Pathological response and tumor stroma immunogenic features predict long-term survival in non-small cell lung cancer after neoadjuvant chemotherapy.

PURPOSE: Major pathological response (MPR) has become a surrogate endpoint for overall survival (OS) in non-small cell lung cancer (NSCLC) after neoadjuvant therapy, however, the prognostic histologic features and optimal N descriptor after neoadjuvant therapy are poorly defined. METHODS: We retrospectively analyzed data from 368 NSCLC patients who underwent surgery after neoadjuvant chemotherapy (NAC) from January 2010 to December 2020. The percentage of residual viable tumors in the primary tumor, lymph nodes (LN), and inflammation components within the tumor stroma were comprehensively reviewed. The primary endpoint was OS. RESULTS: Of the 368 enrolled patients, 12.0% (44/368) achieved MPR in the primary tumor, which was associated with significantly better OS (HR, 0.36 0.17-0.77, p = 0.008) and DFS (HR = 0.59, 0.36-0.92, p = 0.038). In patients who did not have an MPR, we identified an immune-activated phenotype in primary tumors, characterized by intense tumor-infiltrating lymphocyte or multinucleated giant cell infiltration, that was associated with similar OS and DFS as patients who had MPR. Neoadjuvant pathologic grade (NPG), consisting of MPR and immune-activated phenotype, identified 30.7% (113/368) patients that derived significant OS (HR 0.28, 0.17-0.46, p < 0.001) and DFS (HR 0.44, 0.31-0.61, p < 0.001) benefit from NAC. Moreover, the combination of NPG and the number of positive LN stations (nS) in the multivariate analysis had a higher C-index (0.711 vs. 0.663, p < 0.001) than the ypTNM Stage when examining OS. CONCLUSION: NPG integrated with nS can provide a simple, practical, and robust approach that may allow for better stratification of patients when evaluating neoadjuvant chemotherapy in clinical practice.

Treatment patterns and clinical outcomes of patients with resectable non-small cell lung cancer receiving neoadjuvant immunochemotherapy: A large-scale, multicenter, real-world study (NeoR-World).

BACKGROUND: Neoadjuvant immunotherapy has ushered in a new era of perioperative treatment for resectable non-small cell lung cancer (NSCLC). However, large-scale data for verifying the efficacy and optimizing the therapeutic strategies of neoadjuvant immunochemotherapy in routine clinical practice are scarce. METHODS: NeoR-World (NCT05974007) was a multicenter, retrospective cohort study involving patients who received neoadjuvant immunotherapy plus chemotherapy or chemotherapy alone in routine clinical practice from 11 medical centers in China between January 2010 and March 2022. Propensity score matching was performed to address indication bias. RESULTS: A total of 408 patients receiving neoadjuvant immunochemotherapy and 684 patients receiving neoadjuvant chemotherapy were included. The pathologic complete response (pCR) and major pathologic response (MPR) rates of the real-world neoadjuvant immunochemotherapy cohort were 32.8% and 58.1%, respectively. Notably, patients with squamous cell carcinoma exhibited significantly higher pCR and MPR rates than those with adenocarcinoma (pCR, 39.2% vs 16.5% [P < .001]; MPR, 66.6% vs 36.5% [P < .001]), whereas pCR and MPR rates were comparable among patients receiving different neoadjuvant cycles. In addition, the 2-year rates of disease-free survival (DFS) and overall survival (OS) rate were 82.0% and 93.1%, respectively. Multivariate analyses identified adjuvant therapy as an independent prognostic factor for DFS (hazard ratio [HR], 0.51; 95% confidence interval [CI], 0.29-0.89; P = .018) and OS (HR, 0.28; 95% CI, 0.13-0.58; P < .001). A significantly longer DFS with adjuvant therapy was observed in patients with non-pCR or 2 neoadjuvant cycles. We observed significant benefits in pCR rate (32.4% vs 6.4%; P < .001), DFS (HR, 0.50; 95% CI, 0.38-0.68; P < .001) and OS (HR, 0.61; 95% CI, 0.40-0.94; P = .024) with immunotherapy plus chemotherapy compared to chemotherapy alone both in the primary propensity-matched cohort and across most key subgroups. CONCLUSIONS: The study validates the superior efficacy of neoadjuvant immunochemotherapy over chemotherapy alone for NSCLC. Adjuvant therapy could prolong DFS in patients receiving neoadjuvant immunochemotherapy, and patients with non-pCR or those who underwent 2 neoadjuvant cycles were identified as potential beneficiaries of adjuvant therapy.

