Tracing tumor heterogeneity of pleomorphic carcinoma of the lung.

BACKGROUND: Pulmonary pleomorphic carcinoma (PPC) is an aggressive and highly heterogeneous non-small-cell lung carcinoma whose underlying biology is still poorly understood. METHODS: Forty-two tumor areas from 20 PPC patients were microdissected, including 39 primary tumors and 3 metastases and the histologically distinct components were subjected to whole exome sequencing (WES) separately. We further performed in silico analysis of microdissected bulk RNAseq and methylation data of 28 samples from 14 PPC patients. We validated our findings using immunohistochemistry. RESULTS: The epithelial and the sarcomatoid components of PPCs shared a large number of genomic alterations. Most mutations in cancer driver genes were clonal and truncal between the two components of PPCs suggesting a common ancestor. The high number of alterations in the RTK-RAS pathway suggests that it plays an important role in the evolution of PPC. The metastases morphologically and genetically resembled the epithelial or the sarcomatoid components of the tumor. The transcriptomic and epigenetic profiles of the sarcomatoid components of PPCs with matched squamous-like or adenocarcinoma-like components differed from each other and they shared more similarities to their matched epithelial components. NCAM1/CD56 was preferentially expressed in the sarcomatoid component of squamous-like PPCs, whereas CDH1/E-Cadherin expression was downregulated in the sarcomatoid component of most PPCs. CONCLUSION: LUAD-like PPCs are mainly driven by RTK-RAS signaling, whereas epithelial-mesenchymal transition programs as highlighted by increased NCAM1 and decreased CDH1 expression govern the epithelial-sarcomatoid transition between the clonally related tumor components. Several alterations in PPCs pinpoint therapeutic opportunities.

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency.

BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES: This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS: This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

Prognostic impact of tumour mutational burden in resected stage I and II lung adenocarcinomas from a European Thoracic Oncology Platform Lungscape cohort.

BACKGROUND: The primary objective of this study is to evaluate tumor mutational burden (TMB), its associations with selected clinicopathological and molecular characteristics as well as its clinical significance, in a retrospective cohort of surgically resected stage I-II lung adenocarcinomas, subset of the ETOP Lungscape cohort. METHODS: TMB was evaluated on tumor DNA extracted from resected primary lung adenocarcinomas, based on FoundationOne®CDx (F1CDx) genomic profiling, centrally performed at the University Hospital Zurich. The F1CDx test sequences the complete exons of 324 cancer-related genes and detects substitutions, insertions and deletions (indels), copy number alterations and gene rearrangements. In addition, the genomic biomarkers TMB and microsatellite instability (MSI) are analyzed. RESULTS: In the Lungscape cohort, TMB was assessed in 78 surgically resected lung adenocarcinomas from two Swiss centers (62 % males, 55 %/45 % stage I/II). Median TMB was 7.6 Muts/Mb, with TMB high (≥10 Muts/Mb) in 40 % of cases (95 %CI:29 %-52 %). The most frequently mutated genes were TP53/KRAS/EGFR/MLL2 detected in 58 %/38 %/33 %/30 % of samples, respectively. TMB was significantly higher among males (TMB high: 50 % vs 23 % in females, p = 0.032), as well as among current/former smokers (TMB high: 44 % vs 8 % in never smokers, p = 0.023). Furthermore, TMB was significantly higher in TP53 mutated than in non-mutated patients (TMB high: 60 % vs 12 %, p < 0.001), while it was higher in EGFR non-mutated patients compared to EGFR mutated (TMB high: 48 % vs 23 %, p = 0.049). At a median follow-up time of 56.1 months (IQR:38.8-72.0), none of the three outcome variables (OS, RFS, TTR) differed significantly by TMB status (all p-values > 5 %). This was also true when adjusting for clinicopathological characteristics. CONCLUSIONS: While presence of TP53 mutations and absence of EGFR mutations are associated with high TMB, increased TMB had no significant prognostic impact in patients with resected stage I/II lung adenocarcinoma beyond T and N classification, in both unadjusted and adjusted analyses.

