Clonality Analysis for the Relationship between the Pulmonary Combined Neuroendocrine Carcinoma and "the So-Called Reported Histologic Transformation".

Histologic transformation (HT) is common following targeted therapy in adenocarcinoma. However, whether the transformed tumor is a new component or a combined neuroendocrine carcinoma (C-NEC) remains controversial. We aimed to explore the relationship between pulmonary C-NEC and HT. Macro-dissection was performed on different components of surgically resected C-NEC samples. Molecular alterations and clonal evolution were analyzed using whole exome sequencing (WES). The gene statuses for TP53 and RB1 were determined using immunohistochemistry (IHC) and WES to analyze the relationship between C-NEC and reported HT. Sixteen combined small-cell lung cancer patients and five combined large-cell neuroendocrine carcinoma patients were enrolled. The frequency of p53 and Rb inactivation, assessed using IHC in NEC and non-NEC components, was 76.2/76.2% and 66.7/61.9%, respectively. The expression consistency between the components was 81.0 and 85.7% for p53 and Rb, respectively. The frequencies of TP53, RB1, and EGFR mutations, assessed using WES in NEC and non-NEC components, were 81.0/81.0%, 28.6/28.6%, and 42.9/42.9%, respectively. The concordance rates for TP53, RB1, and EGFR were 90.5, 71.4, and 90.5%, respectively. The consistency rate between IHC and WES was 81.0 and 61.9% for TP53 and RB1, respectively. The different components had a common clonal origin for the 21 C-NECs in the clonal analysis, consistent with previous studies on HT. Our study shows that IHC is more sensitive for Rb detection and C-NEC, and the reported HT may be due to differences in evaluations between pathologist and clinicians. Assessing the p53/Rb and EGFR status for such cases would help in recognizing potential transformation cases or uncovering potential combined components.

Molecular genetic characteristics of thymic epithelial tumors with distinct histological subtypes.

BACKGROUND: Due to the low incidence and histological heterogeneity, the molecular features and underlying carcinogenic mechanisms of thymic epithelial tumors (TETs) are yet to be fully elucidated, especially for different subtypes of TETs. METHODS: Tumor tissue samples of 43 TETs with distinct histological subtypes were collected. We analyzed the molecular characteristics in different subtypes based on whole exome sequencing data. RESULTS: The mutational profiles of the different subtypes of TETs varied. Compared with thymomas, thymic carcinomas (TCs) had a higher mutation frequency of MYO16 (33% vs. 3%, p = 0.024) and a lower frequency of ZNF729 mutations (0% vs. 35%, p = 0.044). No significant difference was observed in the median tumor mutation burden across different subtypes. The value of copy number variation burden, weighted genome instability index, and the number of amplified segments were all higher in TCs than thymomas, and they also tended to be higher in B3 thymoma than in non-B3 thymomas, while they had no significant differences between B3 thymoma and TCs. Clustering analyses revealed that Wnt, MAPK, Hedgehog, AMPK, and cell junction assembly signaling pathways were exclusively enriched in non-B3 thymomas, lysine degradation pathway in B3 thymoma, and extracellular matrix-receptor (ECM-receptor) interaction, positive regulation of cell cycle process, and activation of innate immune response pathways in TCs. CONCLUSIONS: This study revealed distinct molecular landscapes of different subtypes of TETs, suggesting diverse pathogenesis of non-B3 thymomas, B3 thymomas, and TCs. Our findings warrant further validation in future large-scale studies and may provide a theoretical basis for potential personalized therapeutic strategies.

An innovative single-base extension method for synchronous detection of point mutations and MSI status in colorectal cancer.

