Affinity Measurement of Non-covalent Interactions of the Covalent KRAS G12C GDP Inhibitor MRTX849 to RAS Isoforms Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is an optical effect at an electron-rich surface that enables affinity measurements of biomolecules in real time. It is label free and versatile, not limited to proteins, nucleic acids, and small molecules. SPR is a widely accepted method to measure not only affinity of molecular interactions but also association and dissociation rates of such interactions. In this chapter, we describe a general method to measure the affinity of a small molecule drug, MRTX849, to GDP bound HRAS, KRAS, and NRAS.

A Dasatinib-Induced Chylothorax Persisting After the Discontinuation of Dasatinib.

Dasatinib is a second-generation tyrosine kinase inhibitor with activity on the BCR-ABL fusion gene. It is used in the treatment of BCR-ABL positive chronic myeloid leukaemia and has the known side effect of pleural effusion. This usually involves exudate with the presence of lymphocytes. A more uncommon side effect is the development of a chylothorax. We describe a case of a 67-year-old man with chronic myeloid leukaemia, for which treatment with dasatinib, with the presence of a right-sided chylothorax. The man presented at an outpatient consultation due to complaints of exertional dyspnoea. Radiographic imaging showed the presence of a right-sided pleural effusion. Several punctures were performed with an evacuation of 4,900 mL bloody chylous fluid in total. Each puncture revealed an increased concentration of triglycerides on the fluid, hence confirming a recurring chylothorax. A pleurodesis was performed as a final therapy. Bosutinib was substituted for dasatinib eight months prior to initial admission, making this the first reported persisting dasatinib-induced chylothorax after discontinuation. The pathophysiology has not yet been fully elucidated; it is suspected to involve an interaction on the PDGFR-ß pathway. LEARNING POINTS: Dasatinib is a rare cause of chylothorax.Chylothorax should be included in the differential diagnosis in patients developing pleural effusions when treated with dasatinib.The cornerstone of the treatment is tapering the dose or discontinuing the dasatinib therapy and changing it to another tyrosine kinase inhibitor.

Structure of the SHOC2-MRAS-PP1C complex provides insights into RAF activation and Noonan syndrome.

SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions.

Tolypocladamides A-G: Cytotoxic Peptaibols from Tolypocladium inflatum.

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.

Tumor-Draining Lymph Node Reconstruction Promotes B Cell Activation During E0771 Mouse Breast Cancer Growth.

Lymph node metastasis is associated with tumor aggressiveness and poor prognosis in patients. Despite its significance in cancer progression, how immune cells in the tumor-draining lymph node (TDLN) participate in cancer immune regulation remains poorly understood. It has been reported that both anti-tumor and exhausted tumor-specific T cells can be induced in the TDLNs; however, B cell activation and maturation in the TDLN has received far less attention. In our studies using C57BL/6 mouse syngeneic E0771 breast cancer or B16F10 melanoma cell lines, tumor-associated antigens were found colocalized with the follicular dendritic cells (FDCs) in the germinal centers (GCs), where antigen-specific B cell maturation occurs. LN conduits and the subcapsular sinus (SCS) macrophages are two major routes of antigen trafficking to FDCs. Tumor growth induced LN conduit expansion in the B cell zone and disrupted the SCS macrophage layer, facilitating both the entry of tumor-associated antigens into the B cell zone and access to FDCs located in the GCs. Regional delivery of clodronate liposome specifically depleted SCS macrophages in the TDLN, increasing GC formation, and promoting tumor growth. Our study suggests that TDLN reconstruction creates a niche that favors B cell activation and maturation during tumor growth.

Azithromycin for treatment of hospitalised COVID-19 patients: a randomised, multicentre, open-label clinical trial (DAWn-AZITHRO).