NSCLC Subtyping in Conventional Cytology: Results of the International Association for the Study of Lung Cancer Cytology Working Group Survey to Determine Specific Cytomorphologic Criteria for Adenocarcinoma and Squamous Cell Carcinoma.

INTRODUCTION: Accurate subtyping of NSCLC into lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) is the cornerstone of NSCLC diagnosis. Cytology samples reveal higher rates of classification failures, that is, subtyping as non-small cell carcinoma-not otherwise specified (NSCC-NOS), as compared with histology specimens. This study aims to identify specific algorithms on the basis of known cytomorphologic features that aid accurate and successful subtyping of NSCLC on cytology. METHODS: A total of 13 expert cytopathologists participated anonymously in an online survey to subtype 119 NSCLC cytology cases (gold standard diagnoses being LUAD in 80 and LUSC in 39) enriched for nonkeratinizing LUSC. They selected from 23 predefined cytomorphologic features that they used in subtyping. Data were analyzed using machine learning algorithms on the basis of random forest method and regression trees. RESULTS: From 1474 responses recorded, concordant cytology typing was achieved in 53.7% (792 of 1474) responses. NSCC-NOS rates on cytology were similar among gold standard LUAD (36%) and LUSC (38%) cases. Misclassification rates were higher in gold standard LUSC (17.6%) than gold standard LUAD (5.5%; p < 0.0001). Keratinization, when present, recognized LUSC with high accuracy. In its absence, the machine learning algorithms developed on the basis of experts' choices were unable to reduce cytology NSCC-NOS rates without increasing misclassification rates. CONCLUSIONS: Suboptimal recognition of LUSC in the absence of keratinization remains the major hurdle in improving cytology subtyping accuracy with such cases either failing classification (NSCC-NOS) or misclassifying as LUAD. NSCC-NOS seems to be an inevitable morphologic diagnosis emphasizing that ancillary immunochemistry is necessary to achieve accurate subtyping on cytology.

Integration of transbronchial cryobiopsy into multidisciplinary board decision: a single center analysis of one hundred consecutive patients with interstitial lung disease.

BACKGROUND: Transbronchial cryobiopsy in the evaluation of patients with interstitial lung diseases (ILD) is expected to reduce the need for surgical lung biopsy (SLB). OBJECTIVE: To evaluate the diagnostic value of cryobiopsy in combination with bronchoalveolar lavage (BAL), radiologic and clinical data in patients with ILD. METHODS: Between 08/15 and 01/20 patients with ILD underwent cryobiopsy if they: did not have (i) an usual interstitial pneumonia (UIP)-pattern on CT, (ii) predominant ground-glass opacities suggesting alveolitis, (iii) findings suggestive of sarcoidosis on CT, or if they had (i) a CT showing UIP-pattern, but had findings suggesting alternative diagnosis than idiopathic pulmonary fibrosis (IPF), or (ii) had previous non-diagnostic conventional transbronchial forceps biopsy. Histological findings were integrated into the multidisciplinary team discussion (MDTD) and a diagnostic consensus was sought. RESULTS: One hundred patients underwent cryobiopsy. In 88/100 patients, cryobiopsy was representative with diagnostic findings in 45/88 and non-specific histological findings in 43/88 patients. In 25/43 with non-specific findings, a consensus diagnosis was reached after MDTD integrating BAL, radiologic and clinical data; eight of the remaining 18 patients with non-specific findings were referred to SLB. In 12/100 patients cryobiopsy was not representative and three of these patients were also referred to SLB. In 7/11 patients (64%) SLB was diagnostic. Complications of cryobiopsy included pneumothorax (14%) and locally controlled bleeding (24%). CONCLUSIONS: The diagnostic yield of cryobiopsy was 70%:45% of cryobiopsies were diagnostic based on histology alone and an additional 25% provided non-specific, but valuable findings allowing a consensus diagnosis after MDTD. Our data demonstrate that the diagnostic value of cryobiopsy is high if combined with BAL, radiologic and clinical data.

