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Background and objective The switch/sucrose nonfermentable chromatin-remodeling (SWI/SNF) complex is a pivotal chromatin remodeling complex, and the genomic alterations (GAs) of the SWI/SNF complex are observed in several cancer types, correlating with multiple biological features of tumor cells. However, their role in liver metastasis of non-small cell lung cancer (NSCLC) remains unclear. Our study aims to investigate the role and potential mechanisms underlying NSCLC liver metastasis induced by the GAs of SWI/SNF complex. Methods The GAs of SWI/SNF complex in NSCLC cell lines (H1299, H23 and H460) were identified by whole-exome sequencing (WES). ARID1A knockout H1299 cell was constructed with the CRISPR/Cas9 technology. The mouse model of liver metastasis from NSCLC was established to simulate lung cancer liver metastasis and observe the metastasis rate under different gene mutation conditions. RNA sequencing and Western blot were conducted for differential gene expression analysis. Immunohistochemistry (IHC) analysis was used to assess protein expression levels of SWI/SNF-regulated target molecules in mouse liver metastases. Results WES analysis revealed intracellular gene mutations. The animal experiments demonstrated a correlation between the GAs of SWI/SNF complex and a higher liver metastasis rate in immunodeficient mice. Transcriptome sequencing and Western blot analysis showed upregulated expression of ALDH1A1 and APOBEC3B in SWI/SNF-mut cells, particularly in ARID1A-deficient H460 and H1299 sgARID1A cells. IHC staining of mouse liver metastases further demonstrated elevated expression of ALDH1A1 in the H460 and H1299 sgARID1A group. Conclusion This study underscores the critical role of the GAs of SWI/SNF complex, such as ARID1A and SMARCA4, in promoting liver metastasis of lung cancer cells. The GAs of SWI/SNF complex may promote liver-specific metastasis by upregulating ALDH1A1 and APOBEC3B expression, providing novel insights into the molecular mechanisms underlying lung cancer liver metastasis.

Abstract Background Age is an independent prognostic factor for small cell lung cancer (SCLC). We aimed to construct a nomogram survival prediction for elderly SCLC patients based on the Surveillance, Epidemiology, and End Results (SEER) database. Methods A total of 2851 elderly SCLC patients from the SEER database were selected as a primary cohort, which were randomly divided into a training cohort and an internal validation cohort. Additionally, 512 patients from two institutions in China were identified as an external validation cohort. We used univariate and multivariate to determine the independent prognostic factors and establish a nomogram to predict survival. The value of the nomogram was evaluated by calibration plots, concordance index (C-index) and decision curve analysis (DCA). Results Ten independent prognostic factors were determined and integrated into the nomogram. Calibration plots showed an ideal agreement between the nomogram predicted and actual observed probability of survival. The C-indexes of the training and validation groups for cancer-specific survival (CSS) (0.757 and 0.756, respectively) based on the nomogram were higher than those of the TNM staging system (0.631 and 0.638, respectively). Improved AUC value and DCA were also obtained in comparison with the TNM model. The risk stratification system can significantly distinguish individuals with different survival risks. Conclusion We constructed and externally validated a nomogram to predict survival for elderly patients with SCLC. Our novel nomogram outperforms the traditional TNM staging system and provides more accurate prediction for the prognosis of elderly SCLC patients.

Background As yet, there is no unified method of treatment for the evaluation and management of gastric low-grade intraepithelial neoplasia (LGIN) worldwide. Methods Patients with gastric LGIN who had been treated with Helicobacter pylori eradication were gathered retrospectively. Based on several relevant characteristics described and analyzed by LASSO regression analysis and multivariable logistic regression, a prediction nomogram model was established. C-index, the area under the receiver operating characteristic curve (AUC), calibration plot, and decision curve analysis (DCA) were adopted to evaluate the accuracy and reliability of the model. Results A total of 309 patients with LGIN were randomly divided into the training groups and the validation groups. LASSO regression analysis and multivariable logistic regression identified that 6 variables including gender, size, location, borderline, number, and erosion were independent risk factors. The nomogram model displayed good discrimination with a C-index of .765 (95% confidence interval: .702-.828). The accuracy and reliability of the model were also verified by an AUC of .764 in the training group and .757 in the validation group. Meanwhile, the calibration curve and the DCA suggested that the predictive nomogram had promising accuracy and clinical utility. Conclusions A predictive nomogram model was constructed and proved to be clinically applicable to identify high-risk groups with possible pathologic upgrade in patients with gastric LGIN. Since it is regarded that strengthening follow-up or endoscopic treatment of high-risk patients may contribute to improving the detection rate or reducing the incidence of gastric cancer, the predictive nomogram model provides a reliable basis for the treatment of LGIN.

