Establishing mesothelioma patient-derived organoid models from malignant pleural effusions.

OBJECTIVES: Pleural mesothelioma is a cancer arising in the cells that line the lungs and chest wall with poor survival and poor response to first-line therapy. Organoid models of cancer can faithfully recapitulate the genetic and histopathological characteristics of individualized tumors and have potential to be used for precision medicine, however methods of establishing patient-derived mesothelioma organoids have not been well established in the published literature. MATERIALS AND METHODS: Long-term mesothelioma patient-derived organoids were established from ten malignant pleural effusion fluids. Mesothelioma patient-derived organoids were compared to the corresponding biopsy tissue specimens using immunohistochemistry labelling for select diagnostic markers and the TruSight Oncology-500 sequencing assay. Cell viability in response to the chemotherapeutic drug cisplatin was assessed. RESULTS: We established five mesothelioma patient-derived organoid cultures from ten malignant pleural effusion fluids collected from nine individuals with pleural mesothelioma. Mesothelioma patient-derived organoids typically reflected the histopathological and genomic features of patients' matched biopsy specimens and displayed cytotoxic sensitivity to cisplatin in vitro. CONCLUSION: This is the first study of its kind to establish long-term mesothelioma organoid cultures from malignant pleural effusions and report on their utility to test individuals' chemotherapeutic sensitivities ex vivo.

Real-world prevalence of PD-L1 expression in non-small cell lung cancer: an Australia-wide multi-centre retrospective observational study.

An investigator-initiated, Australia-wide multi-centre retrospective observational study was undertaken to investigate the real-world prevalence of programmed death ligand-1 (PD-L1) expression in non-small cell lung carcinoma (NSCLC). Multiple centres around Australia performing PD-L1 immunohistochemistry (IHC) were invited to participate. Histologically confirmed NSCLC of any stage with a PD-L1 IHC test performed for persons aged ≥18 years between 1 January 2018 and 1 January 2020, and eligible for review, were identified at each centre, followed by data extraction and de-identification, after which data were submitted to a central site for collation and analysis. In total data from 6690 eligible PD-L1 IHC tests from histologically (75%) or cytologically (24%) confirmed NSCLC of any stage were reviewed from persons with a median age of 70 years, 43% of which were female. The majority (81%) of tests were performed using the PD-L1 IHC SP263 antibody with the Ventana BenchMark Ultra platform and 19% were performed using Dako PD-L1 IHC 22C3 pharmDx assay. Reported PD-L1 tumour proportion score (TPS) was ≥50% for 30% of all tests, with 62% and 38% scoring PD-L1 ≥1% and <1%, respectively. Relative prevalence of clinicopathological features with PD-L1 scores dichotomised to <50% and ≥50%, or to <1% and ≥1%, were examined. Females scored ≥1% slightly more often than males (64% vs 61%, respectively, p=0.013). However, there was no difference between sexes or age groups (<70 or ≥70 years) where PD-L1 scored ≥50%. Specimens from patients with higher stage (III/IV) scored ≥1% or ≥50% marginally more often compared to specimens from patients with lower stage (I/II) (p≤0.002). Proportions of primary and metastatic specimens did not differ where PD-L1 TPS was ≥1%, however more metastatic samples scored TPS ≥50% than primary samples (metastatic vs primary; 34% vs 27%, p<0.001). Cytology and biopsy specimens were equally reported, at 63% of specimens, to score TPS ≥1%, whereas cytology samples scored TPS ≥50% slightly more often than biopsy samples (34% vs 30%, respectively, p=0.004). Resection specimens (16% of samples tested) were reported to score TPS ≥50% or ≥1% less often than either biopsy or cytology samples (p<0.001). There was no difference in the proportion of tests with TPS ≥1% between PD-L1 IHC assays used, however the proportion of tests scored at TPS ≥50% was marginally higher for 22C3 compared to SP263 (34% vs 29%, respectively, p<0.001). These real-world Australian data are comparable to some previously published global real-world data, with some differences noted.

GM-CSF antibodies in artificial stone associated silicoproteinosis: A case report and literature review.