Deciphering the genetic landscape of pulmonary lymphomas.

Pulmonary lymphoid malignancies comprise various entities, 80% of them are pulmonary marginal zone B-cell lymphomas (PMZL). So far, little is known about point mutations in primary pulmonary lymphomas. We characterized the genetic landscape of primary pulmonary lymphomas using a customized high-throughput sequencing gene panel covering 146 genes. Our cohort consisted of 28 PMZL, 14 primary diffuse large B-cell lymphomas (DLBCL) of the lung, 7 lymphomatoid granulomatoses (LyG), 5 mature small B-cell lymphomas and 16 cases of reactive lymphoid lesions. Mutations were detected in 22/28 evaluable PMZL (median 2 mutation/case); 14/14 DLBCL (median 3 mutations/case) and 4/7 LyG (1 mutation/case). PMZL showed higher prevalence for mutations in chromatin modifier-encoding genes (44% of mutant genes), while mutations in genes related to the NF-κB pathway were less common (24% of observed mutations). There was little overlap between mutations in PMZL and DLBCL. MALT1 rearrangements were more prevalent in PMZL than BCL10 aberrations, and both were absent in DLBCL. LyG were devoid of gene mutations associated with immune escape. The mutational landscape of PMZL differs from that of extranodal MZL of other locations and also from splenic MZL. Their landscape resembles more that of nodal MZL, which also show a predominance of mutations of chromatin modifiers. The different mutational composition of pulmonary DLBCL compared to PMZL suggests that the former probably do not present transformations. DLBCL bear more mutations/case and immune escape gene mutations compared to LyG, suggesting that EBV infection in LyG may substitute for mutations.

Deciphering the clonal relationship between glandular and squamous components in adenosquamous carcinoma of the lung using whole exome sequencing.

Adenosquamous carcinoma of the lung (ASC) is a rare subtype of non-small cell lung cancer, consisting of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) components. ASC shows morphological characteristics of classic LUAD and LUSC but behaves more aggressively. Although ASC can serve as a model of lung cancer heterogeneity and transdifferentiation, its genomic background remains poorly understood. In this study, we sought to explore the genomic landscape of macrodissected LUAD and LUSC components of three ASC using whole exome sequencing (WES). Identified truncal mutations included the pan-cancer tumor-suppressor gene TP53 but also EGFR, BRAF, and MET, which are characteristic for LUAD but uncommon in LUSC. No truncal mutation of classical LUSC driver mutations were found. Both components showed unique driver mutations that did not overlap between the three ASC. Mutational signatures of truncal mutations differed from those of the branch mutations in their descendants LUAD and LUSC. Most common signatures were related to aging (1, 5) and smoking (4). Truncal chromosomal copy number aberrations shared by all three ASC included losses of 3p, 15q and 19p, and an amplified region in 5p. Furthermore, we detected loss of STK11 and SOX2 amplification in ASC, which has previously been shown to drive transdifferentiation from LUAD to LUSC in preclinical mouse models. Conclusively, this is the first study using WES to elucidate the clonal evolution of ASC. It provides strong evidence that the LUAD and LUSC components of ASC share a common origin and that the LUAD component appears to transform to LUSC.

Robust assessment of tumor mutational burden in cytological specimens from lung cancer patients.