BACKGROUND: An accurate genotyping analysis is one of the critical prerequisites for patients with colorectal cancer receiving matched therapies. Conventional genotyping analysis is currently used to detect either gene mutations or MSI status, delaying the detection of critical tumor biomarkers and thus the optimal time for treatment. An assay that analyzes both biomarkers in a streamlined process is eagerly needed. METHODS: We developed an assay combining Multiplex PCR Amplification, Single-base Extension and capillary electrophoresis (CE) analysis (MASE-CE) for synchronous detection of KRAS/NRAS/BRAF mutations and MSI status. In a 190 colorectal cancer cohort, we identified seven somatic mutations in KRAS, NRAS and BRAF as well as five MSI loci (D2S123/D5S346/D17S250/BAT-25/BAT-26) simultaneously. KRAS/NRAS/BRAF mutations were detected by NGS and MASE-CE, and MSI status were detected by PCR-CE and MASE-CE methods. RESULTS: The MASE-CE method showed high consistency with NGS for mutation detection (Kappa value ≥0.8) and PCR-CE (Kappa value = 0.79). In addition, the limits of detection (LOD) of MASE-CE assay for MSI and somatic mutation were 5% and 2%, respectively. CONCLUSIONS: In somatic mutation detection and MSI detection, the LOD of MASE-CE assay was superior to that of qPCR and NGS. MASE-CE assay is a highly sensitive, time-saving and specimen-saving method, which can greatly avoid the cumbersome testing process and provide clinical decision for doctors in time.

Lynch syndrome pre-screening and comprehensive characterization in a multi-center large cohort of Chinese patients with colorectal cancer.

OBJECTIVE: Lynch syndrome (LS) pre-screening methods remain under-investigated in colorectal cancers (CRCs) in Asia. Here, we aimed to systematically investigate LS pre-screening and comprehensively characterize LS CRCs. METHODS: Microsatellite instability (MSI) and germline variants of DNA mismatch repair (MMR) genes were examined in 406 deficient MMR (dMMR) and 250 proficient MMR CRCs. The genetic differences between LS and sporadic CRCs were studied with whole exome sequencing analysis. RESULTS: The incidence of dMMR in Chinese patients with CRCs was 13.8%. Consistency analysis between MMR immunohistochemistry (IHC) and MSI testing showed the kappa value was 0.758. With next-generation sequencing (NGS), germline variants were detected in 154 CRCs. Finally, 88 patients with CRC were identified as having LS by Sanger sequencing. Among them, we discovered 21 previously unreported pathogenic germline variants of MMR genes. Chinese patients with LS, compared with sporadic CRCs, tended to be early-onset, right-sided, early-stage and mucinous. Overall, the performance of MMR IHC and MSI testing for LS pre-screening was comparable: the area under the ROC curve for dMMR, MSI-H, and MSI-H/L was 0.725, 0.750, and 0.745, respectively. dMMR_MSI-H LS and sporadic CRCs showed substantial differences in somatic genetic characteristics, including different variant frequencies of APC, CREBBP, and KRAS, as well as different enriched pathways of VEGF, Notch, TGFßR, mTOR, ErbB, and Rac protein signal transduction. CONCLUSIONS: MMR IHC and MSI testing were effective methods for LS pre-screening. The revealed clinical and somatic genetic characteristics in LS CRCs may have the potential to improve the performance of LS pre-screening in combination with dMMR/MSI.

A nomogram to predict the recurrence-free survival and analyze the utility of chemotherapy in stage IB non-small cell lung cancer.

BACKGROUND: Large part of patients of stage IB non-small cell lung cancer (IB NSCLC) may suffer recurrence after surgery. This study is to determine risk factors and establish a nomogram for postoperative recurrence and to provide a reference for adjuvant chemotherapy selection in patients with stage IB NSCLC. METHODS: A total of 394 patients with postoperative stage IB NSCLC who visited Fujian Medical University Union Hospital between January 2010 and June 2016 were selected. Patients were divided into training and validation cohorts based on the time of diagnosis. Independent risk factors were identified using a Cox proportional hazards regression model. A nomogram was created to predict recurrence-free survival (RFS) and was validated with an independent cohort. The predictive ability of the nomogram was evaluated using the concordance index (C-index) and calibration curve. RFS between the high- and low-risk groups was determined using Kaplan-Meier curves, and subgroup analysis of chemotherapy was performed. RESULTS: Visceral pleura invasion, micropapillary structures, tumor size, preoperative serum carcinoembryonic antigen (CEA) level, preoperative serum cytokeratin-19 fragments (Cyfra21-1) level, and postoperative histology were identified as independent risk factors for stage IB NSCLC recurrence. Discrimination of the nomogram showed good prognostic accuracy and clinical applicability, with a C-index of 0.827 and 0.866 in the training and validation cohorts, respectively. The difference in RFS between the high- and low-risk groups in both cohorts was significant (P<0.05). Finally, a significant difference was observed on whether high-risk group should accept postoperative chemotherapy (P<0.05). CONCLUSIONS: This nomogram can predict postoperative recurrence probability in patients with stage IB NSCLC, and can select patients with risk factors who need adjuvant chemotherapy.

