Clinical insights into small cell lung cancer: Tumor heterogeneity, diagnosis, therapy, and future directions.

Small cell lung cancer (SCLC) is characterized by rapid growth and high metastatic capacity. It has strong epidemiologic and biologic links to tobacco carcinogens. Although the majority of SCLCs exhibit neuroendocrine features, an important subset of tumors lacks these properties. Genomic profiling of SCLC reveals genetic instability, almost universal inactivation of the tumor suppressor genes TP53 and RB1, and a high mutation burden. Because of early metastasis, only a small fraction of patients are amenable to curative-intent lung resection, and these individuals require adjuvant platinum-etoposide chemotherapy. Therefore, the vast majority of patients are currently being treated with chemoradiation with or without immunotherapy. In patients with disease confined to the chest, standard therapy includes thoracic radiotherapy and concurrent platinum-etoposide chemotherapy. Patients with metastatic (extensive-stage) disease are treated with a combination of platinum-etoposide chemotherapy plus immunotherapy with an anti-programmed death-ligand 1 monoclonal antibody. Although SCLC is initially very responsive to platinum-based chemotherapy, these responses are transient because of the development of drug resistance. In recent years, the authors have witnessed an accelerating pace of biologic insights into the disease, leading to the redefinition of the SCLC classification scheme. This emerging knowledge of SCLC molecular subtypes has the potential to define unique therapeutic vulnerabilities. Synthesizing these new discoveries with the current knowledge of SCLC biology and clinical management may lead to unprecedented advances in SCLC patient care. Here, the authors present an overview of multimodal clinical approaches in SCLC, with a special focus on illuminating how recent advancements in SCLC research could accelerate clinical development.

Small cells - big issues: biological implications and preclinical advancements in small cell lung cancer.

Current treatment guidelines refer to small cell lung cancer (SCLC), one of the deadliest human malignancies, as a homogeneous disease. Accordingly, SCLC therapy comprises chemoradiation with or without immunotherapy. Meanwhile, recent studies have made significant advances in subclassifying SCLC based on the elevated expression of the transcription factors ASCL1, NEUROD1, and POU2F3, as well as on certain inflammatory characteristics. The role of the transcription regulator YAP1 in defining a unique SCLC subset remains to be established. Although preclinical analyses have described numerous subtype-specific characteristics and vulnerabilities, the so far non-existing clinical subtype distinction may be a contributor to negative clinical trial outcomes. This comprehensive review aims to provide a framework for the development of novel personalized therapeutic approaches by compiling the most recent discoveries achieved by preclinical SCLC research. We highlight the challenges faced due to limited access to patient material as well as the advances accomplished by implementing state-of-the-art models and methodologies.

Expression patterns of novel immunotherapy targets in intermediate- and high-grade lung neuroendocrine neoplasms.

BACKGROUND: Advancements in immunotherapeutic approaches only had a modest impact on the therapy of lung neuroendocrine neoplasms (LNENs). Our multicenter study aimed to investigate the expression patterns of novel immunotherapy targets in intermediate- and high-grade LNENs. METHODS: The expressions of V-domain Ig suppressor of T cell activation (VISTA), OX40L, Glucocorticoid-induced TNF receptor (GITR), and T cell immunoglobulin and mucin domain 3 (TIM3) proteins were measured by immunohistochemistry in surgically resected tumor samples of 26 atypical carcinoid (AC), 49 large cell neuroendocrine lung cancer (LCNEC), and 66 small cell lung cancer (SCLC) patients. Tumor and immune cells were separately scored. RESULTS: Tumor cell TIM3 expression was the highest in ACs (p < 0.001), whereas elevated tumor cell GITR levels were characteristic for both ACs and SCLCs (p < 0.001 and p = 0.011, respectively). OX40L expression of tumor cells was considerably lower in ACs (vs. SCLCs; p < 0.001). Tumor cell VISTA expression was consistently low in LNENs, with no significant differences across histological subtypes. ACs were the least immunogenic tumors concerning immune cell abundance (p < 0.001). Immune cell VISTA and GITR expressions were also significantly lower in these intermediate-grade malignancies than in SCLCs or in LCNECs. Immune cell TIM3 and GITR expressions were associated with borderline prognostic significance in our multivariate model (p = 0.057 and p = 0.071, respectively). CONCLUSIONS: LNEN subtypes have characteristic and widely divergent VISTA, OX40L, GITR, and TIM3 protein expressions. By shedding light on the different expression patterns of these immunotherapy targets, the current multicenter study provides support for the future implementation of novel immunotherapeutic approaches.