BACKGROUND AND OBJECTIVES: Azithromycin was rapidly adopted as a repurposed drug to treat coronavirus disease 2019 (COVID-19) early in the pandemic. We aimed to evaluate its efficacy in patients hospitalised for COVID-19. METHODS: In a series of randomised, open-label, phase 2 proof-of-concept, multicentre clinical trials (Direct Antivirals Working against the novel coronavirus (DAWn)), several treatments were compared with standard of care. In 15 Belgian hospitals, patients hospitalised with moderate to severe COVID-19 were allocated 2:1 to receive standard of care plus azithromycin or standard of care alone. The primary outcome was time to live discharge or sustained clinical improvement, defined as a two-point improvement on the World Health Organization (WHO) ordinal scale sustained for at least 3 days. RESULTS: Patients were included between April 22 and December 17, 2020. When 15-day follow-up data were available for 160 patients (56% of preset cohort), an interim analysis was performed at request of the independent Data Safety and Monitoring Board. Subsequently, DAWn-AZITHRO was stopped for futility. In total, 121 patients were allocated to the treatment arm and 64 patients to the standard-of-care arm. We found no effect of azithromycin on the primary outcome with a hazard ratio of 1.044 (95% CI 0.772-1.413; p=0.7798). None of the predefined subgroups showed significant interaction as covariates in the Fine-Gray regression analysis. No benefit of azithromycin was found on any of the short- and longer-term secondary outcomes. CONCLUSION: Time to clinical improvement is not influenced by azithromycin in patients hospitalised with moderate to severe COVID-19.

Development of Ice-Shelf Estuaries Promotes Fractures and Calving.

As the global climate warms, increased surface meltwater production on ice shelves may trigger ice-shelf collapse and enhance global sea-level rise. The formation of surface rivers could help prevent ice-shelf collapse if they can efficiently evacuate meltwater. Here, we present observations of the evolution of a surface river into an ice-shelf estuary atop the Petermann Ice Shelf in northwest Greenland, and identify a second estuary at the nearby Ryder Ice Shelf. This surface hydrology process can foster fracturing and enhance calving. At the Petermann estuary, sea ice was observed converging at the river mouth upstream, indicating a flow reversal. Seawater persists in the estuary, after the surrounding icescape is frozen. Along the base of Petermann estuary, linear fractures were initiated at the calving front and propagated upstream along the channel. Similar fractures along estuary channels shaped past large rectilinear calving events at the Petermann and Ryder Ice Shelves. Increased surface melting in a warming world will enhance fluvial incision, promoting estuary development, longitudinal fracturing orthogonal to ice-shelf fronts, and increase rectilinear calving. Estuaries could develop in Antarctica within the next half-century, resulting in increased calving and accelerating both ice loss and global sea-level rise.

Development of a High-throughput NanoBRET Screening Platform to Identify Modulators of the RAS/RAF Interaction.

Activating mutations in RAS are found in approximately 30% of human cancers, resulting in the delivery of a persistent signal to critical downstream effectors that drive tumorigenesis. RAS-driven malignancies respond poorly to conventional cancer treatments and inhibitors that target RAS directly are limited; therefore, the identification of new strategies and/or drugs to disrupt RAS signaling in tumor cells remains a pressing therapeutic need. Taking advantage of the live-cell bioluminescence resonance energy transfer (BRET) methodology, we describe the development of a NanoBRET screening platform to identify compounds that modulate binding between activated KRAS and the CRAF kinase, an essential effector of RAS that initiates ERK cascade signaling. Using this strategy, libraries containing synthetic compounds, targeted inhibitors, purified natural products, and natural product extracts were evaluated. These efforts resulted in the identification of compounds that inhibit RAS/RAF binding and in turn suppress RAS-driven ERK activation, but also compounds that have the deleterious effect of enhancing the interaction to upregulate pathway signaling. Among the inhibitor hits identified, the majority were compounds derived from natural products, including ones reported to alter KRAS nanoclustering (ophiobolin A), to impact RAF function (HSP90 inhibitors and ROS inducers) as well as some with unknown targets and activities. These findings demonstrate the potential for this screening platform in natural product drug discovery and in the development of new therapeutic agents to target dysregulated RAS signaling in human disease states such as cancer.

Projected land ice contributions to twenty-first-century sea level rise.