OBJECTIVES: Tumor mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint inhibitor therapy. While the feasibility of TMB analysis on formalin-fixed paraffin-embedded (FFPE) samples has been thoroughly evaluated, only limited analyses have been performed on cytological samples, and no dedicated study has investigated concordance of TMB between different sample types. Here, we assessed TMB on matched histological and cytological samples from lung cancer patients and evaluated the accuracy of TMB estimation in these sample types. MATERIALS AND METHODS: We analyzed mutations and resulting TMB in FFPE samples and matched ethanol-fixed cytological smears (n = 12 matched pairs) by using a targeted next-generation sequencing assay (Oncomine™ Tumor Mutational Load). Two different variant allele frequency (VAF) thresholds were used to estimate TMB (VAF = 5% or 10%). RESULTS: At 5% VAF threshold, 73% (107/147) of mutations were concordantly detected in matched histological and cytological samples. Discordant variants were mainly unique to FFPE samples (34/40 discordant variants) and mostly C:G > T:A transitions with low allelic frequency, likely indicating formalin fixation artifacts. Increasing the VAF threshold to 10% clearly increased the number of concordantly detected mutations in matched histological and cytological samples to 96% (100/106 mutations), and drastically reduced the number of FFPE-only mutations (from 34 to 4 mutations). In contrast, cytological samples showed consistent mutation count and TMB values at both VAF thresholds. Using FFPE samples, 2 out of 12 patients were classified as TMB-high at VAF cutoff of 5% but TMB-low at 10%, whereas cytological specimens allowed consistent patient classification independently from VAF cutoff. CONCLUSION: Our results show that cytological smears provide more consistent TMB values due to high DNA quality and lack of formalin-fixation induced artifacts. Therefore, cytological samples should be the preferred sample type for robust TMB estimation.

A case report of a cystic fibrosis patient with repeated isolation of Trichosporon mycotoxinivorans identified by a novel short-extraction method.

BACKGROUND: Trichosporon mycotoxinivorans is a recently described yeast-like fungal organism and its association as a pathogen for patients with cystic fibrosis (CF) was reported previously. We show the clinical course of a CF patient over 9 years as well as the applications of modern molecular and proteomic identification techniques of this rare fungus. CASE PRESENTATION: We present the case of a 32-year-old male CF patient with sputum cultures continuously positive with the anamorphic yeast T. mycotoxinivorans during 9 years. Furthermore, susceptibility testing of T. mycotoxinivorans to different antifungals were performed. In addition, a rapid identification method of this novel fungal pathogen with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied using a simple extraction protocol. CONCLUSIONS: Our case presentation confirms T. mycotoxinivorans as a potential emerging pathogen in patients with CF. However, our CF patient showed mild symptoms over a very long time period of 9 years. A short MALDI-TOF MS procedure allows reliable and rapid identification of T. mycotoxinivorans and therefore should facilitate further study on the clinical relevance and epidemiology of this unusual fungal organism.

The microtubule-depolymerizing agent ansamitocin P3 programs dendritic cells toward enhanced anti-tumor immunity.

In addition to direct tumor cell cytotoxicity, chemotherapy can mediate tumor reduction through immune modulation of the tumor microenvironment to promote anti-tumor immunity. Mature dendritic cells (DCs) play key roles in priming robust immune responses in tumor-bearing hosts. Here, we screened a panel of 21 anticancer agents with defined molecular targets for their ability to induce direct maturation of DCs. We identified ansamitocin P3, a microtubule-depolymerizing agent, as a potent inducer of phenotypic and functional maturation of DCs. Exposure of both murine spleen-derived and human monocyte-derived DCs to ansamitocin P3 triggered up-regulation of maturation markers and production of pro-inflammatory cytokines, resulting in an enhanced T cell stimulatory capacity. Local administration of ansamitocin P3 induced maturation of skin Langerhans cells in vivo and promoted antigen uptake and extensive homing of tumor-resident DCs to tumor-draining lymph nodes. When used as an adjuvant in a specific vaccination approach, ansamitocin P3 dramatically increased activation of antigen-specific T cells. Finally, we demonstrate that ansamitocin P3, due to its immunomodulatory properties, acts in synergy with antibody-mediated blockade of the T cell inhibitory receptors PD-1 and CTLA-4. The combination treatment was most effective and induced durable growth inhibition of established tumors. Mechanistically, we observed a reduced regulatory T cell frequency and improved T cell effector function at the tumor site. Taken together, our study unravels an immune-based anti-tumor mechanism exploited by microtubule-depolymerizing agents, including ansamitocin P3, and paves the way for future clinical trials combining this class of agents with immunotherapy.