Potential subtype-specific therapeutic approaches in small cell lung cancer.

PURPOSE OF REVIEW: Small cell lung cancer (SCLC) remains one of the most aggressive thoracic malignancies with an especially dismal prognosis. While the detection of various targetable driver mutations and immune checkpoints have revolutionized the treatment of non-small cell lung cancer (NSCLC), there has been only modest therapeutic innovation over the past decades in SCLC. In this review, we aim to provide a brief summary on the clinical relevance of recent research findings, which could soon pave the way towards a more personalized and targeted management of SCLC patients. RECENT FINDINGS: Substantial research on the biological and molecular heterogeneity of SCLC has been conducted in the last years. Recent results from comprehensive profiling studies have shown that unique major SCLC subtypes can be distinguished based on the relative expression of key transcription regulators (ASCL1, NEUROD1, POU2F3) or distinct inflammatory features. Understanding the differing molecular characteristics of these distinct subtypes has resulted in the identification of specific therapeutic vulnerabilities. SUMMARY: The recently introduced molecular SCLC subtype classification represents a substantial progress towards a personalized and more efficacious approach in SCLC. The consequences of this paradigm shift provide hope for improved patient care and clinical outcomes in this exceptionally lethal thoracic malignancy.

Pseudomonas aeruginosa infection correlates with high MFI donor-specific antibody development following lung transplantation with consequential graft loss and shortened CLAD-free survival.

BACKGROUND: Donor-specific antibodies (DSAs) are common following lung transplantation (LuTx), yet their role in graft damage is inconclusive. Mean fluorescent intensity (MFI) is the main read-out of DSA diagnostics; however its value is often disregarded when analyzing unwanted post-transplant outcomes such as graft loss or chronic lung allograft dysfunction (CLAD). Here we aim to evaluate an MFI stratification method in these outcomes. METHODS: A cohort of 87 LuTx recipients has been analyzed, in which a cutoff of 8000 MFI has been determined for high MFI based on clinically relevant data. Accordingly, recipients were divided into DSA-negative, DSA-low and DSA-high subgroups. Both graft survival and CLAD-free survival were evaluated. Among factors that may contribute to DSA development we analyzed Pseudomonas aeruginosa (P. aeruginosa) infection in bronchoalveolar lavage (BAL) specimens. RESULTS: High MFI DSAs contributed to clinical antibody-mediated rejection (AMR) and were associated with significantly worse graft (HR: 5.77, p < 0.0001) and CLAD-free survival (HR: 6.47, p = 0.019) compared to low or negative MFI DSA levels. Analysis of BAL specimens revealed a strong correlation between DSA status, P. aeruginosa infection and BAL neutrophilia. DSA-high status and clinical AMR were both independent prognosticators for decreased graft and CLAD-free survival in our multivariate Cox-regression models, whereas BAL neutrophilia was associated with worse graft survival. CONCLUSIONS: P. aeruginosa infection rates are elevated in recipients with a strong DSA response. Our results indicate that the simultaneous interpretation of MFI values and BAL neutrophilia is a feasible approach for risk evaluation and may help clinicians when to initiate DSA desensitization therapy, as early intervention could improve prognosis.

C-Myc protein expression indicates unfavorable clinical outcome in surgically resected small cell lung cancer.