Background This study aimed to explore the potentially predictive role and dynamic changes of immune checkpoints on T cell subsets in patients with breast cancer receiving neoadjuvant chemotherapies. Methods Fluorescent multiplex immunohistochemistry (mIHC) was used to stain CD4, CD8, PD1, TIM3, and cytokeratins simultaneously in paired breast cancer samples before and after neoadjuvant therapies (NAT) in a prospective cohort (n = 50). Singleplex IHC was conducted to stain for CD3 in 100 cases with inclusion of extra retrospective 50 cases. Cell levels were correlated with clinicopathological parameters and pathological complete response (pCR). Results In pretreatment tumors, the percentages of infiltrating CD8+, PD1+, PD1+CD8+, and the ratio of PD1+CD8+/CD8+ cells, were higher in pCR than non‐pCR patients in either the stromal or intratumoral area, but PD1+CD4+, TIM3+CD4+, TIM3+CD8+ cells and CD4+/CD8+ ratio was not. Multivariate analyses showed that the percentage of intratumoral CD8+ cells (OR, 1.712; 95% CI: 1.052–2.786; P = 0.030) and stromal PD1+CD8+/CD8+ ratio (OR, 1.109; 95% CI: 1.009–1.218; P = 0.032) were significantly associated with pCR. Dynamically, reduction in the percentages of PD1+, CD8+ and PD1+CD8+ cells after therapy strongly correlated with pCR. Notably, incremental percentages of PD1+CD8+ cells, rather than TIM3+CD8+, were shown in tumors from non‐pCR patients after NAT. CD3 staining confirmed the percentage of T cells were associated with pCR. Conclusions PD1+CD8+ rather than TIM3+CD8+ cells are main predictive components within tumor‐infiltrating T cells in NAT breast cancer patients. Dynamically incremental levels of PD1+CD8+ cells occurred in non‐pCR cases after NAT, suggesting the combination of chemotherapy with PD1 inhibition might benefit these patients. Key points Significant findings of the study PD1+CD8+, rather than TIM3+CD8+, T cells are the main component to predict the response of neoadjuvant therapies in breast cancer. What this study adds Incremental levels of PD1+CD8+ T cells in non‐pCR post‐NAT tumors suggest PD1 inhibition might benefit in the neoadjuvant setting.

A 53‐year‐old man diagnosed with disease stage IIIB pulmonary adenocarcinoma underwent chemotherapy and radiotherapy in the first‐line setting. After disease progression, he received targeted therapy because of subsequent detection of EGFR exon 19 del mutation. Following an increase in his adrenal metastases, a combination of immunotherapy and antiangiogenic therapy (sintilimab plus bevacizumab) was commenced. After one month, imaging showed that the adrenal metastases had shrunk and a progression‐free survival (PFS) of 6.0 months was achieved. In this case, we showed that the PD1 inhibitor sintilimab plus bevacizumab was effective in a refactory advanced EGFR‐mutant NSCLC with positive PD‐L1 expression. Key points Our case report provides clinical evidence of the durable response of a patient with advanced EGFR‐mutant lung adenocarcinoma to a combination of immunotherapy and anti‐angiogenic agent, sintilimab and bevacizumab, as subsequent‐line therapy. Sintilimab and bevacizumab combination therapy was well‐tolerated and effective, resulting in dramatic tumor reduction and improvement in clinical symptoms.