Multi-omics analysis of intra-tumoural and inter-tumoural heterogeneity in pancreatic ductal adenocarcinoma.

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is associated with the tumour heterogeneity. To explore intra- and inter-tumoural heterogeneity in PDAC, we analysed the multi-omics profiles of 61 PDAC lesion samples, along with the matched pancreatic normal tissue samples, from 19 PDAC patients. Haematoxylin and Eosin (H&E) staining revealed that diversely differentiated lesions coexisted both within and across individual tumours. Whole exome sequencing (WES) of samples from multi-region revealed diverse types of mutations in diverse genes between cancer cells within a tumour and between tumours from different individuals. The copy number variation (CNV) analysis also showed that PDAC exhibited intra- and inter-tumoural heterogeneity in CNV and that high average CNV burden was associated poor prognosis of the patients. Phylogenetic tree analysis and clonality/timing analysis of mutations displayed diverse evolutionary pathways and spatiotemporal characteristics of genomic alterations between different lesions from the same or different tumours. Hierarchical clustering analysis illustrated higher inter-tumoural heterogeneity than intra-tumoural heterogeneity of PDAC at the transcriptional levels as lesions from the same patients are grouped into a single cluster. Immune marker genes are differentially expressed in different regions and tumour samples as shown by tumour microenvironment (TME) analysis. TME appeared to be more heterogeneous than tumour cells in the same patient. Lesion-specific differentially methylated regions (DMRs) were identified by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Furthermore, the integration analysis of multi-omics data showed that the mRNA levels of some genes, such as PLCB4, were significantly correlated with the gene copy numbers. The mRNA expressions of potential PDAC biomarkers ZNF521 and KDM6A were correlated with copy number alteration and methylation, respectively. Taken together, our results provide a comprehensive view of molecular heterogeneity and evolutionary trajectories of PDAC and may guide personalised treatment strategies in PDAC therapy.

Inhibition of cotranslational translocation by apratoxin S4: Effects on oncogenic receptor tyrosine kinases and the fate of transmembrane proteins produced in the cytoplasm.

Receptor tyrosine kinases (RTKs) have become major targets for anticancer therapy. However, resistance and signaling pathway redundancy has been problematic. The marine-derived apratoxins act complementary to direct kinase inhibitors by downregulating the levels of multiple of these receptors and additionally prevent the secretion of growth factors that act on these receptors by targeting Sec61α, therefore interfering with cotranslational translocation. We have profiled the synthetic, natural product-inspired apratoxin S4 against panels of cancer cells characterized by differential sensitivity to RTK inhibitors due to receptor mutations, oncogenic KRAS mutations, or activation of compensatory pathways. Apratoxin S4 was active at low-nanomolar to sub-nanomolar concentrations against panels of lung, head and neck, bladder, and pancreatic cancer cells, concomitant with the downregulation of levels of several RTKs, including EGFR, MET and others. However, the requisite concentration to inhibit certain receptors varied, suggesting some differential substrate selectivity in cellular settings. This selectivity was most pronounced in breast cancer cells, where apratoxin S4 selectively targeted HER3 over HER2 and showed greater activity against ER+ and triple negative breast cancer cells than HER2+ cancer cells. Depending on the breast cancer subtype, apratoxin S4 differentially downregulated transmembrane protein CDCP1, which is linked to metastasis and invasion in breast cancer and modulates EGFR activity. We followed the fate of CDCP1 through proteomics and found that nonglycosylated CDCP1 associates with chaperone HSP70 and HUWE1 that functions as an E3 ubiquitin ligase and presumably targets CDCP1, as well as potentially other substrates inhibited by apratoxins, for proteasomal degradation. By preventing cotranslational translocation of VEGF and other proangiogenic factors as well as VEGFR2 and other receptors, apratoxins also possess antiangiogenic activity, which was validated in endothelial cells where downregulation of VEGFR2 was observed, extending the therapeutic scope to angiogenic diseases.

The genomic and immunologic profiles of pure pulmonary sarcomatoid carcinoma in Chinese patients.