Development and validation of a nomogram based on preoperative variables for predicting recurrence-free survival in stage IA lung adenocarcinoma.

BACKGROUND: This study aimed to establish a nomogram for predicting risk of recurrence and provide a model for decision-making between lobectomy and sublobar resection in patients with stage IA lung adenocarcinoma. METHODS: Patients diagnosed with stage IA lung adenocarcinoma (LUAD) between December 2010 and October 2018 from Cancer Hospital Chinese Academy of Medical Sciences were included. Patients were randomly assigned to training and validation cohorts, accounting for 70% and 30% of the total cases, respectively. We collected laboratory variables before surgery. Univariate and multivariate analyses were performed in the training cohort to identify variables significantly associated with recurrence-free survival (RFS) which were subsequently used to construct a nomogram. Validation was conducted in both cohorts. A receiver operating characteristic curve was used to determine the optional cutoff values of the scores calculated from the nomogram. Patients were then divided into low- and high-risk groups. Survival was performed to determine if the nomogram could guide the operation method. RESULTS: A total of 543 patients were included in this study. Gender, albumin level, carcinoembryonic antigen level and cytokeratin-19-fragment level were included in the nomogram. In both cohorts, the nomogram stratified the patients into high- and low-risk groups in terms of RFS. In particular, there was a significant difference in RFS between lobectomy and sublobar resection in the high-risk group. CONCLUSIONS: Gender, albumin level, carcinoembryonic antigen level and cytokeratin-19-fragment level are valuable markers in predicting recurrence and can guide surgical practice in patients with stage IA LUAD.

Fibroblast Activation Protein Targeting Probe with Gly-Pro Sequence for PET of Glioblastoma.

As an important cancer-associated fibroblast-specific biomarker, fibroblast activation protein (FAP) has become an attractive target for tumor diagnosis and treatment. However, most FAP-based radiotracers showed inadequate uptake and short retention in tumors. In this study, we designed and synthesized a novel FAP ligand (DOTA-GPFAPI-04) through assembling three functional moieties: a quinoline-based FAP inhibitor for specifically targeting FAP, a FAP substrate Gly-Pro as a linker for increasing the FAP protein interaction, and a 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) chelator for radiolabeling with different radionuclides. The FAP targeting ability of DOTA-GPFAPI-04 was investigated by molecular docking studies. DOTA-GPFAPI-04 was then radiolabeled with 68Ga to give [68Ga]Ga-DOTA-GPFAPI-04 for positron emission tomography (PET) imaging of glioblastoma. [68Ga]Ga-DOTA-GPFAPI-04 exhibited a purity of >98% and high stability analyzed by radio-HPLC in saline and mouse serum. Cell uptake studies demonstrated the targeting specificity of the probe. Further in vivo pharmacokinetic studies in normal mice demonstrated the quick clearance of the probe. Moreover, compared with the widely studied [68Ga]Ga-FAPI-04, [68Ga]Ga-DOTA-GPFAPI-04 showed much higher U87MG tumor uptake values (4.467 ± 0.379 for [68Ga]Ga-DOTA-GPFAPI-04 and 1.267 ± 0.208% ID/g for [68Ga]Ga-FAPI-04 at 0.5 h post-injection, respectively). The area under the curve based on time-activity curve (TAC) analysis for tumor radioactivity in small animal models was 422.5 for [68Ga]Ga-DOTA-GPFAPI-04 and 98.14 for [68Ga]Ga-FAPI-04, respectively, demonstrating that the former had longer tumor retention time. The tumor-to-muscle (T/M) ratio for [68Ga]Ga-DOTA-GPFAPI-04 reached 9.15 in a U87MG xenograft animal model. PET imaging and blocking assays showed that [68Ga]Ga-DOTA-GPFAPI-04 had specific tumor uptake. In summary, this study demonstrates the successful synthesis and evaluation of a novel FAPI targeting probe, [68Ga]Ga-DOTA-GPFAPI-04, with a Gly-Pro sequence. It shows favorable in vivo glioblastoma imaging properties and relatively long tumor retention, highlighting DOTA-GPFAPI-04 as a promising molecular scaffold for developing FAP targeting tumor theranostic agents.