Immune characteristics were reported correlated to benefit neoadjuvant chemotherapy (NAC) in breast cancer, yet integration of comprehensive genomic alterations and T-cell receptors (TCR) to predict efficacy of NAC needs further investigation. This study simultaneously analyzed TMB (Tumor Mutation Burden), TCRs, and TILs (tumor infiltrating lymphocyte) in breast cancers receiving NAC was conducted in a prospective cohort (n = 22). The next-generation sequencing technology-based analysis of genomic alterations and TCR repertoire in paired breast cancer samples before and after NAC was conducted in a prospective cohort (n = 22). Fluorescent multiplex immunohistochemistry was used to stain CD4, CD8, PD1, TIM3, and cytokeratins simultaneously in those paired samples. TMB in pretreatment tumor tissues and TCR diversity index are higher in non-pCR patients than in pCR patients (10.6 vs. 2.3; p = 0.043) (2.066 vs. 0.467; p = 0.010). TMB and TCR diversity index had linear correlation (y = 5.587x − 0.881; r = 0.522, p = 0.012). Moreover, infiltrating T cells are significantly at higher presence in pCR versus non-pCR patients. Dynamically, the TMB reduced significantly after therapy in non-pCR patients (p = 0.010) but without TCR index change. The CDR3 peptide AWRSAGNYNEQF is the most highly expressed in pre-NAC samples of pCR patients and in post-NAC samples of non-pCR patients. In addition to pCR, high clonality of TCR and high level of CD8+ expression are associated with disease-free survival (DFS). TCR index and TMB have significant interaction and may guide neo-adjuvant treatment in operable breast cancers. Response to NAC in tumors with high TCR clonality may be attributable to high infiltration and expansion of tumor-specific CD8 positive effector cells.

Abstract Background Intratumoral epidermal growth factor receptor (EGFR) mutational heterogeneity is yet controversial in non-small cell lung cancer (NSCLC) patients. Single-cell analysis provides the genetic profile of single cancer cells and an in-depth understanding of the heterogeneity of a tumor. Methods Firstly, single H1975 cells harboring the EGFR L858R mutation were submitted to flow cytometry isolation, nested polymerase chain reaction (nested-PCR) amplification, and direct DNA sequencing to assess the feasibility of single-cell direct DNA sequencing. Then, the single cells of patients with lung adenocarcinoma receiving gefitinib were captured by laser capture microdissection and analyzed by the above methods to identify the intratumoral heterogeneity of the EGFR L858R mutant. Three patients with progression-free survival (PFS) > 14 months were categorized as the long PFS group, and 3 patients with PFS

Next-generation sequencing (NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously. Therefore, it represents a promising tool for the analysis of molecular targets for the initial diagnosis of disease, monitoring of disease progression, and identifying the mechanism of drug resistance. On behalf of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology (CSCO) and the China Actionable Genome Consortium (CAGC), the present expert group hereby proposes advisory guidelines on clinical applications of NGS technology for the analysis of cancer driver genes for precision cancer therapy. This group comprises an assembly of laboratory cancer geneticists, clinical oncologists, bioinformaticians, pathologists, and other professionals. After multiple rounds of discussions and revisions, the expert group has reached a preliminary consensus on the need of NGS in clinical diagnosis, its regulation, and compliance standards in clinical sample collection. Moreover, it has prepared NGS criteria, the sequencing standard operation procedure (SOP), data analysis, report, and NGS platform certification and validation.

Abstract Background Gastric cancer (GC) is a common cancer in Asia and currently lacks a targeted therapy approach. Mesothelin (MSLN) has been reported to be expressed in GC tissue and could be targeted by chimeric antigen receptor (CAR) T cells. Mesothelin targeting CAR-T has been reported in mesothelioma, lung cancer, breast cancer, and pancreas cancer. However, the feasibility of using anti-MSLN CAR T cells to treat GC remains to be studied. Methods We verified MSLN expression in primary human GC tissues and GC cell lines and then redirected T cells with a CAR containing the MSLN scFv (single-chain variable fragment), CD3ζ, CD28, and DAP10 intracellular signaling domain (M28z10) to target MSLN. We evaluated the function of these CAR T cells in vitro in terms of cytotoxicity, cytokine secretion, and surface phenotype changes when they encountered MSLN+ GC cells. We also established four different xenograft GC mouse models to assess in vivo antitumor activity. Results M28z10 T cells exhibited strong cytotoxicity and cytokine-secreting ability against GC cells in vitro. In addition, cell surface phenotyping suggested significant activation of M28z10 T cells upon target cell stimulation. M28z10 T cells induced GC regression in different xenograft mouse models and prolonged the survival of these mice compared with GFP-transduced T cells in the intraperitoneal and pulmonary metastatic GC models. Importantly, peritumoral delivery strategy can lead to improved CAR-T cells infiltration into tumor tissue and significantly suppress the growth of GC in a subcutaneous GC model. Conclusion These results demonstrate that M28z10 T cells possess strong antitumor activity and represent a promising therapeutic approach to GC.