BACKGROUND: By being highly involved in the tumor evolution and disease progression of small cell lung cancer (SCLC), Myc family members (C-Myc, L-Myc, and N-Myc) might represent promising targetable molecules. Our aim was to investigate the expression pattern and prognostic relevance of these oncogenic proteins in an international cohort of surgically resected SCLC tumors. METHODS: Clinicopathological data and surgically resected tissue specimens from 104 SCLC patients were collected from two collaborating European institutes. Tissue sections were stained by immunohistochemistry (IHC) for all three Myc family members and the recently introduced SCLC molecular subtype-markers (ASCL1, NEUROD1, POU2F3, and YAP1). RESULTS: IHC analysis showed C-Myc, L-Myc, and N-Myc positivity in 48%, 63%, and 9% of the specimens, respectively. N-Myc positivity significantly correlated with the POU2F3-defined molecular subtype (r = 0.6913, p = 0.0056). SCLC patients with C-Myc positive tumors exhibited significantly worse overall survival (OS) (20 vs. 44 months compared to those with C-Myc negative tumors, p = 0.0176). Ultimately, in a multivariate risk model adjusted for clinicopathological and treatment confounders, positive C-Myc expression was confirmed as an independent prognosticator of impaired OS (HR 1.811, CI 95% 1.054-3.113, p = 0.032). CONCLUSIONS: Our study provides insights into the clinical aspects of Myc family members in surgically resected SCLC tumors. Notably, besides showing that positivity of Myc family members varies across the patients, we also reveal that C-Myc protein expression independently correlates with worse survival outcomes. Further studies are warranted to investigate the role of Myc family members as potential prognostic and predictive markers in this hard-to-treat disease.

Dual targeting of BCL-2 and MCL-1 in the presence of BAX breaks venetoclax resistance in human small cell lung cancer.

BACKGROUND: No targeted drugs are currently available against small cell lung cancer (SCLC). BCL-2 family members are involved in apoptosis regulation and represent therapeutic targets in many malignancies. METHODS: Expression of BCL-2 family members in 27 SCLC cell lines representing all known four SCLC molecular subtypes was assessed by qPCR, Western blot and mass spectrometry-based proteomics. BCL-2 and MCL-1 inhibition (venetoclax and S63845, respectively) was assessed by MTT assay and flow cytometry and in mice bearing human SCLC tumours. Drug interactions were calculated using the Combenefit software. Ectopic BAX overexpression was achieved by expression plasmids. RESULTS: The highest BCL-2 expression levels were detected in ASCL1- and POU2F3-driven SCLC cells. Although sensitivity to venetoclax was reflected by BCL-2 levels, not all cell lines responded consistently despite their high BCL-2 expression. MCL-1 overexpression and low BAX levels were both characteristic for venetoclax resistance in SCLC, whereas the expression of other BCL-2 family members did not affect therapeutic efficacy. Combination of venetoclax and S63845 resulted in significant, synergistic in vitro and in vivo anti-tumour activity and apoptosis induction in double-resistant cells; however, this was seen only in a subset with detectable BAX. In non-responding cells, ectopic BAX overexpression sensitised to venetoclax and S63845 and, furthermore, induced synergistic drug interaction. CONCLUSIONS: The current study reveals the subtype specificity of BCL-2 expression and sheds light on the mechanism of venetoclax resistance in SCLC. Additionally, we provide preclinical evidence that combined BCL-2 and MCL-1 targeting is an effective approach to overcome venetoclax resistance in high BCL-2-expressing SCLCs with intact BAX.

Protein Expression of immune checkpoints STING and MHCII in small cell lung cancer.