PURPOSE: The distribution of genetic mutations differs between pure pulmonary sarcomatoid carcinoma (PSC) and biphasic PSC; however, most of the enrolled cases in previous studies are biphasic PSC. The current study aimed to investigate the genomic and immunologic profiles of pure PSC in the Chinese population. MATERIALS AND METHODS: Next-generation sequencing analysis of a panel of 1021 genes was performed on surgical specimens of 58 pure PSCs. The tumor mutational burden (TMB) was calculated from 0.69 megabases (Mbs) of sequenced DNA. PD-L1 expression was evaluated by immunohistochemistry. Microsatellite instability (MSI) was evaluated by fluorescence-labeled microsatellite marker polymerase chain reaction followed by capillary electrophoresis fragment size analysis. RESULTS: The top mutational genes of pure PSC were TP53 (74 %, 43/58), KRAS (24 %, 14/58), SMARCA4 (14 %, 8/58), MET (12 %, 7/58), EGFR (10 %, 6/58), MLL4 (10 %, 6/58), NF1 (10 %, 6/58), NOTCH4 (10 %, 6/58), and TERT (10 %, 6/58). The median TMB was 8.6 mutations/Mb; 37.9 % of cases (22/58) had a TMB > 10 mutations/Mb and 12.1 % of cases (7/58) had a TMB > 20 mutations/Mb. The median TMB was higher in TP53-mutant tumors than in wild-type tumors (10.1 versus 7.2 mutations/Mb, p = 0.019). Thirty-five patients had microsatellite-stable pure PSC, and four patients carried MSI-H tumors. The MSI status was independent of MET exon 14 status. Twenty-six patients (45 %) had PD-L1-positive tumors (≥1%) and 14 (24 %) had high PD-L1 expression (≥50 %). CONCLUSION: In our cohort, 45 % of patients with pure PSC harbored at least one actionable alteration. More importantly, more than 60 % of patients (65.5 %, 38/58) had either MSI-H, PD-L1-positive, or high-TMB tumors, and these might derive survival benefits from immune checkpoint inhibitors.

Identifying prognostic signatures in the microenvironment of prostate cancer.

BACKGROUND: An increasing number of studies has indicated that the tumor microenvironment (TME), an important component of tumor tissue, has clinicopathological significance in predicting disease outcome and therapeutic efficacy. However, little evidence in prostate cancer (PCa) is available. METHODS: The cohort of TCGA-PRAD (n=477) was used in this study. Based on the proportion of 22 types of immune cells calculated by CIBERSORT, the TME was classified by K-means clustering and differentially expressed genes (DEGs) were determined. The TMEscore was calculated based on cluster signature genes, which were obtained from DEGs by the random forest method, and the samples were classified into two subtypes. Analyses of somatic mutation and copy number variation (CNVs) were further conducted to identify the genetic characteristics of the two subtypes. Correlation analysis was performed to explore the correlation between TMEscore and the tumor response to immune checkpoint inhibitors (ICIs) as well as the prognosis of PCa. RESULTS: Based on the distribution of infiltrating immune cells in the TME, we constructed the TMEscore model and classified PCa samples into high and low TMEscore groups. Survival analysis indicated that the high TMEscore group had significantly better survival outcome than the low TMEscore group. Correlation analysis showed a significantly positive correlation between TMEscore and the known prognostic factors of PCa. CONCLUSIONS: Our study indicates that the TMEscore could be a potential prognostic biomarker in PCa. A comprehensive description of the characteristics of TME may help predict the response to therapies and provide new treatment strategies for PCa patients.

Development and interpretation of a pathomics-based model for the prediction of microsatellite instability in Colorectal Cancer.