The land ice contribution to global mean sea level rise has not yet been predicted1 using ice sheet and glacier models for the latest set of socio-economic scenarios, nor using coordinated exploration of uncertainties arising from the various computer models involved. Two recent international projects generated a large suite of projections using multiple models2-8, but primarily used previous-generation scenarios9 and climate models10, and could not fully explore known uncertainties. Here we estimate probability distributions for these projections under the new scenarios11,12 using statistical emulation of the ice sheet and glacier models. We find that limiting global warming to 1.5 degrees Celsius would halve the land ice contribution to twenty-first-century sea level rise, relative to current emissions pledges. The median decreases from 25 to 13 centimetres sea level equivalent (SLE) by 2100, with glaciers responsible for half the sea level contribution. The projected Antarctic contribution does not show a clear response to the emissions scenario, owing to uncertainties in the competing processes of increasing ice loss and snowfall accumulation in a warming climate. However, under risk-averse (pessimistic) assumptions, Antarctic ice loss could be five times higher, increasing the median land ice contribution to 42 centimetres SLE under current policies and pledges, with the 95th percentile projection exceeding half a metre even under 1.5 degrees Celsius warming. This would severely limit the possibility of mitigating future coastal flooding. Given this large range (between 13 centimetres SLE using the main projections under 1.5 degrees Celsius warming and 42 centimetres SLE using risk-averse projections under current pledges), adaptation planning for twenty-first-century sea level rise must account for a factor-of-three uncertainty in the land ice contribution until climate policies and the Antarctic response are further constrained.

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane.

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

Usefulness of surgical lung biopsies after cryobiopsies when pathological results are inconclusive or show a pattern suggestive of a nonspecific interstitial pneumonia.

BACKGROUND: Although increasing data supports the use of transbronchial lung cryobiopsies (TBLCs) for the diagnosis of diffuse parenchymal lung diseases (DPLDs), its role as an alternative to surgical lung biopsy (SLB) is still under debate. The aim of this study was to assess the benefit of additional SLBs performed in selected patients after TBLCs. METHOD: We conducted a multicentric Belgian prospective trial in which SLBs were performed after TBLCs when the pathological diagnosis was uncertain or if a nonspecific interstitial pneumonia (NSIP) pattern was observed hypothesizing that SLB could provide additional information and that a co-existent UIP pattern could be missed. RESULTS: Eighty-one patients with TBLCs performed for a DPLD were included in the study between April 2015 and December 2019. A specific histological diagnosis was obtained in 52 patients (64%) whereas no pathological diagnosis following TBLCs was obtained in 13 patients (16%) and a pattern suggestive of a NSIP was observed in 16 patients (20%). Fourteen out of these 29 patients had SLBs after TBLCs. SLBs showed a UIP pattern in 11 (79%), a pattern suggestive of a hypersensitivity pneumonitis in two (14%) and a NSIP pattern in one patient (7%). Among the 16 patients with pathological NSIP following TBLCs, six underwent a SLBs showing a UIP in five and confirming a NSIP in one patient only. A retrospective pathological analysis of patients having both procedures showed a lower diagnostic confidence and agreement among pathologists for TBLCs compared to SLBs. Major factors underlying the added value of SLBs were the bigger size of the sample as well as the subpleural localization of the biopsies. CONCLUSIONS: TBLCs are useful in the setting of DPLDs with a good diagnostic yield. However, our study suggests that SLB provides critical additional information in case TBLCs are inconclusive or show a pattern suggestive of a NSIP, questioning the accuracy of TBLC to adequately identify this histological pattern.

The genomic landscape of nonsmall cell lung carcinoma in never smokers.

Lung cancer is the number one cause of cancer-related death worldwide with cigarette smoking as its major risk factor. Although the incidence of lung cancer in never smokers is rising, this subgroup of patients is underrepresented in genomic studies of lung cancer. Here, we assembled a prospective cohort of 46 never-smoking, nonsmall cell lung cancer (NSCLC) patients and performed whole-exome and low-coverage whole-genome sequencing on tumors and matched germline DNA. We observed fewer somatic mutations, genomic breakpoints and a smaller fraction of the genome with chromosomal instability in lung tumors from never smokers compared to smokers. The lower number of mutations, enabled us to identify TSC22D1 as a potential driver gene in NSCLC. On the other hand, the frequency of mutations in actionable genes such as EGFR and ERBB2 and of amplifications in MET were higher, while the mutation rate of TP53, which is a negative prognostic factor, was lower in never smokers compared to smokers. Together, these observations suggest a more favorable prognosis for never smokers with NSCLC. Classification of somatic mutations into six-substitution type patterns or into 96-substitution type signatures revealed distinct clusters between smokers and never smokers. Particularly, we identified in never smokers signatures related to aging, homologous recombination damage and APOBEC/AID activity as the most important underlying processes of NSCLC. This further indicates that second-hand smoking is not driving NSCLC pathogenesis in never smokers.

Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder.

Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (KRas). KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a fragment binder to KRasG12D and used 1H 15N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR to characterize its binding site as a pocket adjacent to the switch 2 region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a Ras ligand previously described to bind the same pocket. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.

Structures of N-terminally processed KRAS provide insight into the role of N-acetylation.

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

Lymph Node Subcapsular Sinus Macrophages as the Frontline of Lymphatic Immune Defense.

Lymphatic vessels collect and transport lymph and pathogens to the draining lymph node (LN) to generate proper immune protection. A layer of macrophages that strategically line the LN subcapsular sinus (SCS) is directly exposed to the afferent lymph and are denoted as SCS macrophages. These macrophages are the frontline of immune defense that interact with lymph-borne antigens. The importance of these macrophages in limiting the spread of pathogens has been demonstrated in both viral and bacterial infection. In anti-microbial responses, these macrophages can directly or indirectly activate other LN innate immune cells to fight against pathogens, as well as activate T cells or B cells for adaptive immunity. As the first layer of immune cells embracing the tumor-derived antigens, SCS macrophages also actively participate in cancer immune regulation. Recent studies have shown that the LNs' SCS macrophage layer is interrupted in disease models. Despite their importance in fighting the spread of pathogens and in activating anti-tumor immunity, the mechanism and the immunological functional consequences for their disruption are not well-understood. Understanding the mechanism of these macrophages will enhance their capability for therapeutic targeting.

Deciphering the sexual diploid members of the Boechera suffrutescens complex (Brassicaceae, Boechereae).

Boechera is a model genus that is of particular interest for understanding apomixis due to the presence of numerous apomictic diploid lineages that are tightly correlated with hybridisation events. Boechera includes many narrowly distributed endemics and apomictic hybrid lineages that obscure morphological boundaries amongst taxa. In this study, we focus on the Boechera suffrutescens complex, a phylogenetically well-supported but taxonomically complex north-western United States clade whose diploid species currently include the widespread B. suffrutescens and two narrowly distributed serpentine endemics, B. constancei and B. rollei. Using a 15-locus microsatellite dataset, we infer ploidy and sexual vs. apomictic reproduction for all individuals and then assess species limits for all sexual diploid samples. Our results support the recognition of B. rollei and B. constancei as distinct species and reveal three divergent sexual diploid lineages within B. suffrutescens sensu lato. The latter three lineages exhibit geographic, genetic and morphological coherence and consequently warrant recognition at the species rank. These include Boechera suffrutescens s.s., which is restricted to Idaho and eastern Oregon, Boechera botulifructa, a newly described species distributed along the Cascade Mountain Province from Lassen County, California north to Deschutes County, Oregon and the heretofore dismissed species Boechera duriuscula (basionym ≡ Arabis duriuscula), which occurs along the Sierra Nevada Province from Plumas County southwards to Fresno County, California. Our data also reveal substructure in B. constancei that is likely attributable to the highly fragmented distribution of its serpentine habitat. This refined taxonomic framework for the B. suffrutescens complex enhances Boechera as a model system, adds to our knowledge of speciation in edaphically extreme environments and provides information on ongoing conservation efforts for these taxa.

Does nivolumab for progressed metastatic lung cancer fulfill its promises? An efficacy and safety analysis in 20 general hospitals.