BACKGROUND: SCLC is an aggressive malignancy where immunotherapies show limited efficacy. We aimed to characterize the SCLC microenvironment according to the expression patterns of SCLC subtype markers and novel immune checkpoints to identify therapeutic vulnerabilities. METHODS: We included SCLC tissue samples from 219 surgically resected, limited-stage patients in this cross-sectional study. We performed immunohistochemistry for STING and MHCII, as well as for the novel subtype markers (ASCL1, NEUROD1, POU2F3, YAP1). Moreover, we assessed CD45 + , CD8 + and CD68 + immune cell infiltration. RESULTS: 36% of SCLC tumors showed significant stromal or intraepithelial CD45 + immune cell infiltration. These patients exhibited significantly increased overall survival (OS) (vs. patients with immune-deserted tumors). High CD8 expression was associated with increased median OS. We found STING expression on cancer-associated fibroblasts in the stroma and on T-cells and macrophages in both tumorous and stromal compartments. STING expression positively correlated with immune cell infiltration. Increased STING-positivity in tumor nests was an independent favorable prognosticator for OS. ASCL1 was the most frequently expressed subtype-specific protein. Concomitant expression of three or four subtype-defining markers was seen in 13.8% of the included samples, whereas 24.1% of the cases were classified as quadruple negative tumors. YAP1 expression was associated with increased immune infiltrates. Tumor cell MHCII expression positively correlated with immune cell infiltration and with STING- and YAP1 expressions. CONCLUSIONS: STING and MHCII are expressed in SCLC. The majority of immune-infiltrated SCLCs exhibit increased STING expression. Immune infiltration and STING expression are prognostic in limited-stage SCLC, making STING a potential therapeutic target.

Thoracic irradiation as consolidation therapy in patients with extensive-stage small cell lung cancer.

PURPOSE OF REVIEW: Small cell lung cancer (SCLC) is marked by an exceptionally high proliferative rate and poor prognosis. Given its high propensity to metastasize, nearly two-thirds of SCLC patients are diagnosed with extensive-stage (ES) disease when surgery is not a treatment option anymore. Over several decades, only minimal changes have been made in the therapeutic armamentarium of ES-SCLC. Recently, however, several new therapeutic avenues were defined, thus renewing the hope for patients with this recalcitrant cancer. Here, we present an overview of the most current therapeutic advances in ES-SCLC focusing in particular on consolidative thoracic radiation therapy (cTRT) and chemo-immunotherapy. RECENT FINDINGS: The incorporation of immunotherapy in the standard-of-care of ES-SCLC patients and the resulting outcomes are both a remarkable hallmark of progress and a disappointment. Indeed, chemo-immunotherapy with or without cTRT and prophylactic cranial irradiation contributes to longer survival outcomes with minimal toxicity rates in well selected and properly monitored patients. Nevertheless, the gain in overall survival is still modest relative to that seen in many other solid tumors. SUMMARY: Despite the encouraging results, further clinical trials are needed to determine the efficacy and safety of these therapeutic approaches, and moreover, to identify new predictive biomarkers of response.

HUNCHEST-II contributes to a shift to earlier-stage lung cancer detection: final results of a nationwide screening program.

OBJECTIVES: The introduction of low-dose CT (LDCT) altered the landscape of lung cancer (LC) screening and contributed to the reduction of mortality rates worldwide. Here we report the final results of HUNCHEST-II, the largest population-based LDCT screening program in Hungary, including the screening and diagnostic outcomes, and the characteristics of the LC cases. METHODS: A total of 4215 high-risk individuals aged between 50 and 75 years with a smoking history of at least 25 pack-years were assigned to undergo LDCT screening. Screening outcomes were determined based on the volume, growth, and volume doubling time of pulmonary nodules or masses. The clinical stage distribution of screen-detected cancers was compared with two independent practice-based databases consisting of unscreened LC patients. RESULTS: The percentage of negative and indeterminate tests at baseline were 74.2% and 21.7%, respectively, whereas the prevalence of positive LDCT results was 4.1%. Overall, 76 LC patients were diagnosed throughout the screening rounds (1.8% of total participants), out of which 62 (1.5%) patients were already identified in the first screening round. The overall positive predictive value of a positive test was 58%. Most screen-detected malignancies were stage I LCs (60.7%), and only 16.4% of all cases could be classified as stage IV disease. The percentage of early-stage malignancies was significantly higher among HUNCHEST-II screen-detected individuals than among the LC patients in the National Koranyi Institute of Pulmonology's archive or the Hungarian Cancer Registry (p < 0.001). CONCLUSIONS: HUNCHEST-II demonstrates that LDCT screening for LC facilitates early diagnosis, thus arguing in favor of introducing systematic LC screening in Hungary. CLINICAL RELEVANCE STATEMENT: HUNCHEST-II is the so-far largest population-based low-dose CT screening program in Hungary. A positive test's overall positive predictive value was 58%, and most screen-detected malignancies were early-stage lesions. These results pave the way for expansive systematic screening in the region. KEY POINTS: • Conducted in 18 medical facilities, HUNCHEST-II is the so far largest population-based low-dose CT screening program in Hungary. • The vast majority of screen-detected malignancies were early-stage lung cancers, and the overall positive predictive value of a positive test was 58%. • HUNCHEST-II facilitates early diagnosis, thus arguing in favor of introducing systematic lung cancer screening in Hungary.