Microsatellite instability (MSI) has been approved as a pan-cancer biomarker for immune checkpoint blockade (ICB) therapy. However, current MSI identification methods are not available for all patients. We proposed an ensemble multiple instance deep learning model to predict microsatellite status based on histopathology images, and interpreted the pathomics-based model with multi-omics correlation. Methods: Two cohorts of patients were collected, including 429 from The Cancer Genome Atlas (TCGA-COAD) and 785 from an Asian colorectal cancer (CRC) cohort (Asian-CRC). We established the pathomics model, named Ensembled Patch Likelihood Aggregation (EPLA), based on two consecutive stages: patch-level prediction and WSI-level prediction. The initial model was developed and validated in TCGA-COAD, and then generalized in Asian-CRC through transfer learning. The pathological signatures extracted from the model were analyzed with genomic and transcriptomic profiles for model interpretation. Results: The EPLA model achieved an area-under-the-curve (AUC) of 0.8848 (95% CI: 0.8185-0.9512) in the TCGA-COAD test set and an AUC of 0.8504 (95% CI: 0.7591-0.9323) in the external validation set Asian-CRC after transfer learning. Notably, EPLA captured the relationship between pathological phenotype of poor differentiation and MSI (P < 0.001). Furthermore, the five pathological imaging signatures identified from the EPLA model were associated with mutation burden and DNA damage repair related genotype in the genomic profiles, and antitumor immunity activated pathway in the transcriptomic profiles. Conclusions: Our pathomics-based deep learning model can effectively predict MSI from histopathology images and is transferable to a new patient cohort. The interpretability of our model by association with pathological, genomic and transcriptomic phenotypes lays the foundation for prospective clinical trials of the application of this artificial intelligence (AI) platform in ICB therapy.

Concomitant genetic alterations are associated with response to EGFR targeted therapy in patients with lung adenocarcinoma.

BACKGROUND: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are considered to be more effective than chemotherapy in the treatment of EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, in addition to EGFR-sensitive mutations, the genetic factors that affect the prognosis of patients who receive TKI treatment are not yet clear. METHODS: The clinical data of 36 NSCLC patients with EGFR mutation who received TKI treatment were retrospectively analyzed. Liquid re-biopsy with next generation sequencing (NGS) analysis was performed to analyze genetic alterations and potential resistance mechanisms. RESULTS: All of the patients harbored actionable sensitive EGFR mutations by NGS, with the major types being 19del or 21L858R (52.78%, 19/36 and 55.56%, 20/36, respectively). The 3 most frequent accompanying somatic mutations were TP53 (12, 48.4%), KRAS (7, 19.44%) and PIK3CA (3, 8.33%). Concomitant mutations were present in 16 patients (44.44%). The occurrence of co-mutation was found to be significantly related to a history of smoking [87.5% (7 of 8) vs. 32.14% (9 of 28); Pearson chi-square, P=0.005]. Patients who received EGFR-TKIs treatment (P=0.0079) or third-generation EGFR-TKIs only (P=0.0468) had better progression-free survival (PFS). Concomitant mutations were significantly related to lower objective response rates (43.75% vs. 80.0%; P=0.024) and poorer PFS (P<0.001). Patients with concomitant genetic alterations had a worse response after receiving EGFR-TKIs treatment (P=0.0033). CONCLUSIONS: Our research underscores the importance of using multiple molecular profiles. Concomitant genetic alterations were significantly associated with response to EGFR targeted therapy in NSCLC. Therefore, research on multi-drug or sequential therapy to address the covariation that drives drug resistance is urgently needed.

Molecular gene mutation profiles, TMB and the impact of prognosis in Caucasians and east Asian patients with lung adenocarcinoma.

BACKGROUND: The difference in molecular gene mutation profile, tumor mutational burden (TMB) and their prognostic effects in lung adenocarcinoma between different ethnic groups are still unknown. A retrospective analysis was used to investigate the differences in lung adenocarcinoma driver gene mutations, TMB, and their impact on prognosis across different ethnic groups. METHODS: The incidence of epidermal growth factor receptor (EGFR) mutations and follow-up data of 647 Chinese lung adenocarcinoma patients were compared with the data from 522 Caucasian patients in The Cancer Genome Atlas (TCGA) database. Moreover, a comprehensive analysis was performed to compare the differences in gene mutation frequency, signaling pathway variation, and TMB using the whole-exome sequencing (WES) data of Chinese patients with that of Caucasian patients. RESULTS: A comparison of tumor signaling pathways and gene mutation profiles between Caucasians and Chinese revealed ethnic variations in the incidence of mutations in TGF-ß and RTK-RAS signaling pathways, with P values of 0.012 and 0.016, respectively. In the Caucasian population, the mutations in 5 signaling pathways and 18 genes were all significantly correlated with TMB, whereas in the Chinese population, only mutations in the Notch pathway and 6 genes were found to be associated with TMB-high. EGFR mutations showed a better prognosis in Chinese patients with lung adenocarcinoma, while the opposite was found in Caucasians patients. CONCLUSIONS: Variations in the incidence of mutations in signaling pathways involved in lung adenocarcinoma and the correlation of the signaling pathways with TMB may exist across different ethnic groups.