Background and objective Uridine-diphosphoglucuronosyl transferase 1A1 (UGT1A1), UGT1A1*28 polymorphism can reduce UGT1A1 enzymatic activity, which may lead to severe toxicities in patients who receive irinotecan. This study tries to build a fragment analysis method to detect UGT1A1*28 polymorphism. Methods A total of 286 blood specimens from the lung cancer patients who were hospitalized in Guangdong General Hospital between April 2014 to May 2015 were detected UGT1A1*28 polymorphism by fragment analysis method. Results Comparing with Sanger sequencing, precision and accuracy of the fragment analysis method were 100%. Of the 286 patients, 236 (82.5% harbored TA6/6 genotype, 48 (16.8%) TA 6/7 genotype and 2 (0.7%) TA7/7 genotype. Conclusion Our data suggest hat the fragment analysis method is robust for detecting UGT1A1*28 polymorphism in clinical practice. It’s simple, time-saving, and easy-to-carry.

Abstract Background Multiple iterations of chimeric antigen receptors (CARs) have been developed, mainly focusing on intracellular signaling modules. However, the effect of non-signaling extracellular modules on the expansion and therapeutic efficacy of CARs remains largely undefined. Methods We generated two versions of CAR vectors, with or without a hinge domain, targeting CD19, mesothelin, PSCA, MUC1, and HER2, respectively. Then, we systematically compared the effect of the hinge domains on the growth kinetics, cytokine production, and cytotoxicity of CAR T cells in vitro and in vivo. Results During in vitro culture period, the percentages and absolute numbers of T cells expressing the CARs containing a hinge domain continuously increased, mainly through the promotion of CD4+ CAR T cell expansion, regardless of the single-chain variable fragment (scFv). In vitro migration assay showed that the hinges enhanced CAR T cells migratory capacity. The T cells expressing anti-CD19 CARs with or without a hinge had similar antitumor capacities in vivo, whereas the T cells expressing anti-mesothelin CARs containing a hinge domain showed enhanced antitumor activities. Conclusions Hence, our results demonstrate that a hinge contributes to CAR T cell expansion and is capable of increasing the antitumor efficacy of some specific CAR T cells. Our results suggest potential novel strategies in CAR vector design.

Chimeric antigen receptor (CAR) T cell immunotherapies have shown remarkable efficacy in treating multiple types of hematological malignancies but are not sufficiently effective at treating solid tumors. NKG2D is a strong activating receptor for NK cells and a co-stimulatory receptor for T cells. NKG2D signal transduction depends on DNAX-activating protein 10 (DAP10). Here, we introduced the cytoplasmic domain of DAP10 into the second-generation CARs M28z and G28z to generate M28z10 and G28z10, which target mesothelin (MSLN) and glypican 3 (GPC3), respectively. T cells expressing M28z10 or G28z10 showed enhanced and prolonged effector function against MSLN+ lung cancer or GPC3+ hepatocellular carcinoma cell lines in culture and secreted elevated levels of cytokines, including IL-2, IFN-γ, granzyme B, and GM-CSF. In addition, M28z10 CAR-T cells showed greater anti-tumor activity than those expressing M28z in both A549 cell line xenografts and human lung cancer patient-derived xenografts (PDX). Similarly, G28z10 exhibited higher efficacy in causing tumor regression than did G28z in hepatocellular carcinoma PDX. Therefore, our results show that DAP10 signaling contributes to the function of CAR-T cells in both lung cancer and hepatocellular carcinoma and can enhance the efficacy of CAR-T cells.