Laser ablation-inductively coupled plasma-mass spectrometry analysis reveals differences in chemotherapeutic drug distribution in surgically resected pleural mesothelioma.

AIMS: Pleural mesothelioma (PM) is a highly aggressive thoracic tumour with poor prognosis. Although reduced tissue drug accumulation is one of the key features of platinum (Pt) resistance, little is known about Pt distribution in human PM. METHODS: We assessed Pt levels of blood samples and surgically resected specimens from 25 PM patients who had received neoadjuvant Pt-based chemotherapy (CHT). Pt levels and tissue distributions were measured by laser ablation-inductively coupled plasma-mass spectrometry and correlated with clinicopathological features. RESULTS: In surgically resected PM specimens, mean Pt levels of nontumourous (fibrotic) areas were significantly higher (vs tumourous regions, P = 0.0031). No major heterogeneity of Pt distribution was seen within the tumourous areas. Pt levels correlated neither with the microvessel area nor with apoptosis rate in the tumourous or nontumourous regions. A significant positive correlation was found between serum and both full tissue section and tumourous area mean Pt levels (r = 0.532, P = 0.006, 95% confidence interval [95% CI] 0.161-0.771 and r = 0.415, P = 0.039, 95% CI 0.011-0.702, respectively). Furthermore, a significant negative correlation was detected between serum Pt concentrations and elapsed time from the last cycle of CHT (r = -0.474, P = 0.017, 95% CI -0.738--0.084). Serum Pt levels correlated negatively with overall survival (OS) (P = 0.029). CONCLUSIONS: There are major differences in drug distribution between tumourous and nontumourous areas of PM specimens. Serum Pt levels significantly correlate with full section and tumourous area average Pt levels, elapsed time from the last CHT cycle, and OS. Further studies investigating clinicopathological factors that modulate tissue Pt concentration and distribution are warranted.

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

The tissue distribution and prognostic relevance of subtype-specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small-cell lung cancer (SCLC). The expression of subtype-specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype-specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC-A (ASCL1-dominant), SCLC-AN (combined ASCL1/NEUROD1), SCLC-N (NEUROD1-dominant), and SCLC-P (POU2F3-dominant), IHC and cluster analyses identified a quadruple-negative SCLC subtype (SCLC-QN). No unique YAP1-subtype was found. The highest overall survival rates were associated with non-neuroendocrine subtypes (SCLC-P and SCLC-QN) and the lowest with neuroendocrine subtypes (SCLC-A, SCLC-N, SCLC-AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard-of-care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype-specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1-subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Nationwide lung cancer screening with low-dose computed tomography: implementation and first results of the HUNCHEST screening program.