Whole exome sequencing (WES) analysis of transformed small cell lung cancer (SCLC) from lung adenocarcinoma (LUAD).

BACKGROUND: Histologic transformation of non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is a rare mechanism of acquired resistance to epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors. However, the SCLC transformation has also been observed in non. EGFR: mutant NSCLC. In these cases, whether SCLC initially co-exists with NSCLC or originates from initial NSCLC remains to be determined. METHODS: Whole exome sequencing was performed on 10 samples from 5 patients with SCLC transformation from lung adenocarcinoma (LUAD), a main subtype of NSCLC. Somatic mutations and copy number variations (CNVs) were analyzed to explore the differences between initial LUAD and transformed SCLC, as well as the origin of transformed SCLC. RESULTS: After SCLC transformation, the mutation spectrum changed, with decreased C>T and increased C>A. Compared with initial LUAD, the CNV burden of transformed SCLC was greatly increased (39.0 vs. 61.1, Wilcoxon P=0.4). The higher the CNV burden of LUAD, the shorter the time to SCLC transformation was observed to be; and the higher the CNV burden of transformed SCLC, the shorter the overall survival (OS) after transformation. Clonal evolution analysis showed different clonal components between initial LUAD and transformed SCLC. CONCLUSIONS: The transformation of LUAD into SCLC may be promoted by CNV events rather than mutational events. CNV burden was associated with the time to SCLC transformation and with the OS of patients following SCLC transformation. Transformed SCLC did not evolve directly from the initial LUAD but branched off from LUAD before the time of initial diagnosis.

Prognostic impact of tumor mutation burden and the mutation in KIAA1211 in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a highly aggressive lung cancer subtype with poor survival and limited treatment options. Sequencing results have revealed gene mutations associated with SCLC, however, the correlation between the genomic alterations and clinical prognosis of SCLC is yet unclear. METHODS: Targeted next-generation sequencing of 62 cancer related genes was performed on 53 SCLC samples. The correlations between clinical outcomes and genomic alterations were analyzed. RESULTS: 38/62 (61.3%) candidate genes harbored some alterations, while all the SCLC samples carried at least 3 gene mutations. The most common nonsynonymous mutations included ERBB2 (95.9%), CREBBP (95.9%), and TP53 (77.6%). The median nonsynonymous tumor mutation burden (TMB) was 21.7 mutations/Mb (rang, 9.3-55.9). High TMB (> 21 mutations/Mb) was good prognostic factor in overall survival (OS) (21.7 vs. 10.4 months, P = 0.012). Multivariate analysis showed that high TMB was an independent prognostic factor. The overall survival (OS) of patients carrying KIAA1211 mutation was significantly longer than those with wild-type KIAA1211 (P < 0.001). CONCLUSIONS: The current study highlights the potential role of genomic alterations for the prognosis of SCLC. Higher TMB was associated with a better prognosis, and KIAA1211 might be a good prognostic factor in SCLC.

Immunogenicity of Del19 EGFR mutations in Chinese patients affected by lung adenocarcinoma.

BACKGROUND: Mutant peptides presented by cancer cells are superior vaccine candidates than self peptides. The efficacy of mutant K-Ras, P53 and EGFR (Epidermal Growth Factor Receptor) peptides have been tested as cancer vaccines in pancreatic, colorectal, and lung cancers. The immunogenicity of EGFR Del19 mutations, frequent in Chinese lung adenocarcinoma patients, remains unclear. RESULTS: We predicted the HLA binding epitopes of Del19 mutations of EGFR in Chinese lung adenocarcinoma patients with NetMHC software. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the EGFR-reactive IgG in lung cancer patients. Del19 mutations may be presented by multiple HLA Class I molecules, with delE746_A750 presented by 37.5% of Chinese population. For HLA Class II molecules, Del19 mutations of EGFR may be presented by multiple HLA-DRB1 molecules, with delE746_A750 presented by 58.1% of Chinese population. Serum reactivity to wild type EGFR protein was significantly higher in patients with Del19 EGFR mutations than those with EGFR L858R point mutation or with EGFR wild type genotype. CONCLUSIONS: These findings suggest that Del19 mutations of EGFR, with an estimated frequency of 40% in Chinese lung adenocarcinoma patients, may serve as unique targets for immunotherapy in Chinese lung cancer patients.

