Clinical implications of PD-L1 expression and pathway-related molecular subtypes in advanced Asian colorectal cancer patients.

The expression level of PD-L1 does not accurately predict the prognosis of advanced colorectal cancer (CRC) patients, but it still reflects the tumor microenvironment to some extent. By stratifying PD-L1 status, gene subtypes in PD-L1 positivity-related pathological pathways were analyzed for their relationship to MSI or TMB to provide more individualized treatment options for CRCs. A total of 752 advanced CRCs were included, and their genomic variance was measured by a targeted next generation sequencing panel in this study. MSI and TMB were both measured by NGS, while PD-L1 expression level was measured using the PD-L1 colon 22C3 pharmDx kit. We found RTK/RAS pathway was positively related to high PD-L1 expression, with BRAF V600E and most KRAS mutations (G12 and G13) subtypes showing a significant correlation. Conversely, the Wnt and p53 pathways were negatively related to high PD-L1 expression, with APC C-terminal alterations and other non-inactivation mutations in TP53 making a primary contribution with significant statistical significance. Major subtypes showing a significantly higher proportion of TMB-H or MSI-H were irrespective of PD-L1 status. These findings demonstrate pathological pathways associated with high PD-L1 expression, suggesting that pathway-induced oncogenic constructive PD-L1 upregulation may be the reason for the corresponding patients' primary resistance to immune checkpoint inhibitors (ICIs), rather than a lack of pre-existing immune responses.

PD-L1 mRNA derived from tumor-educated platelets as a potential immunotherapy biomarker in non-small cell lung cancer.

Background: To date, the role of programmed death ligand-1 (PD-L1) messenger RNA (mRNA) derived from tumor-educated platelets (TEPs) has not been well investigated in patients with advanced non-small cell lung cancer (NSCLC). A few reports have examined whether mRNA in TEPs can predict the clinical responses of patients with advanced NSCLC following immunotherapy. This study aimed to identify novel biomarkers to improve the clinical benefits and outcomes of NSCLC patients. Methods: Advanced NSCLC patients receiving a combination of immunotherapy and chemotherapy, or immunotherapy alone as a first- or second-line treatment at the Fudan University Shanghai Cancer Center were enrolled in this study. All the patients had wild-type epidermal growth factor receptor/anaplastic lymphoma kinase. The patients were enrolled in clinical trials for immune checkpoint inhibitors (ICIs), including nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, and camrelizumab. Tumoral PD-L1 expression was tested by immunohistochemistry (PD-L1 22C3 pharmDx kit, Agilent, Santa Clara, CA, USA) in archived tissue samples, when available, to calculate the tumor proportion scores (TPSs). RNA and exosomal RNA of blood were isolated before immunotherapy using the Yunying RNA extraction kit (Yunying Medicine, Shanghai, China). The concentration and quality of the RNA was determined using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). Finally, we analyzed the predictive value of TEP-derived PD-L1 mRNA expression and association with the level of the tumoral PD-L1 expression. Results: In total, 72 patients were enrolled in this study. Most of the patients were male (n=54, 75.0%), had adenocarcinoma (n=49, 68.1%). We found there was no significant correlation between the TEP-derived mRNA of PD-L1 and tumoral PD-L1 expression based on the results of the Pearson Correlation test (r=-0.19, P=0.233). Based on the median of PD-L1 mRNA, 72 patients were divided into a high PD-L1 group and a low PD-L1 group. We found that 19 patients (44.4%) responded to immunotherapy [partial response or progression-free survival (PFS) >6 months] in the high PD-L1 group, but only five patients (13.9%) responded to immunotherapy in the low PD-L1 group (P<0.01). The median PFS of the low PD-L1 group was lower than that of the high PD-L1 group (2.8 vs. 8.3 months, P<0.001). For the patients who were treated with immunotherapy alone (n=64), a similar PFS advantage was observed in the high PD-L1 group (2.8 vs. 8.0 months, P=0.002). Conclusions: This article presented the first data on TEP-derived PD-L1 mRNA in advanced NSCLC patients following immunotherapy and showed the potential advantage of using it as the surrogate biomarker for predicting the PFS and overall survival of patients following immunotherapy.

Unraveling EGFR-TKI resistance in lung cancer with high PD-L1 or TMB in EGFR-sensitive mutations.