Pulmonary alveolar proteinosis (PAP) is a rare lung disease where there is accumulation of surfactant in the alveoli. It can be classified based on the underlying aetiology into three categories: primary, secondary and congenital. Autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF-Ab) are a key diagnostic feature of autoimmune PAP. High intensity occupational exposure and inhalation of toxic particles such as silica can cause a form of secondary PAP called acute silicoproteinosis. We describe a 26-year-old stone benchtop fabricator with silicoproteinosis following daily exposure to high levels of silica who had elevated serum GM-CSF-Ab. We discuss the role of GM-CSF-Ab in cases of PAP with occupational inhalational exposure and the challenges in its interpretation.

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.

Lineage-restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

A Grading System for Invasive Pulmonary Adenocarcinoma: A Proposal From the International Association for the Study of Lung Cancer Pathology Committee.

INTRODUCTION: A grading system for pulmonary adenocarcinoma has not been established. The International Association for the Study of Lung Cancer pathology panel evaluated a set of histologic criteria associated with prognosis aimed at establishing a grading system for invasive pulmonary adenocarcinoma. METHODS: A multi-institutional study involving multiple cohorts of invasive pulmonary adenocarcinomas was conducted. A cohort of 284 stage I pulmonary adenocarcinomas was used as a training set to identify histologic features associated with patient outcomes (recurrence-free survival [RFS] and overall survival [OS]). Receiver operating characteristic curve analysis was used to select the best model, which was validated (n = 212) and tested (n = 300, including stage I-III) in independent cohorts. Reproducibility of the model was assessed using kappa statistics. RESULTS: The best model (area under the receiver operating characteristic curve [AUC] = 0.749 for RFS and 0.787 for OS) was composed of a combination of predominant plus high-grade histologic pattern with a cutoff of 20% for the latter. The model consists of the following: grade 1, lepidic predominant tumor; grade 2, acinar or papillary predominant tumor, both with no or less than 20% of high-grade patterns; and grade 3, any tumor with 20% or more of high-grade patterns (solid, micropapillary, or complex gland). Similar results were seen in the validation (AUC = 0.732 for RFS and 0.787 for OS) and test cohorts (AUC = 0.690 for RFS and 0.743 for OS), confirming the predictive value of the model. Interobserver reproducibility revealed good agreement (k = 0.617). CONCLUSIONS: A grading system based on the predominant and high-grade patterns is practical and prognostic for invasive pulmonary adenocarcinoma.

Australian consensus statement for best practice ROS1 testing in advanced non-small cell lung cancer.

Lung cancer is the most commonly diagnosed malignancy and the leading cause of death from cancer globally. Diagnosis of advanced non-small cell lung cancer (NSCLC) is associated with 5-year relative survival of 3.2%. ROS proto-oncogene 1 (ROS1) is an oncogenic driver of NSCLC occurring in up to 2% of cases and commonly associated with younger age and a history of never or light smoking. Results of an early trial with the tyrosine kinase inhibitor (TKI) crizotinib that inhibits tumours that harbour ROS1 rearrangements have shown an objective response rate (ORR) of 72% (95% CI 58-83%), median progression free survival (PFS) of 19.3 months (95% CI 15.2-39.1 months) and median overall survival (OS) of 51.4 months (95% CI 29.3 months to not reached). Therefore, with the availability of highly effective ROS1-targeted TKI therapy, upfront molecular testing for ROS1 status alongside EGFR and ALK testing is recommended for all patients with NSCLC. We review the tissue requirements for ROS1 testing by immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) and we present a testing algorithm for advanced NSCLC and consider how the future of pathology testing for ROS1 may evolve.

Immunocytochemistry for predictive biomarker testing in lung cancer cytology.