OBJECTIVES: In patients with refractory or recurrent non-small-cell lung cancer (NSCLC) after first line chemotherapy, phase III trials showed superiority of nivolumab, an IgG4 programmed death-1 immune-checkpoint-inhibitor antibody, over docetaxel. We evaluated case mix, effectiveness and safety of nivolumab upon implementation in general practice. MATERIALS AND METHODS: In 20 general hospitals, all consecutive NSCLC patients treated with nivolumab within the medical need program (inclusion period 12 months) in Flanders - Belgium were evaluated. RESULTS: There were 267 patients, Eastern Cooperative Oncology Group (ECOG) score was 2 in 24% and 0-1 in 76%. In 48%, two or more systemic regimens were given before nivolumab. The median overall survival was 7.8 months (95% confidence interval (CI) 6.3-9.3). At one year, the overall survival rate was 36.5±0.34%. Median progression-free survival was 3.7 months (95% CI 2.9-4.5). An objective response was obtained in 23.2%. ECOG score 2 and presence of liver metastasis strongly correlated with worse survival (p<0.00001). Treatment related adverse events grade 3 or 4 were reported in 21%, colitis (4%) and pneumonitis (7%) were most frequent. CONCLUSION: Upon implementation of nivolumab therapy in general hospitals, the case mix was characterized by a more heavily pretreated population with a substantial fraction of patients with ECOG score 2. The median overall survival is slightly inferior to what was published in the randomized phase III trials. An ECOG score 2 and the presence of liver metastasis correlated strongly with a worse survival. We report a high prevalence of serious adverse events.

Same-day dual radial artery puncture examination in patients requiring percutaneous coronary intervention and the incidence of radial artery occlusion.

BACKGROUND: We sought to investigate the rate of radial artery occlusion (RAO) after same-day dual radial artery puncture. The trans-radial arterial approach (TRA) for diagnostic and interventional procedures has risen significantly in the United States. Although becoming more commonly performed, TRA is not without risk, and a potential complication is RAO. The rate of RAO after same-day dual artery puncture is unknown. METHODS: A retrospective analysis of 27 patients who underwent same-day dual radial artery puncture for percutaneous coronary intervention (PCI) at our institution (Providence Heart Institute in Southfield, MI, USA) from November 2011 to December 2013 were included after initially presenting for cardiac catheterization at a non-PCI-capable facility. The study patients were asked to follow up for evaluation of the radial artery, including obtaining a duplex ultrasound evaluation. RESULTS: The mean age of the patients was 65 years old with 66% of the patients being male. Of the 27 study participants, there were no symptoms reported that were related to RAO. Overall, one (3.7%) patient had an absent radial pulse. The modified Allen's test was normal in all of the patients with a mean return of palmar flush time of 4 seconds. Duplex ultrasound revealed subtotal RAO in four (14.8%) patients and no patients experienced total occlusion following the intervention. CONCLUSION: Dual radial artery puncture appears to be a well-tolerated and viable strategy in patients that are transferred to a PCI-capable hospital for coronary interventions.

Role of Transbronchial Lung Cryobiopsies in Diffuse Parenchymal Lung Diseases: Interest of a Sequential Approach.

Background. Transbronchial lung cryobiopsies (TBLCs) are a promising diagnostic tool in the setting of diffuse parenchymal lung diseases (DPLDs). However, no comparison with surgical lung biopsy (SLB) in the same patient is available. Methods. The diagnostic yield and safety data of TBLCs, as well as the result of SLB performed after TBLCs, were analysed in a multicentric Belgian study. A SLB was performed after TBLCs in absence of a definite pathological diagnosis or if a NSIP pattern was observed without related condition identified following multidisciplinary discussion. Results. Between April 2015 and November 2016, 30 patients were included. Frequent complications included pneumothorax (20%) and bleeding (severe 7%, moderate 33%, and mild 53%). There was no mortality. The overall diagnostic yield was 80%. A SLB was performed in six patients (three without definite histological pattern and three with an NSIP). The surgical biopsy changed the pathological diagnosis into a UIP pattern in five patients and confirmed a NSIP pattern in one patient. Conclusion. TBLCs are useful in the diagnostic work-up of DPLDs avoiding a SLB in 80% of the patients. However, surgical biopsies, performed as a second step after TBLCs because of an indefinite diagnosis or a NSIP pattern, provide additional information supporting the interest of a sequential approach in these patients.