune responses. The cDC1 (conventional dendritic cell 1) subset has been shown to be essential for antitumor responses and response to immunotherapy, but its precise role in humans is largely unexplored. Using a multidisciplinary approach, we demonstrate that human cDC1 play an important role in the antitumor immune response through their capacity to produce type III interferon (IFN-λ). By analyzing a large cohort of breast primary tumors and public transcriptomic datasets, we observed specific production of IFN-λ1 by cDC1. In addition, both IFN-λ1 and its receptor were associated with favorable patient outcomes. We show that IFN-III promotes a TH1 microenvironment through increased production of IL-12p70, IFN-γ, and cytotoxic lymphocyte-recruiting chemokines. Last, we showed that engagement of TLR3 is a therapeutic strategy to induce IFN-III production by tumor-associated cDC1. These data provide insight into potential IFN- or cDC1-targeting antitumor therapies.

How do cells sense DNA lesions?

DNA is exposed to both endogenous and exogenous DNA damaging agents that chemically modify it. To counteract the deleterious effects exerted by DNA lesions, eukaryotic cells have evolved a network of cellular pathways, termed DNA damage response (DDR). The DDR comprises both mechanisms devoted to repair DNA lesions and signal transduction pathways that sense DNA damage and transduce this information to specific cellular targets. These targets, in turn, impact a wide range of cellular processes including DNA replication, DNA repair and cell cycle transitions. The importance of the DDR is highlighted by the fact that DDR inactivation is commonly found in cancer and causes many different human diseases. The protein kinases ATM and ATR, as well as their budding yeast orthologs Tel1 and Mec1, act as master regulators of the DDR. The initiating events in the DDR entail both DNA lesion recognition and assembly of protein complexes at the damaged DNA sites. Here, we review what is known about the early steps of the DDR.

Translating Systems Medicine Into Clinical Practice: Examples From Pulmonary Medicine With Genetic Disorders, Infections, Inflammations, Cancer Genesis, and Treatment Implication of Molecular Alterations in Non-small-cell Lung Cancers and Personalized Medicine.

Non-small-cell lung cancers (NSCLC) represent 85% of all lung cancers, with adenocarcinoma as the most common subtype. Since the 2000's, the discovery of molecular alterations including epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements together with the development of specific tyrosine kinase inhibitors (TKIs) has facilitated the development of personalized medicine in the management of this disease. This review focuses on the biology of molecular alterations in NSCLC as well as the diagnostic tools and therapeutic alternatives available for each targetable alteration. Rapid and sensitive methods are essential to detect gene alterations, using tumor tissue biopsies or liquid biopsies. Massive parallel sequencing or Next Generation Sequencing (NGS) allows to simultaneously analyze numerous genes from relatively low amounts of DNA. The detection of oncogenic fusions can be conducted using fluorescence in situ hybridization, reverse-transcription polymerase chain reaction, immunohistochemistry, or NGS. EGFR mutations, ALK and ROS1 rearrangements, MET (MET proto-oncogenereceptor tyrosine kinase), BRAF (B-Raf proto-oncogen serine/threonine kinase), NTRK (neurotrophic tropomyosin receptor kinase), and RET (ret proto-oncogene) alterations are described with their respective TKIs, either already authorized or still in development. We have herein paid particular attention to the mechanisms of resistance to EGFR and ALK-TKI. As a wealth of diagnostic tools and personalized treatments are currently under development, a close collaboration between molecular biologists, pathologists, and oncologists is crucial.

Human Tumor-Infiltrating Dendritic Cells: From in Situ Visualization to High-Dimensional Analyses.

The interaction between tumor cells and the immune system is considered to be a dynamic process. Dendritic cells (DCs) play a pivotal role in anti-tumor immunity owing to their outstanding T cell activation ability. Their functions and activities are broad ranged, triggering different mechanisms and responses to the DC subset. Several studies identified in situ human tumor-infiltrating DCs by immunostaining using a limited number of markers. However, considering the heterogeneity of DC subsets, the identification of each subtype present in the immune infiltrate is essential. To achieve this, studies initially relied on flow cytometry analyses to provide a precise characterization of tumor-associated DC subsets based on a combination of multiple markers. The concomitant development of advanced technologies, such as mass cytometry or complete transcriptome sequencing of a cell population or at a single cell level, has provided further details on previously identified populations, has unveiled previously unknown populations, and has finally led to the standardization of the DCs classification across tissues and species. Here, we review the evolution of tumor-associated DC description, from in situ visualization to their characterization with high-dimensional technologies, and the clinical use of these findings specifically focusing on the prognostic impact of DCs in cancers.

Local unwinding of double-strand DNA ends by the MRX complex promotes Exo1 processing activity.

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs), which involves different nucleases including the Mre11-Rad50-Xrs2 (MRX) complex and the Exonuclease 1 (Exo1). The characterization of a novel mutation in Mre11 causing accelerated DSB resection has allowed to show that MRX facilitates DNA end processing by Exo1 through local unwinding of double-stranded DNA ends.