With an escalating number of predictive biomarkers emerging in non-small cell lung carcinoma (NSCLC), immunohistochemistry (IHC) is being used as a rapid and cost-effective tool for the screening and detection of many of these markers. In particular, robust IHC assays performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue are widely used as surrogate markers for ALK and ROS1 rearrangements and for detecting programmed death ligand 1 (PD-L1) expression in patients with advanced NSCLC; in addition, they have become essential for treatment decisions. Cytology samples represent the only source of tumor in a significant proportion of patients with inoperable NSCLC, and there is increasing demand for predictive biomarker testing on them. However, the wide variation in the types of cytology samples and their preparatory methods, the use of alcohol-based fixatives that interfere with immunochemistry results, the difficulty in procurement of cytology-specific controls, and the uncertainty regarding test validity have resulted in underutilization of cytology material for predictive immunocytochemistry (ICC), and most cytopathologists limit such testing to FFPE cell blocks (CBs). The purpose of this review is to: 1) analyze various preanalytical, analytical, and postanalytical factors influencing ICC results; 2) discuss measures for validation of ICC protocols; and 3) summarize published data on predictive ICC for ALK, ROS1, EGFR gene alterations and PD-L1 expression on lung cancer cytology. Based on our experience and from a review of the literature, we conclude that cytology specimens are in principal suitable for predictive ICC, but proper optimization and rigorous quality control for high-quality staining are essential, particularly for non-CB preparations.

Deep multi-region whole-genome sequencing reveals heterogeneity and gene-by-environment interactions in treatment-naive, metastatic lung cancer.

Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.

Best Practices Recommendations for Diagnostic Immunohistochemistry in Lung Cancer.

Since the 2015 WHO classification was introduced into clinical practice, immunohistochemistry (IHC) has figured prominently in lung cancer diagnosis. In addition to distinction of small cell versus non-small cell carcinoma, patients' treatment of choice is directly linked to histologic subtypes of non-small cell carcinoma, which pertains to IHC results, particularly for poorly differentiated tumors. The use of IHC has improved diagnostic accuracy in the classification of lung carcinoma, but the interpretation of IHC results remains challenging in some instances. Also, pathologists must be aware of many interpretation pitfalls, and the use of IHC should be efficient to spare the tissue for molecular testing. The International Association for the Study of Lung Cancer Pathology Committee received questions on practical application and interpretation of IHC in lung cancer diagnosis. After discussions in several International Association for the Study of Lung Cancer Pathology Committee meetings, the issues and caveats were summarized in terms of 11 key questions covering common and important diagnostic situations in a daily clinical practice with some relevant challenging queries. The questions cover topics such as the best IHC markers for distinguishing NSCLC subtypes, differences in thyroid transcription factor 1 clones, and the utility of IHC in diagnosing uncommon subtypes of lung cancer and distinguishing primary from metastatic tumors. This article provides answers and explanations for the key questions about the use of IHC in diagnosis of lung carcinoma, representing viewpoints of experts in thoracic pathology that should assist the community in the appropriate use of IHC in diagnostic pathology.

Sex-Dependent Staging in Non-Small-Cell Lung Cancer; Analysis of the Effect of Sex Differences in the Eighth Edition of the Tumor, Node, Metastases Staging System.

INTRODUCTION: Non-small-cell lung cancer (NSCLC) has disproportionately negative outcomes in men compared with women. The importance of the relationship between sex and tumor, node, metastases (TNM) staging system remains unknown. The objective of this study was to investigate the effect of sex on NSCLC survival for each stage in the eighth edition of the TNM staging system in NSCLC. PATIENTS AND METHODS: Two cohorts treated surgically with curative intent between 2000 and 2010 were analyzed. The primary cohort was from Australia with a second population set from the Surveillance, Epidemiology, and End Results (SEER) database. Univariate and multivariate analyses of putative and validated prognostic factors were undertaken to investigate sex-dependent prognostication with detailed analyses of sex differences in each TNM stage. The primary outcome was disease-specific survival (DSS) at 5 years. RESULTS: Inclusion criteria were met by 555 patients in the Australian cohort, 335 men (60.4%) and 220 (39.6%) women; and 47,706 patients from the SEER cohort, 24,671 men (51.7%) and 23,035 women (48.3%). Five-year DSS was significantly worse for men in multivariate analyses for the Australian (hazard ratio [HR], 1.44; 95% confidence interval [CI], 1.04-1.98; P = .026) and SEER (HR, 1.24; 95% CI, 1.20-1.28; P < .001) cohorts. Detailed analysis of TNM stage sex differences revealed a consistent pattern of men having worse survival than women across stages in both cohorts. CONCLUSION: The poorer survival in men with NSCLC presents research and clinical communities with an important challenge. This study's findings suggest that for men and women diagnosed with NSCLC, and managed surgically, stage-specific outcomes should be quoted separately and consideration to a rapid prognostic score with sex combined with staging as a key element.