OBJECTIVES: Lung cancer (LC) kills more people than any other cancer in Hungary. Hence, there is a clear rationale for considering a national screening program. The HUNCHEST pilot program primarily aimed to investigate the feasibility of a population-based LC screening in Hungary, and determine the incidence and LC probability of solitary pulmonary nodules. METHODS: A total of 1890 participants were assigned to undergo low-dose CT (LDCT) screening, with intervals of 1 year between procedures. Depending on the volume, growth, and volume doubling time (VDT), screenings were defined as negative, indeterminate, or positive. Non-calcified lung nodules with a volume > 500 mm3 and/or a VDT < 400 days were considered positive. LC diagnosis was based on histology. RESULTS: At baseline, the percentage of negative, indeterminate, and positive tests was 81.2%, 15.1%, and 3.7%, respectively. The frequency of positive and indeterminate LDCT results was significantly higher in current smokers (vs. non-smokers or former smokers; p < 0.0001) and in individuals with COPD (vs. those without COPD, p < 0.001). In the first screening round, 1.2% (n = 23) of the participants had a malignant lesion, whereas altogether 1.5% (n = 29) of the individuals were diagnosed with LC. The overall positive predictive value of the positive tests was 31.6%. Most lung malignancies were diagnosed at an early stage (86.2% of all cases). CONCLUSIONS: In terms of key characteristics, our prospective cohort study appears consistent to that of comparable studies. Altogether, the results of the HUNCHEST pilot program suggest that LDCT screening may facilitate early diagnosis and thus curative-intent treatment in LC. KEY POINTS: • The HUNCHEST pilot study is the first nationwide low-dose CT screening program in Hungary. • In the first screening round, 1.2% of the participants had a malignant lesion, whereas altogether 1.5% of the individuals were diagnosed with lung cancer. • The overall positive predictive value of the positive tests in the HUNCHEST screening program was 31.6%.

Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives.

Well-characterized archival formalin-fixed paraffin-embedded (FFPE) tissues are of much value for prospective biomarker discovery studies, and protocols that offer high throughput and good reproducibility are essential in proteomics. Therefore, we implemented efficient paraffin removal and protein extraction from FFPE tissues followed by an optimized two-enzyme digestion using suspension trapping (S-Trap). The protocol was then combined with TMTpro 16plex labeling and applied to lung adenocarcinoma patient samples. In total, 9585 proteins were identified, and proteins related to the clinical outcome were detected. Because acetylation is known to play a major role in cancer development, a fast on-trap acetylation protocol was developed for studying endogenous lysine acetylation, which allows identification and localization of the lysine acetylation together with quantitative comparison between samples. We demonstrated that FFPE tissues are equivalent to frozen tissues to study the degree of acetylation between patients. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with isobaric labeling and for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives. The MS proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020157, PXD021986, and PXD021964.

Current therapy of KRAS-mutant lung cancer.

KRAS mutations are the most frequent gain-of-function alterations in patients with lung adenocarcinoma (LADC) in the Western world. Although they have been identified decades ago, prior efforts to target KRAS signaling with single-agent therapeutic approaches such as farnesyl transferase inhibitors, prenylation inhibition, impairment of KRAS downstream signaling, and synthetic lethality screens have been unsuccessful. Moreover, the role of KRAS oncogene in LADC is still not fully understood, and its prognostic and predictive impact with regards to the standard of care therapy remains controversial. Of note, KRAS-related studies that included general non-small cell lung cancer (NSCLC) population instead of LADC patients should be very carefully evaluated. Recently, however, comprehensive genomic profiling and wide-spectrum analysis of other co-occurring genetic alterations have identified unique therapeutic vulnerabilities. Novel targeted agents such as the covalent KRAS G12C inhibitors or the recently proposed combinatory approaches are some examples which may allow a tailored treatment for LADC patients harboring KRAS mutations. This review summarizes the current knowledge about the therapeutic approaches of KRAS-mutated LADC and provides an update on the most recent advances in KRAS-targeted anti-cancer strategies, with a focus on potential clinical implications.