A strategy to reduce the false-positive rate after low-dose computed tomography in lung cancer screening: A multicenter prospective cohort study.

BACKGROUND: The ability of lung cancer screening to manage pulmonary nodules was limited because of the high false-positive rate in the current mainstream screening method, low-dose computed tomography (LDCT). We aimed to reduce overdiagnosis in Chinese population. METHODS: Lung cancer risk prediction models were constructed using data from a population-based cohort in China. Independent clinical data from two programs performed in Beijing and Shandong, respectively, were used as the external validation set. Multivariable logistic regression models were used to estimate the probability of lung cancer incidence in the whole population and in smokers and nonsmokers. RESULTS: In our cohort, 1,016,740 participants were enrolled between 2013 and 2018. Of 79,581 who received LDCT screening, 5165 participants with suspected pulmonary nodules were allocated into the training set, of which, 149 lung cancer cases were diagnosed. In the validation set, 1815 patients were included, and 800 developed lung cancer. The ages of patients and radiologic factors of nodules (calcification, density, mean diameter, edge, and pleural involvement) were included in our model. The area under the curve (AUC) values of the model were 0.868 (95% CI: 0.839-0.894) in the training set and 0.751 (95% CI: 0.727-0.774) in the validation set. The sensitivity and specificity were 70.5% and 70.9%, respectively, which could reduce the 68.8% false-positive rate in simulated LDCT screening. There was no substantial difference between smokers' and nonsmokers' prediction models. CONCLUSION: Our models could facilitate the diagnosis of suspected pulmonary nodules, effectively reducing the false-positive rate of LDCT for lung cancer screening.

Development of a Novel HER2-Targeted Peptide Probe for Dual-Modal Imaging of Tumors.

Human epidermal growth factor receptor 2 (HER2) may serve as a valid target for diagnosis of cancer. The probes with capability for near-infrared window one region II (NIR-II) and positron emission tomography (PET) dual-modal imaging are highly desired for HER2-positive tumor detection. Herein, three HER2-targeted peptides were designed and further modified with indocyanine green (ICG) and 2,2',2″,2″-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA), which were used for NIR-II imaging and complexation with 68Ga for PET. Among the resulted probes (DOTA-ZC01-ICG, DOTA-KSP-ICG, and DOTA-ZC02-ICG), NIR-II imaging revealed that DOTA-ZC02-ICG had the best tumor imaging performance in SKOV3 tumor-bearing mice. The highest T/N ratio (5.4) was achieved at 4 h post-injection. Furthermore, DOTA-ZC02-ICG was radiolabeled with 68Ga to generate [68Ga]-DOTA-ZC02-ICG for PET, and it clearly delineated at 0.5, 1, and 2 h post-injection. The tumor uptake reached 1.9 %ID/g at 0.5 h, and the tumor uptakes were significantly inhibited in the blocking study (p < 0.05). Overall, it provides a promising technique for tumor dual-modal imaging and a new molecular scaffold for developing HER2-targeted theranostic agents.

Novel HER2-Targeted Peptide for NIR-II Imaging of Tumor.

Molecular targets serve a crucial role in drug development. Herein, we discovered a novel peptide that can specifically target the human epidermal growth factor receptor 2 (HER2) and thus named it Herceptide. In our study, Herceptide was conjugated to the near-infrared fluorescent dye indocyanine green (ICG) to obtain a probe, ICG-Herceptide. Importantly, specific binding to HER2 was revealed by molecular docking, surface plasmon resonance analysis, and competition assays. The probe showed high binding affinity (KD = 1.03 nM) and fast binding property (kon = 0.44 min-1). In vivo near-infrared window two (NIR-II, 1000-1700 nm) imaging in HER2-overexpressed SKOV3 tumor-bearing mice demonstrated a high tumor-to-normal tissue signal ratio (T/N = 7.3) at 8 h postinjection. In the blocking study, ICG-Herceptide coinjected with Herceptide only showed a weak tumor signal. In other HER2 high-expression tumors, such as non-small-cell lung cancer A549 and gastric cancer MKN45, the tumor-to-normal tissue signal ratios (T/N) were 4.1 and 4.7, respectively. In contrast, HER2 low-expression tumor MDAMB231 shows no imaging contrast between the tumor and normal tissues. Furthermore, tumor resection was successfully performed under the guidance of the ICG-Herceptide-based NIR-II imaging in subcutaneous SKOV3 mice models. The biocompatibility study indicated that the probe had no observable toxicity to cells and tissues. Overall, these results demonstrate that ICG-Herceptide is a promising optical probe for the diagnosis and localization of HER2-overexpressing tumors. Moreover, Herceptide is a novel HER2-targeting peptide and can be further used for developing theranostic agents.