Concomitant EGFR Mutation and EML4-ALK Rearrangement in Lung Adenocarcinoma Is More Frequent in Multifocal Lesions.

BACKGROUND: The coexistence of epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement in patients with multifocal lung adenocarcinomas (LUAC) constitutes a rare molecular subtype of lung cancer. We aimed to investigate the intertumoral heterogeneity of pathologic and genetic characteristics of multifocal LUAC with EGFR/ALK co-alterations. PATIENTS AND METHODS: A total of 1059 LUAC patients who underwent resection were investigated to screen for EGFR or ALK alterations using amplification refractory mutation system polymerase chain reaction and immunohistochemistry/fluorescence in situ hybridization. Molecular testing was extensively performed in patients with synchronous multifocal LUAC. Clonal evolution analysis was implemented using next-generation sequencing. RESULTS: A total of 97 multiple synchronous lesions were observed among 1059 LUAC patients. Patients with at least 1 sample harboring EGFR mutation or ALK rearrangement were 62.89% (61/97) and 14.43% (14/97), respectively. Patients with concomitant EGFR and ALK alterations were 4.71% (4/97). Comparatively, patients with unifocal LUAC harboring EGFR mutation, ALK rearrangement, and EGFR/ALK co-alterations were 58.25% (570/962), 6.44% (62/962), and 0.83% (8/962), respectively. The prevalence of EGFR/ALK co-alterations in the multifocal LUAC was significantly higher than that in the unifocal LUAC (4.71% (4/97) vs. 0.83% (8/962)). Furthermore, we present 4 cases of EGFR/ALK co-altered multifocal LUAC with different morphological and molecular patterns. In addition to radiographic, pathological, and molecular testing results, clonal evolutional analysis could also be used to distinguish intertumoral heterogeneity. CONCLUSION: The results highlight the importance of distinguishing synchronous primary tumors from intrapulmonary metastases, and of assessing the relative abundance of EGFR mutation and ALK rearrangement in patients with multifocal adenocarcinomas with EGFR/ALK co-alterations.

Developing a New qPCR-Based System for Screening Mutation.

An accurate genotyping analysis is one of the critical prerequisites for lung cancer targeted therapy. Here, a quantitative polymerase chain reaction (qPCR)-based mutation detection system, mutation-selected amplification-specific system PCR (MASS-PCR), is developed. The specific primers and probes used in MASS-PCR exactly match with the mutant sequence that only allows mutant gene to emit the fluorescence peak. To determine the sensitivity of MASS-PCR, 717 lung cancer specimens, 61 formalin-fixed paraffin-embedded (FFPE) tissues, and 656 fresh reaction tissues are collected and undergo mutation detection of lung cancer driver genes (EGFR, KRAS, BRAF, HER2, MET, ALK, and ROS1). These samples are divided into two groups. Mutations in Group I, which has 631 fresh reaction tissues, are analyzed by MASS-PCR and the amplification refractory mutation system PCR (ARMS-PCR). While group II samples, 25 fresh reaction tissues and 61 FFPE tissues, are screened through MASS-PCR and next-generation sequencing (NGS). All results are verified by direct sequencing. MASS-PCR shows high consistency with ARMS-PCR (kappa value > 0.733) and NGS (kappa value = 0.79) (P < 0.001). For the samples with inconsistent MASS-PCR and ARMS-PCR results, DS results more likely support the MASS-PCR results. These data suggest that MASS-PCR is a convenient, accurate, and economical method for the detection of lung cancer driver gene mutations in clinical practice.

Development of apratoxin S10 (Apra S10) as an anti-pancreatic cancer agent and its preliminary evaluation in an orthotopic patient-derived xenograft (PDX) model.

Despite the significant progress in the field of cancer therapeutics, the incidence of pancreatic cancer (PC) has continuously increased. One possible mechanism for this increasing burden is impaired drug delivery and drug resistance resulting from a unique tumor microenvironment and genetic mutations. Apratoxins are potent anticancer agents and cotranslational translocation inhibitors with potential therapeutic applications to treat cancers with active secretory pathways. Here, we developed apratoxin S10 (Apra S10) as an anti-pancreatic cancer agent which potently inhibited the growth of both established and patient-derived primary pancreatic cancer cells. We validated its mechanism of action on pancreatic cancer cells by demonstrating the downregulation of multiple receptor tyrosine kinases and inhibition of growth factor and cytokine secretion. Apra S10 also inhibited a number of cytokines secreted by stromal cells, suggesting that Apra S10 not only inhibited pancreatic cancer cell secretion, but also reduced the level of factors secreted by other cell types active within the tumor microenvironment. As Apra S10 tissue distribution indicated its high enrichment in pancreas tissue, an orthotopic pancreatic patient-derived xenograft mouse model that closely mimics the human pancreatic tumor microenvironment was for the first time used in apratoxin studies. Apra S10 showed promising antitumor effect in this pancreatic cancer model and this effect was mediated through anti-proliferation properties.