Pulmonary loose tumor tissue fragments and spread through air spaces (STAS): Invasive pattern or artifact? A critical review.

The concept of loose tumor tissue fragments as a pattern of invasion in lung carcinoma has recently been proposed and is included in the 2015 WHO fascicle on the classification of lung tumors, so-called "spread through airs paces" or STAS. This inclusion is controversial, as there are significant data to support that this histologic finding represents an artifact of tissue handling and processing rather than a pattern of invasion. These data are summarized in this review. These data are summarized in this review and support the conclusion that the inclusion of STAS in the WHO classification for lung cancer as a pattern of invasion was premature and erroneous. In our opinion, these tumor cell clusters or loose cells appear to be simply an artifact, although one which may or may not pinpoint to a high-grade tumor with discohesive cells and adverse prognosis.

PD-L1 Immunohistochemistry Comparability Study in Real-Life Clinical Samples: Results of Blueprint Phase 2 Project.

OBJECTIVES: The Blueprint (BP) Programmed Death Ligand 1 (PD-L1) Immunohistochemistry Comparability Project is a pivotal academic/professional society and industrial collaboration to assess the feasibility of harmonizing the clinical use of five independently developed commercial PD-L1 immunohistochemistry assays. The goal of BP phase 2 (BP2) was to validate the results obtained in BP phase 1 by using real-world clinical lung cancer samples. METHODS: BP2 were conducted using 81 lung cancer specimens of various histological and sample types, stained with all five trial-validated PD-L1 assays (22C3, 28-8, SP142, SP263, and 73-10); the slides were evaluated by an international panel of pathologists. BP2 also assessed the reliability of PD-L1 scoring by using digital images, and samples prepared for cytological examination. PD-L1 expression was assessed for percentage (tumor proportional score) of tumor cell (TC) and immune cell areas showing PD-L1 staining, with TCs scored continuously or categorically with the cutoffs used in checkpoint inhibitor trials. RESULTS: The BP2 results showed highly comparable staining by the 22C3, 28-8 and SP263 assays; less sensitivity with the SP142 assay; and higher sensitivity with the 73-10 assay to detect PD-L1 expression on TCs. Glass slide and digital image scorings were highly concordant (Pearson correlation >0.96). There was very strong reliability among pathologists in TC PD-L1 scoring with all assays (overall intraclass correlation coefficient [ICC] = 0.86-0.93), poor reliability in IC PD-L1 scoring (overall ICC = 0.18-0.19), and good agreement in assessing PD-L1 status on cytological cell block materials (ICC = 0.78-0.85). CONCLUSION: BP2 consolidates the analytical evidence for interchangeability of the 22C3, 28-8, and SP263 assays and lower sensitivity of the SP142 assay for determining tumor proportion score on TCs and demonstrates greater sensitivity of the 73-10 assay compared with that of the other assays.

The tumor suppressor Hic1 maintains chromosomal stability independent of Tp53.

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.

Interobserver Variation among Pathologists and Refinement of Criteria in Distinguishing Separate Primary Tumors from Intrapulmonary Metastases in Lung.

Multiple tumor nodules are seen with increasing frequency in clinical practice. On the basis of the 2015 WHO classification of lung tumors, we assessed the reproducibility of the comprehensive histologic assessment to distinguish second primary lung cancers (SPLCs) from intrapulmonary metastases (IPMs), looking for the most distinctive histologic features. An international panel of lung pathologists reviewed a scanned sequential cohort of 126 tumors from 48 patients and recorded an agreed set of histologic features, including tumor typing and predominant pattern of adenocarcinoma, thereby opining whether the case was SPLC, IPM, or a combination thereof. Cohen κ statistics of 0.60 on overall assessment of SPLC or IPM indicated a good agreement. Likewise, there was good agreement (κ score 0.64, p < 0.0001) between WHO histologic pattern in individual cases and SPLC or IPM status, but the proportions diversified for histologic pattern and SPLC or IPM status (McNemar test, p < 0.0001). The strongest associations for distinguishing between SPLC and IPM were observed for nuclear pleomorphism, cell size, acinus formation, nucleolar size, mitotic rate, nuclear inclusions, intraalveolar clusters, and necrosis. Conversely, the associations for lymphocytosis, mucin content, lepidic growth, vascular invasion, macrophage response, clear cell change, acute inflammation keratinization, and emperipolesis did not reach significance with tumor extent. Comprehensive histologic assessment is recommended for distinguishing SPLC from IPM with good reproducibility among lung pathologists. In addition to main histologic type and predominant patterns of histologic subtypes, nuclear pleomorphism, cell size, acinus formation, nucleolar size, and mitotic rate strongly correlate with pathologic staging status.