A non-metabolic function of hexokinase 2 in small cell lung cancer: promotes cancer cell stemness by increasing USP11-mediated CD133 stability.

BACKGROUND: Maintenance of cancer stem-like cell (CSC) stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression. As a key glycolytic enzyme, hexokinase 2 (HK2) plays an instrumental role in aerobic glycolysis and tumor progression. However, whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer (SCLC) is largely unclear. In this study, we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in SCLC. METHODS: Immunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated counterparts. CSC-like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse model. Immunoprecipitation and mass spectrometry analyses were performed to identify the binding proteins of CD133. The expression levels of CD133-associated and CSC-relevant proteins were evaluated by immunoblotting, immunoprecipitation, immunofluorescence, and immunohistochemistry assay. RNA expression levels of Nanog, POU5F1, Lin28, HK2, Prominin-1 were analyzed through quantitative reverse transcription PCR. Polyubiquitination of CD133 was examined by in vitro or in vivo ubiquitination assay. CD133+ cells were sorted by flow cytometry using an anti-CD133 antibody. RESULTS: We demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated counterparts. HK2 depletion inhibited CSC stemness and promoted CSC differentiation. Mechanistically, non-mitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA levels. The interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin-specific protease 11 (USP11) to CD133, thereby inhibiting CD133 polyubiquitylation and degradation. HK2-mediated upregulation of CD133 expression enhanced the expression of cell renewal regulators, SCLC cell stemness, and tumor growth in mice. In addition, HK2 expression was positively correlated with CD133 expression in human SCLC specimens, and their expression levels were associated with poor prognosis of SCLC patients. CONCLUSIONS: These results revealed a critical non-metabolic function of HK2 in promotion of cancer cell stemness. Our findings provided new insights into the multifaceted roles of HK2 in tumor development.

Whole-genome bisulfite sequencing analysis of circulating tumour DNA for the detection and molecular classification of cancer.

BACKGROUND: Cancer cell-specific variation and circulating tumour DNA (ctDNA) methylation are promising biomarkers for non-invasive cancer detection and molecular classification. Nevertheless, the applications of ctDNA to the early detection and screening of cancer remain highly challenging due to the scarcity of cancer cell-specific ctDNA, the low signal-to-noise ratio of DNA variation, and the lack of non-locus-specific DNA methylation technologies. METHODS: We enrolled three cohorts of breast cancer (BC) patients from two hospitals in China (BC: n = 123; healthy controls: n = 40). We developed a ctDNA whole-genome bisulfite sequencing technology employing robust trace ctDNA capture from up to 200 µL plasma, mini-input (1 ng) library preparation, unbiased genome-wide coverage and comprehensive computational methods. RESULTS: A diagnostic signature comprising 15 ctDNA methylation markers exhibited high accuracy in the early (area under the curve [AUC] of 0.967) and advanced (AUC of 0.971) BC stages in multicentre patient cohorts. Furthermore, we revealed a ctDNA methylation signature that discriminates estrogen receptor status (Training set: AUC of 0.984 and Test set: AUC of 0.780). Different cancer types, including hepatocellular carcinoma and lung cancer, could also be well distinguished. CONCLUSIONS: Our study provides a toolset to generate unbiased whole-genome ctDNA methylomes with a minimal amount of plasma to develop highly specific and sensitive biomarkers for the early diagnosis and molecular subtyping of cancer.

The Deubiquitinase USP13 Maintains Cancer Cell Stemness by Promoting FASN Stability in Small Cell Lung Cancer.