BACKGROUND: Although EGFR-TKI resistance mechanisms in non-small cell lung cancer (NSCLC) have been extensively studied, certain patient subgroups remain with unclear mechanisms. This retrospective study analysed mutation data of NSCLC patients with EGFR-sensitive mutations and high programmed death-ligand 1 (PD-L1) expression or high TMB to identify primary resistance mechanisms. METHODS: Hybrid capture-based next-generation sequencing (NGS) was used to analyse mutations in 639 genes in tumor tissues and blood samples from 339 NSCLC patients. PD-L1 immunohistochemical staining was also performed on the same cell blocks. Molecular and pathway profiles were compared among patient subgroups. RESULTS: TMB was significantly higher in lung cancer patients with EGFR-sensitive mutations and high PD-L1 expression. Compared with the high-expression PD-L1 or high TMB and low-expression or TMB groups, the top 10 genes exhibited differences in both gene types and mutation rates. Pathway analysis revealed a significant mutations of the PI3K signaling pathway in the EGFR-sensitive mutation group with high PD-L1 expression (38% versus 12%, p < 0.001) and high TMB group (31% versus 13%, p < 0.05). Notably, PIK3CA and PTEN mutations emerged as the most important differentially mutated genes within the PI3K signaling pathway. CONCLUSIONS: Our findings reveal that the presence of PI3K signaling pathway mutations may be responsible for inducing primary resistance to EGFR-TKIs in NSCLC patients with EGFR-sensitive mutations along with high PD-L1 expression or high TMB. This finding is of great significance in guiding subsequent precision treatments in NSCLC.

SDH mutations, as potential predictor of chemotherapy prognosis in small cell lung cancer patients.

PURPOSE: Small cell lung cancer (SCLC) is an aggressive and rapidly progressive malignant tumor characterized by a poor prognosis. Chemotherapy remains the primary treatment in clinical practice; however, reliable biomarkers for predicting chemotherapy outcomes are scarce. METHODS: In this study, 78 SCLC patients were stratified into "good" or "poor" prognosis cohorts based on their overall survival (OS) following surgery and chemotherapeutic treatment. Next-generation sequencing was employed to analyze the mutation status of 315 tumorigenesis-associated genes in tumor tissues obtained from the patients. The random forest (RF) method, validated by the support vector machine (SVM), was utilized to identify single nucleotide mutations (SNVs) with predictive power. To verify the prognosis effect of SNVs, samples from the cbioportal database were utilized. RESULTS: The SVM and RF methods confirmed that 20 genes positively contributed to prognosis prediction, displaying an area under the validation curve with a value of 0.89. In the corresponding OS analysis, all patients with SDH, STAT3 and PDCD1LG2 mutations were in the poor prognosis cohort (15/15, 100%). Analysis of public databases further confirms that SDH mutations are significantly associated with worse OS. CONCLUSION: Our results provide a potential stratification of chemotherapy prognosis in SCLC patients, and have certain guiding significance for subsequent precise targeted therapy.

Specific Mutations in APC, with Prognostic Implications in Metastatic Colorectal Cancer.

PURPOSE: Loss-of-function mutations in the adenomatous polyposis coli (APC) gene are common in metastatic colorectal cancer (mCRC). However, the characteristic of APC specific mutations in mCRC is poorly understood. Here, we explored the clinical and molecular characteristics of N-terminal and C-terminal side APC mutations in Chinese patients with mCRC. MATERIALS AND METHODS: Hybrid capture-based next-generation sequencing was performed on tumor tissues from 275 mCRC pati-ents to detect mutations in 639 tumor-associated genes. The prognostic value and gene-pathway difference between APC specific mutations in mCRC patients were analyzed. RESULTS: APC mutations were highly clustered, accounting for 73% of all mCRC patients, and most of them were truncating mutations. The tumor mutation burden of the N-terminal side APC mutations group (n=76) was significantly lower than that of the C-terminal side group (n=123) (p < 0.001), further confirmed by the public database. Survival analysis showed that mCRC patients with N-terminus side APC mutations had longer overall survival than C-terminus side. Tumor gene pathway analysis showed that gene mutations in the RTK/RAS, Wnt and transforming growth factor ß signaling pathways of the C-terminal group were significantly higher than those of the N-terminal group (p < 0.05). Additionally, KRAS, AMER1, TGFBR2, and ARID1A driver mutations were more common in patients with C-terminal side APC mutations. CONCLUSION: APC specific mutations have potential function as mCRC prognostic biomarkers. There are obvious differences in the gene mutation patterns between the C-terminus and N-terminus APC mutations group, which may have certain guiding significance for the subsequent precise treatment of mCRC.

Genomic characterization of thymic epithelial tumors reveals critical genes underlying tumorigenesis and poor prognosis.

Thymic epithelial tumors (TETs) are rare mediastinal tumors whose tumorigenesis mechanism is poorly understood. Characterization of molecular alterations in TETs may contribute to a better understanding of tumorigenesis and prognosis. Hybrid capture-based next-generation sequencing was performed on tumor tissues from 47 TETs (39 thymomas and 8 thymic carcinomas) to detect mutations in 315 tumor-associated genes. In total, 178 nonsynonymous mutations were identified, with a median of 3.79 per tumor in 47 TETs. Higher tumor mutation burden (TMB) level was more common in older TET patients, and significantly associated with the more advanced pathological type, especially in thymic carcinomas (TC) patients. The gene mutation profiles of B1-3, A/AB, and TC patients varied greatly. In the actionable mutations analysis, we found 32 actionable mutations in 24 genes. Among them, NFKBIA and TP53 mutations was the most frequently, which were only identified in TCs. Additionally, TCGA database analysis found that the expression of NFKBIA mRNA in the TCs were significantly higher than thymomas. TET patients with high NFKBIA expression had shorter overall survival compared with patients with low/medium NFKBIA expression, thus providing insights to consider NFKBIA as a potential prognosis biomarker and therapeutic target in TETs.