Profiling the Protein Targets of Unmodified Bio-Active Molecules with Drug Affinity Responsive Target Stability and Liquid Chromatography/Tandem Mass Spectrometry.

Identifying the target proteins of bioactive small molecules is a key step in understanding mode-of-action of the drug and addressing the underlying mechanisms responsible for a particular phenotype. Proteomics has been successfully used to elucidate the target protein profiles of unmodified and ligand-modified bioactive small molecules. In the latter approach, compounds can be modified via click chemistry and combined with activity-based protein profiling. Target proteins are then enriched by performing a pull-down with the modified ligand. Methods that utilize unmodified bioactive small molecules include the cellular thermal shift assay, thermal proteome profiling, stability of proteins from rates of oxidation, and the drug affinity responsive target stability (DARTS) determination (or read-out). This review highlights recent proteomic approaches utilizing data-dependent analysis and data-independent analysis to identify target proteins by DARTS. When combined with liquid chromatography/tandem mass spectrometry, DARTS enables the identification of proteins that bind to drug molecules that leads to a conformational change in the target protein(s). In addition, an effective strategy is proposed for selecting the target protein(s) from within the pool of analyzed candidates. With additional complementary methods, the biologically relevant target proteins that bind to the small bio-active molecules can be further validated.

Comparative analysis of prognostic histopathologic parameters in subtypes of epithelioid pleural mesothelioma.

AIMS: Malignant pleural mesothelioma (MPM) is a rare malignancy with a dismal prognosis. While the epithelioid type is associated with a more favourable outcome, additional factors are needed to further stratify prognosis and to identify patients who can benefit from multimodal treatment. As epithelioid MPM shows remarkable morphological variability, the prognostic role of the five defined morphologies, the impact of the nuclear grading system and the mitosis-necrosis score were investigated in this study. METHODS AND RESULTS: Tumour specimens of 192 patients with epithelioid MPM from five European centres were histologically subtyped. Nuclear grading and mitosis-necrosis score were determined and correlated with clinicopathological parameters and overall survival (OS). Digital slides of 55 independent cases from The Cancer Genome Atlas (TCGA) database were evaluated for external validation. Histological subtypes were collapsed into three groups based on their overlapping survival curves. The tubulopapillary/microcystic group had a significantly longer OS than the solid/trabecular group (732 days versus 397 days, P = 0.0013). Pleomorphic tumours had the shortest OS (173 days). The solid/trabecular variants showed a significant association with high nuclear grade and mitosis-necrosis score. The mitosis-necrosis score was a robust and independent prognostic factor in our patient cohort. The prognostic significance of all three parameters was externally validated in the TCGA cohort. Patients with tubulopapillary or microcystic tumours showed a greater improvement in OS after receiving multimodal therapy than those with solid or trabecular tumours. CONCLUSIONS: Histological subtypes of epithelioid MPM have a prognostic impact, and might help to select patients for intensive multimodal treatment approaches.

JAK-STAT inhibition impairs K-RAS-driven lung adenocarcinoma progression.

Oncogenic K-RAS has been difficult to target and currently there is no K-RAS-based targeted therapy available for patients suffering from K-RAS-driven lung adenocarcinoma (AC). Alternatively, targeting K-RAS-downstream effectors, K-RAS-cooperating signaling pathways or cancer hallmarks, such as tumor-promoting inflammation, has been shown to be a promising therapeutic strategy. Since the JAK-STAT pathway is considered to be a central player in inflammation-mediated tumorigenesis, we investigated here the implication of JAK-STAT signaling and the therapeutic potential of JAK1/2 inhibition in K-RAS-driven lung AC. Our data showed that JAK1 and JAK2 are activated in human lung AC and that increased activation of JAK-STAT signaling correlated with disease progression and K-RAS activity in human lung AC. Accordingly, administration of the JAK1/2 selective tyrosine kinase inhibitor ruxolitinib reduced proliferation of tumor cells and effectively reduced tumor progression in immunodeficient and immunocompetent mouse models of K-RAS-driven lung AC. Notably, JAK1/2 inhibition led to the establishment of an antitumorigenic tumor microenvironment, characterized by decreased levels of tumor-promoting chemokines and cytokines and reduced numbers of infiltrating myeloid derived suppressor cells, thereby impairing tumor growth. Taken together, we identified JAK1/2 inhibition as promising therapy for K-RAS-driven lung AC.