USP13 is significantly amplified in over 20% of lung cancer patients and critical for tumor progression. However, the functional role of USP13 in small cell lung cancer (SCLC) remains largely unclear. In this study, we found that the deubiquitinase USP13 is highly expressed in SCLC tumor samples and positively associated with poor prognosis in multiple cohorts. In vitro and in vivo depletion of USP13 inhibited SCLC cancer stem cells (CSCs) properties and tumorigenesis, and this inhibitory effect was rescued by reconstituted expression of wide type (WT) USP13 but not the enzyme-inactive USP13 mutant. Mechanistically, USP13 interacts with fatty acid synthase (FASN) and enhances FASN protein stability. FASN downregulation suppresses USP13-enhanced cell renewal regulator expression, sphere formation ability, and de novo fatty acids biogenesis. Accordingly, we found FASN expression is upregulated in surgical resected SCLC specimens, positively correlated with USP13, and associated with poor prognosis of SCLC patients. More importantly, the small molecule inhibitor of FASN, TVB-2640, significantly inhibits lipogenic phenotype and attenuates self-renewal ability, chemotherapy resistance and USP13-mediated tumorigenesis in SCLC. Thus, our study highlights a critical role of the USP13-FASN-lipogenesis axis in SCLC cancer stemness maintenance and tumor growth, and reveals a potential combination therapy for SCLC patients.

External validation of the eighth edition of the TNM classification for lung cancer in small cell lung cancer.

OBJECTIVES: The newly released eighth edition of the American Joint Committee on Cancer TNM staging system for lung cancer seeks to improve prognostic accuracy but lacks external validation for small cell lung cancer (SCLC). Moreover, previous studies posed a few questions concerning survival differences for patients with specific site N3 node involvement or single-site metastasis (SSM) in different distant organs. The aim of this study was to validate the eighth edition of the TNM classification for SCLC in an independent multi-institutional cohort from China and answer the questions raised by the previous research. METHODS: Patients with SCLC from four Chinese cancer centers between 2009 and 2019 were reclassified according to the seventh and eighth edition of the TNM classification. Survival was estimated using the Kaplan-Meier method. Comparisons between adjacent categories and stage groups were performed using Cox proportional hazard regression. R2 statistics were calculated to evaluate the discriminating performance of editions. RESULTS: Of 3384 enrolled cases, 3358 had clinical stage, 537 had pathological stage, and 511 had both. Progressive deterioration of survival was observed with advancing of TNM categories and stages both in the seventh and the eighth edition. The eighth edition stages had a higher R2 statistic than the seventh edition (0.207 versus 0.197). Newly defined categories M1b and M1c and stages IIIC, IVA and IVB in the eighth edition discriminated groups with significantly different prognosis. Patients with N3 contralateral supraclavicular nodes had a significantly worse prognosis than those without (p = 0.032). For patients with single-site metastasis, liver involvement showed a worse prognosis compared to brain involvement (p = 0.030). CONCLUSIONS: Our study provided an external validation of the eighth edition of the TNM classification for lung cancer in Chinese patients with SCLC, and confirmed its improved prognostic accuracy compared with the seventh edition. Patients with N3 and M1b might represent heterogeneous populations that warrant further research.

PSC subtyping based on TTF-1 and p40 expression reveals distinct molecular characteristics and therapeutic strategies.

Pulmonary sarcomatoid carcinoma (PSC) is a unique form of poorly differentiated nonsmall cell lung cancer (NSCLC) and is notorious for its highly malignant nature and dismal prognosis. To introduce effective treatment for PSC patients, precise subtyping of PSC is demanding. In our study, TTF-1 and P40 immunohistochemistry (IHC) staining were applied to 56 PSC patients with multiomics data. According to IHC results, we categorized these patients into three subgroups and profiled their molecular contexture using bioinformatic skills. IHC results classified these patients into three subgroups: TTF-1 positive subgroup (n = 27), P40 positive subgroup (n = 15) and double-negative subgroup (n = 14). Spindle cell samples accounted for 35.71% (5/14) of double-negative patients, higher than others (P = .034). The three subgroups were heterogeneous in the genomic alteration spectrum, showing significant differences in the RTK/RAS pathway (P = .004) and the cell cycle pathway (P = .030). The methylation profile of the double-negative subgroup was between the other two subgroups. In similarity analysis, the TTF-1 and p40 subgroups were closely related to LUAD and LUSC, respectively. The TTF-1 positive subgroup had the highest leukocyte fraction (LF) among several cancer types, and the tumor mutation burden (TMB) of the p40 positive subgroup ranked third in the TMB list, suggesting the applicability of immunotherapy for PSC. The study established a new subtyping method of PSC based on IHC results and reveals three subgroups with distinct molecular features, providing evidence for refined stratification in the treatment of PSC.