Promoter hypomethylation of NY-ESO-1, association with clinicopathological features and PD-L1 expression in non-small cell lung cancer.

Cancer-Testis antigens (CTA) are immunogenic molecules with normal tissue expression restricted to testes but with aberrant expression in up to 30% of non-small cell lung cancers (NSCLCs). Regulation of CTA expression is mediated in part through promoter DNA methylation. Recently, immunotherapy has altered treatment paradigms in NSCLC. Given its immunogenicity and ability to be re-expressed through demethylation, NY-ESO-1 promoter methylation, protein expression and its association with programmed death receptor ligand-1 (PD-L1) expression and clinicopathological features were investigated. Lung cancer cell line demethylation resulting from 5-Aza-2'-deoxycytidine treatment was associated with both NY-ESO-1 and PD-L1 re-expression in vitro but not increased chemosensitivity. NY-ESO-1 hypomethylation was observed in 15/94 (16%) of patient samples and associated with positive protein expression (P < 0.0001). In contrast, PD-L1 expression was observed in 50/91 (55%) but strong expression in only 12/91 (13%) cases. There was no association between NY-ESO-1 and PD-L1 expression, despite resultant re-expression of both by 5-Aza-2'-deoxycytidine. Importantly, NY-ESO-1 hypomethylation was found to be an independent marker of poor prognosis in patients not treated with chemotherapy (HR 3.59, P = 0.003) in multivariate analysis. In patients treated with chemotherapy there were no differences in survival associated with NY-ESO-1 hypomethylation. Collectively, these results provided supporting evidence for the potential use of NY-ESO-1 hypomethylation as a prognostic biomarker in stage 3 NSCLCs. In addition, these data highlight the potential to incorporate demethylating agents to enhance immune activation, in tumours currently devoid of immune infiltrates and expression of immune checkpoint genes.

De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features.

EGFR and KRAS mutations do not enrich for the activation of IL-6, JAK1 or phosphorylated STAT3 in resected lung adenocarcinoma.

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) against EGFR mutant lung adenocarcinoma develops after a median of nine to thirteen months. Upregulation of the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway may be a potential source of resistance to EGFR TKIs. We undertook a detailed assessment of the IL-6/JAK1/phosphorylated STAT3 (pSTAT3) pathway in resected lung adenocarcinoma specimens, with special interest in whether the presence of an EGFR mutation enriched for pSTAT3 positivity. Tumours from 143 patients with resected lung adenocarcinoma were assessed. EGFR and KRAS mutation status were scanned for with high-resolution melting and confirmed by polymerase chain reaction. Immunohistochemisty (IHC) was performed for IL-6, gp130, JAK1 and pSTAT3. Two methods for assigning IHC positivity were assessed (the presence of any positivity, and the presence of positivity at an H score >40). We found statistically significant associations between IL-6, JAK1 and pSTAT3 measured by IHC, consistent with the activation of the pathway in clinical specimens. No relationship was demonstrated between members of this pathway and oncogenic mutations in EGFR or KRAS. However, a proportion of tumours with EGFR mutations showed staining for IL-6, JAK1 and pSTAT3. No correlations with clinicopathologic features or survival outcomes were found for IL-6, JAK1 or pSTAT3 staining. The presence of EGFR or KRAS mutations did not enrich for the activation of IL-6, JAK1 or pSTAT3. pSTAT3 may still play a role in resistance to EGFR TKIs in clinical practice.

Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.