Tumor necrosis correlates with PD-L1 and PD-1 expression in lung adenocarcinoma.

Background: Predictive biomarkers for immunotherapy in lung cancer are intensively investigated; however, correlations between PD-L1/PD-1 expressions and clinical features or histopathological tumor characteristics determined on hematoxylin and eosin stained sections have not extensively been studied. Material and methods: We determined PD-L1 expression of tumor cells (TC) and immune cells (IC), and PD-1 expression of IC by immunohistochemistry in 268 lung adenocarcinoma (LADC) patients, and correlated the data with smoking, COPD, tumor grade, necrosis, lepidic growth pattern, vascular invasion, density of stromal IC, and EGFR/KRAS status of the tumors. Results: There was a positive correlation between PD-L1 expression of TC and IC, as well as PD-L1 and PD-1 expression of IC. Tumor necrosis was associated with higher PD-L1 expression of TC and PD-1 expression of IC. A negative correlation was observed between lepidic growth pattern and PD-L1 expression of TC and PD-L1/PD-1 expression of IC. EGFR mutation seemed to negatively correlate with PD-1 expression of IC, but this tendency could not be verified when applying corrections for multiple comparisons. No significant effect of the KRAS mutation on any of the studied variables could be established. Conclusion: Here we first demonstrate that the presence of necrosis correlates with higher PD-L1 expression of TC and PD-1 expression of IC in LADC. Further studies are required to determine the predictive value of this observation in LADC patients receiving immunotherapy.

DNA methylation of microRNA-coding genes in non-small-cell lung cancer patients.

Deregulated DNA methylation leading to transcriptional inactivation of certain genes occurs frequently in non-small-cell lung cancers (NSCLCs). As well as protein-coding genes, microRNA (miRNA)-coding genes may be targets for methylation in NSCLCs; however, the number of known methylated miRNA genes is still small. Thus, we investigated methylation of miRNA genes in primary tumour (TU) samples and corresponding non-malignant lung tissue (NL) samples of 50 NSCLC patients by using methylated DNA immunoprecipitation followed by custom-designed tiling microarray analyses (MeDIP-chip), and 252 differentially methylated probes between TU samples and NL samples were identified. These probes were annotated, which resulted in the identification of 34 miRNA genes with increased methylation in TU samples. Some of these miRNA genes were already known to be methylated in NSCLCs (e.g. those encoding miR-9-3 and miR-124), but methylation of the vast majority of them was previously unknown. We selected six miRNA genes (those encoding miR-10b, miR-1179, miR-137, miR-572, miR-3150b, and miR-129-2) for gene-specific methylation analyses in TU samples and corresponding NL samples of 104 NSCLC patients, and observed a statistically significant increase in methylation of these genes in TU samples (p < 0.0001). In silico target prediction of the six miRNAs identified several oncogenic/cell proliferation-promoting factors (e.g. CCNE1 as an miR-1179 target). To investigate whether miR-1179 indeed targets CCNE1, we transfected miR-1179 gene mimics into CCNE1-expressing NSCLC cells, and observed downregulated CCNE1 mRNA expression in these cells as compared with control cells. Similar effects on cyclin E1 expression were seen in western blot analyses. In addition, we found a statistically significant reduction in the growth of NSCLC cells transfected with miR-1179 mimics as compared with control cells. In conclusion, we identified many methylated miRNA genes in NSCLC patients, and found that the miR-1179 gene is a potential tumour cell growth suppressor in NSCLCs. Overall, our findings emphasize the impact of miRNA gene methylation on the pathogenesis of NSCLCs. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.