Multimodality Treatment of Pulmonary Sarcomatoid Carcinoma: A Review of Current State of Art.

Pulmonary sarcomatoid carcinoma (PSC) is an unconventional non-small-cell lung cancer (NSCLC) that is currently managed under guidelines used for conventional NSCLC and has poor survival. Surgery is the optimal choice for resectable PSC, and the prevalence of mutations in this type of tumor laid the foundation for novel systemic therapies such as targeted therapy and immunotherapy. PSC is resistant to chemotherapy and radiotherapy, and the effects of the 2 therapies are controversial. Targeted therapies have been reported to confer survival benefits, and savolitinib, an oral selective MET tyrosine-kinase inhibitor, has been approved in metastatic patients with MET exon 14 skipping mutations. Expression and positive rate of programmed death ligand 1 in PSC are high; our previous research has also revealed a high mutational burden and a T-cell-inflamed microenvironment of PSC. Correspondingly, immune checkpoint inhibitors have shown preliminary antitumor effects (overall response rates of 40.5% (15/37) and 31.6% (6/19) in two retrospective studies, respectively) in PSC patients. In summary, patients should receive operations at an early stage and multimodality treatments are needed to maximize the benefits of patients with advanced disease.

Ferroptosis Characterization in Lung Adenocarcinomas Reveals Prognostic Signature With Immunotherapeutic Implication.

The iron-dependent cell death named ferroptosis has been implicated in the progression and therapeutic response of several tumors. However, potential role of ferroptosis in lung adenocarcinomas (LUAD) remained less well understood. In TCGA-LUAD cohort, unsupervised clustering was first conducted based on ferroptosis regulators extracted from FerrDb database. Comprehensive correlation analysis and comparisons were performed among ferroptosis subtypes. The ferroptosis-related prognostic (FRP) signature was identified based on filtered features and repeated LASSO and was validated in five independent cohorts. The clinical relevance between the risk score and therapeutic response was further explored by multiple algorithms. qPCR was implemented to verify gene expression. A total of 1,168 LUAD patients and 161 ferroptosis regulators were included in this study. Three ferroptosis subtypes were identified and patients in subtype B had the best prognosis among the three subtypes. Significant differences in immune microenvironment and biological function enrichment were illustrated in distinct subtypes. The Boruta algorithm was conducted on 308 common differentially expressed genes for dimensionality reduction. A total of 56 genes served as input for model construction and a six-gene signature with the highest frequencies of 881 was chosen as FRP. The prognostic significance of FRP was validated in five independent cohorts. High FRP risk score was also linked to increased tumor mutation burden, PD-L1 protein expression and number of neoantigens. Of the FRP genes, 83.3% was abnormally expressed in LUAD cell lines. In conclusion, ferroptosis plays a non-negligible role in LUAD. Exploration of the ferroptosis pattern will enhance the prognostic stratification of individual patients and move toward the purpose of personalized treatment.

Multi-omics profiling of primary small cell carcinoma of the esophagus reveals RB1 disruption and additional molecular subtypes.

Primary small cell carcinoma of the esophagus (PSCCE) is a lethal neuroendocrine carcinoma. Previous studies proposed a genetic similarity between PSCCE and esophageal squamous cell carcinoma (ESCC) but provided little evidence for differences in clinical course and neuroendocrine differentiation. We perform whole-exome sequencing, RNA sequencing and immunohistochemistry profiling on 46 PSCCE cases. Integrated analyses enable the discovery of multiple mechanisms of RB1 disruption in 98% (45/46) of cases. The transcriptomic landscape of PSCCE closely resembles small cell lung cancer (SCLC) but differs from ESCC or esophageal adenocarcinoma (EAC). Distinct gene expression patterns regulated by ASCL1 and NEUROD1 define two molecular subtypes, PSCCE-A and PSCCE-N, which are highly similar to SCLC subtypes. A T cell excluded phenotype is widely observed in PSCCE. In conclusion, PSCCE has genomic alterations, transcriptome features and molecular subtyping highly similar to SCLC but distinct from ESCC or EAC. These observations are relevant to oncogenesis mechanisms and therapeutic vulnerability.