Epigenetic regulation of RCAN1 expression in kidney disease and its role in podocyte injury.

Mounting evidence suggests that epigenetic modification is important in kidney disease pathogenesis. To determine whether epigenetic regulation is involved in HIV-induced kidney injury, we performed genome-wide methylation profiling and transcriptomic profiling of human primary podocytes infected with HIV-1. Comparison of DNA methylation and RNA sequencing profiles identified several genes that were hypomethylated with corresponding upregulated RNA expression in HIV-infected podocytes. Notably, we found only one hypermethylated gene with corresponding downregulated RNA expression, namely regulator of calcineurin 1 (RCAN1). Further, we found that RCAN1 RNA expression was suppressed in glomeruli in human diabetic nephropathy, IgA nephropathy, and lupus nephritis, and in mouse models of HIV-associated nephropathy and diabetic nephropathy. We confirmed that HIV infection or high glucose conditions suppressed RCAN1 expression in cultured podocytes. This suppression was alleviated upon pretreatment with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine, suggesting that RCAN1 expression is epigenetically suppressed in the context of HIV infection and diabetic conditions. Mechanistically, increased expression of RCAN1 decreased HIV- or high glucose-induced nuclear factor of activated T cells (NFAT) transcriptional activity. Increased RCAN1 expression also stabilized actin cytoskeleton organization, consistent with the inhibition of the calcineurin pathway. In vivo, knockout of RCAN1 aggravated albuminuria and podocyte injury in mice with Adriamycin-induced nephropathy. Our findings suggest that epigenetic suppression of RCAN1 aggravates podocyte injury in the setting of HIV infection and diabetic nephropathy.

Pharmacologic control of oxidative stress and inflammation determines whether diabetic glomerulosclerosis progresses or decreases: A pilot study in sclerosis-prone mice.

Diabetic kidney disease (DKD) is characterized by progressive glomerulosclerosis (GS). ROP mice have a sclerosis-prone phenotype. However, they develop severe, rapidly progressive GS when rendered diabetic. Since GS also develops in aged C57Bl6 mice, and can be reversed using bone marrow from young mice which have lower oxidative stress and inflammation (OS/Infl), we postulated that this might also apply to DKD. Therefore, this pilot study asked whether reducing OS/Infl in young adult sclerosis-prone (ROP) diabetic mice leads to resolution of existing GS in early DKD using safe, FDA-approved drugs.After 4 weeks of stable streptozotocin-induced hyperglycemia 8-12 week-old female mice were randomized and treated for 22 weeks as follows: 1) enalapril (EN) (n = 8); 2) pyridoxamine (PYR)+EN (n = 8); 3) pentosan polysulfate (PPS)+EN (n = 7) and 4) PPS+PYR+EN (n = 7). Controls were untreated (non-DB, n = 7) and hyperglycemic (DB, n = 8) littermates. PPS+PYR+EN reduced albuminuria and reversed GS in DB. Treatment effects: 1) Anti-OS/Infl defenses: a) PPS+PYR+EN increased the levels of SIRT1, Nrf2, estrogen receptor α (ERα) and advanced glycation endproduct-receptor1 (AGER1) levels; and b) PYR+EN increased ERα and AGER1 levels. 2) Pro-OS/Infl factors: a) PPS+PYR+EN reduced sTNFR1, b) all except EN reduced MCP1, c) RAGE was reduced by all treatments. In summary, PYR+PPS+EN modulated GS in sclerosis-prone hyperglycemic mice. PYR+PPS+EN also decreased albuminuria, OS/Infl and the sclerosis-prone phenotype. Thus, reducing OS/Infl may reverse GS in early diabetes in patients, and albuminuria may allow early detection of the sclerosis-prone phenotype.