In recent years, immunotherapies, such as those involving chimeric antigen receptor (CAR) T cells, have become increasingly promising approaches to non-small-cell lung cancer (NSCLC) treatment. In this study, we explored the antitumor potential of prostate stem cell antigen (PSCA)-redirected CAR T and mucin 1 (MUC1)-redirected CAR T cells in tumor models of NSCLC. First, we generated patient-derived xenograft (PDX) mouse models of human NSCLC that maintained the antigenic profiles of primary tumors. Next, we demonstrated the expression of PSCA and MUC1 in NSCLC, followed by the generation and confirmation of the specificity and efficacy of PSCA- and MUC1-targeting CAR T cells against NSCLC cell lines in vitro. Finally, we demonstrated that PSCA-targeting CAR T cells could efficiently suppress NSCLC tumor growth in PDX mice and synergistically eliminate PSCA+MUC1+ tumors when combined with MUC1-targeting CAR T cells. Taken together, our studies demonstrate that PSCA and MUC1 are both promising CAR T cell targets in NSCLC and that the combinatorial targeting of these antigens could further enhance the antitumor efficacy of CAR T cells.

Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM) assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC), to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35). p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

Background and objective Epidermal growth factor receptor (EGFR) is the most important therapeutic target in non-small cell lung cancer (NSCLC). EGFR mutations may predict responsiveness to tyrosine kinase inhibitors (TKIs). These mutations are commonly identified using direct sequencing, which is considered the gold standard. But direct sequencing is time-consuming and hyposensitive. In addition, this method requires a lot of tumor specimens. Denaturing highperformance liquid chromatography (DHPLC) is a rapid automated sensitive and specific method in mutant gene detection. The aim of this study is to evaluate DHPLC as a rapid detection method for EGFR mutations in NSCLC tumor specimens. Methods DHPLC was used to evaluate the accuracy and sensitivity of detection the serial dilutions of mutant and wild type EGFR plasma DNA. Frozen tumor specimens of 83 NSCLC patients from various ways had been included, after DNA extraction and polymerase chain reaction (PCR) on EGFR exon 19 and 21, the results from the direct sequencing and DHPLC were compared. Results Mutant plasma DNA can be detected in the serial dilution of 1:100 by DHPLC and 1:10 by direct sequencing respectively. The results from DHPLC showed 22 EGFR mutations were detected in 83 NSCLC patients, and only 19 mutation samples had been conformed by direct sequencing. Moreover, the other 61 samples were deemed as wild type by DHPLC and direct sequencing. The sensitivity and specificity of DHPLC was 100% and 95.13% respectively. The detection of the tumor specimens from CT-guided transthoracic needle lung biopsy, lymph node biopsy and surgical resection all showed high sensitivity and specificity. EGFR mutation has strong correlation with gender and pathologic type, but irrelevant to age and smoking status. Conclusion DHPLC was a precise rapid preliminary screening method for detection of NSCLC EGFR genotype.

Background and objective All the advanced NSCLC patients that received EGFR-TKI therapy will eventually relapse after a period of efficacy. The aim of this study is to investigate the serum biomarkers as potential predictive factors for the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) targeted therapy in advanced non-small cell lung cancer. Methods Twenty self-paired serum samples were collected from 9 advanced NSCLC patients that evaluated as disease control (SD or PR) after gefinitib therapy, at the time points of before and after gefinitib treatment but 2 weeks before being evaluated as disease progress. All samples were pre-separated by WCX microbeads, and then detected on the MALDI-TOF-MS platform of Bruker AutoflexTM. ClinProTools (Version: 2.1) was used to analyze the differentially expressed proteins. Results There were 7 protein peaks (m/z), 3242.09, 8 690.36, 2 952.64, 3 224.04, 1 450.51, 1 887.8 and 3 935.73 found statistically differentially expressed between the self-paired samples. Three proteins (3 242.09, 2 952.64 and 3 224.04) were down-regulated and four proteins (8 690.36, 1 450.51, 1 887.8 and 3 935.73) up-regulated in gefinitib treated sera. Conclusion The data here suggest that several specific protein peaks might indicate gefinitib resistance, yet the identities of these proteins and the mechanisms underlying the responsiveness to gefinitib treatment need further investigation.