Comprehensive Analysis of PD-L1 Expression, Immune Infiltrates, and m6A RNA Methylation Regulators in Esophageal Squamous Cell Carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancer types and represents a threat to global public health. N6-Methyladenosine (m6A) methylation plays a key role in the occurrence and development of many tumors, but there are still few studies investigating ESCC. This study attempts to construct a prognostic signature of ESCC based on m6A RNA methylation regulators and to explore the potential association of these regulators with the tumor immune microenvironment (TIME). Methods: The transcriptome sequencing data and clinical information of 20 m6A RNA methylation regulators in 453 patients with ESCC (The Cancer Genome Atlas [TCGA] cohort, n = 95; Gene Expression Omnibus [GEO] cohort, n = 358) were obtained. The differing expression levels of m6A regulators between ESCC and normal tissue were evaluated. Based on the expression of these regulators, consensus clustering was performed to investigate different ESCC clusters. PD-L1 expression, immune score, immune cell infiltration and potential mechanisms among different clusters were examined. LASSO Cox regression analysis was utilized to obtain a prognostic signature based on m6A RNA methylation modulators. The relationship between the risk score based on the prognostic signature and the TIME of ESCC patients was studied in detail. Results: Six m6A regulators (METTL3, WTAP, IGF2BP3, YTHDF1, HNRNPA2B1 and HNRNPC) were observed to be significantly highly expressed in ESCC tissues. Two molecular subtypes (clusters 1/2) were determined by consensus clustering of 20 m6A modulators. The expression level of PD-L1 in ESCC tissues increased significantly and was significantly negatively correlated with the expression levels of YTHDF2, METL14 and KIAA1429. The immune score, CD8 T cells, resting mast cells, and regulatory T cells (Tregs) in cluster 2 were significantly increased. Gene set enrichment analysis (GSEA) shows that this cluster involves multiple hallmark pathways. We constructed a five-gene prognostic signature based on m6A RNA methylation, and the risk score based on the prognostic signature was determined to be an independent prognostic indicator of ESCC. More importantly, the prognostic value of the prognostic signature was verified using another independent cohort. m6A regulators are related to TIME, and their copy-number alterations will dynamically affect the number of tumor-infiltrating immune cells. Conclusion: Our study established a strong prognostic signature based on m6A RNA methylation regulators; this signature was able to accurately predict the prognosis of ESCC patients. The m6A methylation regulator may be a key mediator of PD-L1 expression and immune cell infiltration and may strongly affect the TIME of ESCC.

PD-L1 and CD47 co-expression predicts survival and enlightens future dual-targeting immunotherapy in non-small cell lung cancer.

BACKGROUD: Recent studies have indicated that programmed cell death-ligand 1 (PD-L1) and cluster of differentiation 47 (CD47) play an essential role in tumor immune evasion and may serve as potential targets for combined immunotherapy. The aim of our study was to evaluate the PD-L1/CD47 expression status in lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD), and explore its survival impact and relevance with the immune microenvironment. METHODS: The specimens from 190 LUSC and 240 LUAD patients who underwent intent-to-treat surgeries were retrospectively collected for immunohistochemistry assays of PD-L1, CD47, cluster of differentiation 8 (CD8), and cluster of differentiation 68 (CD68). RESULTS: A total of 96 (22.3%) and 296 (68.8%) cases were positive for PD-L1 and CD47 expression, respectively, and 80 (18.6%) of them demonstrated the co-expression of PD-L1/CD47. The rate of PD-L1/CD47 co-expression was 23.7% in LUSC, significantly higher than the 14.6% in LUAD (p = 0.018). The median overall survival (OS) for all patients was 55.9 months (range 2.0-146.0 months). The univariate analysis showed that patients with positive CD47 expression (LUSC p = 0.003, LUAD p = 0.036) and PD-L1/CD47 co-expression (LUSC p = 0.023, LUAD p = 0.004) exhibited significantly worse prognosis. The multivariate analysis demonstrated that PD-L1/CD47 co-expression was an independent prognostic factor for OS (LUSC hazard ratio [HR] 1.922, 95% CI 1.245-2.969, p = 0.003; LUAD HR 1.549, 95% CI 1.015-2.364, p = 0.043). PD-L1/CD47 co-expression was associated with high CD8-positive T-lymphocyte density in LUSC (p = 0.004) and LUAD (p = 0.043), and with high CD68-positive macrophage density in LUSC (p = 0.026). CONCLUSIONS: PD-L1/CD47 co-expression was an independent prognostic factor for LUSC and LUAD patients and may serve as a potential predictive biomarker for combined dual-targeting immunotherapy.

Comprehensive Analysis of Ferroptosis Regulators in Lung Adenocarcinomas Identifies Prognostic and Immunotherapy-Related Biomarkers.

Ferroptosis is a newly discovered type of programmed cell death that differs from canonical apoptosis. However, the potential role of ferroptosis in lung adenocarcinoma (LUAD) has not been elaborated. In total, 1,328 samples from databases and 36 ferroptosis regulators were included in this study. By combining random survival forest and principal component analysis algorithms, a robust prognostic ferroptosis-related risk score (FRRS) was constructed, and the performance was validated in three independent datasets. Based on the median risk score, two subgroups were identified. Then, comparisons, including of mutational profiles, functional enrichment analyses and immune components, were conducted between subgroups. An immunotherapy cohort was applied to explore potential therapeutic-related biomarkers. Finally, the clinical utility of FRRS was validated in a proteomic cohort. In the TCGA-LUAD cohort, FRRS was calculated using the expression of 11 selected genes, and patients with high FRRS had a significantly (p < 0.001) worse prognosis than those with low FRRS. Multivariate regression suggested that FRRS was an independent prognostic factor. Functional enrichment analysis indicated that FRRS was mainly involved in cell cycle, metabolic and immune-related pathways. Furthermore, FRRS was shown to be significantly (p < 0.001) associated with the abundance of CD8 T cells and tumor mutation burden (TMB). The combination of TMB and FANCD2 expression, the main contributor to FRRS, substantially increased the precision of predicting the therapeutic response. In conclusion, the present study revealed the potential role of ferroptosis regulators in LUAD and identified ferroptosis-related biomarkers for prognostic and immunotherapeutic predictions.

ERAP2 Is Associated With Immune Infiltration and Predicts Favorable Prognosis in SqCLC.

Background: Immunotherapy has been proven effective among several human cancer types, including Squamous cell lung carcinoma (SqCLC). ERAP2 plays a pivotal role in peptide trimming of many immunological processes. However, the prognostic role of ERAP2 and its relationship with immune cell infiltration in SqCLC remains unclear. Methods: The differential expression of ERAP2 was identified via GEO and TCGA databases. We calculated the impact of ERAP2 on clinical prognosis using the Kaplan-Meier plotter. TIMER was applied to evaluate the abundance of immune cells infiltration and immune markers. SqCLC tissue microarrays containing 190 patients were constructed, and we performed immunohistochemical staining for ERAP2, CD8, CD47, CD68, and PD-L1 to validate our findings in public data. Results: In the GEO SqCLC database, ERAP2 was upregulated in patients with better survival (p=0.001). ERAP2 expression in SqCLC was significantly lower than that of matched normal samples (p<0.05) based on TCGA SqCLC data. Higher expression of ERAP2 was significantly associated with better survival in SqCLC patients from TCGA (p=0.007), KM-plotter (p=0.017), and our tissue microarrays (TMAs) (p=0.026). In univariate and multivariate Cox analysis of SqCLC TMAs, high ERAP2 expression was identified as an independent protective factor for SqCLC patients (Univariate Cox, HR=0.659, range 0.454-0.956, p<0.05. Multivariate Cox, HR=0.578, range 0.385-0.866, p<0.05). In TIMER, ERAP2 was positively correlated with several immune markers (CD274, p=1.27E-04; CD68, p=5.88E-08) and immune infiltrating cells (CD8+ T cell, p=4.09E-03; NK cell, p=1.00E-04). In our cohort, ERAP2 was significantly correlated with CD8+ tumor-infiltrating lymphocytes (TILs) (p=0.0029), and patients with higher ERAP2 expression had a higher percentage of PD-L1 positive patients (p=0.049) and a higher CD8+ TILs level (p=0.036). Conclusions: For the first time, our study demonstrates that higher expression of ERAP2 is tightly associated with the immuno-supportive microenvironment and can predict a favorable prognosis in SqCLC. Meanwhile, ERAP2 may be a promising immunotherapeutic target for patients with SqCLC.