PIK3CA somatic mutations as potential biomarker for immunotherapy in elder or TP53 mutated gastric cancer patients.

Immune checkpoint inhibitors (ICIs) improve overall survival in patients with advanced gastric cancer (GC). However, the molecular characterization of GC in ICIs responders is unclear. A total of 288 advanced GC patients were included in this study. Next-generation sequencing analysis was performed on tumor tissue and paired blood to screen for somatic mutants in 639 tumor-associated genes. We demonstrated that ARID1A, HER2/3/4, KMT2C/2D, LRP1B, PIK3CA, SPTA1, and TP53 mutations were significantly correlated with high tumor mutation burden (TMB) score, as well as HER2 amplification. For HER2 and PIK3CA mutations types, this relationship was statistically significant with age and TP53 mutation status, which was also found in the CDH1 gene. These results were confirmed by sequencing 873 GC cases in the cBioPortal database. PIK3CA mutations appear to be associated with longer survival in elderly population and TP53 mutant subtypes. For the first time, we found that GC patients ≥60 years old or with TP53 mutated type and PIK3CA mutations were associated with higher TMB and better ICI response. Building upon the age and TP53 mutation status, this study suggested a novel stratification approach to GC patients and explored the correlations between genetic somatic mutations and TMB score.

Assessment of PD-L1 mRNA expression in gastrointestinal tumors and the response to immunotherapy.

Background: Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) has been proposed as a predictive biomarker to predict response to immunotherapy. Given the limitations of IHC test in PD-L1 detection, this study aimed to investigate the technical feasibility of using quantitative RT-PCR (qRT-PCR) to replace IHC in PD-L1 detection in gastrointestinal tumors. Materials and methods: The Cancer Genome Atlas database was used to evaluate the relationship between PD-L1 expression in tumor tissue and the patient prognosis. In addition, 52 patients with gastrointestinal cancer were enrolled and divided into the stomach (STAD), colon (COAD), and rectum (READ) adenocarcinoma cohorts. IHC test was used to determine the PD-L1 level of the tissue specimens, and the qRT-PCR test was used to analyze the mRNA expression in both blood and tissue specimens. Moreover, the correlation between blood PD-L1 mRNA expression and immunotherapy efficacy was investigated in additional 15 patients with gastric cancer that further enrolled. Results: The expression level of PD-L1 in tumor tissue is related to the tumor stage of COAD (p-value = 0.001) and primary therapy outcomes in patients with READ (p-value = 0.003) but not significantly correlated to the overall survival (OS) time of patients with gastrointestinal cancer. Moreover, the concordance of PD-L1 mRNA expression level of tissue and paired blood samples is low, despite a weak linear relationship that was found in the STAD cohort (r = 0.43, p-value = 0.049). We further demonstrated that qRT-PCR results in both tissue and blood specimens were numerically but not statistically significant consistent with IHC results (corresponding to a p-value of 0.84 and 0.55, respectively). Remarkably, high PD-L1 expression in blood of patients with STAD shows a better response to immunotherapy (p-value = 0.04), which could be well identified at the relative expression cutoff of 1.5 (sensitivity of 85.7%, specificity of 75.0%, and AUC of 0.82). Conclusions: Our study established a novel strategy for rapidly distinguishing patients with gastrointestinal cancer with the response to immunotherapy and has potential clinical benefits.

Leukocyte CH25H is a potential diagnostic and prognostic marker for lung adenocarcinoma.

Metastasis, a major challenge during the treatment of lung cancer, causes deterioration in patient health outcomes. Thus, to address this problem, this study aimed to explore the role and contribution of Cholesterol 25-Hydroxylase (CH25H) as a potential diagnostic and prognostic marker in lung cancer. Online public databases were used to analyze the expression level, prognostic value, gene-pathway enrichment, and immune infiltration of CH25H in lung cancer patients. The Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was used to analyze and detect the CH25H expression levels in leukocytes from lung cancer patients. The expression level of CH25H was significantly reduced in lung adenocarcinoma (LUAD), which is associated with a higher disease stage, but not in lung squamous cell carcinoma (LUSC). Kaplan-Meier survival analysis indicated that LUAD patients with low CH25H expression had a worse prognosis. Mechanistically, our results showed that in LUAD, CH25H may be a regulatory factor affecting the immune cell infiltration level, and the resultant tumor development. Experimental data showed that low expression of CH25H in leukocytes was significantly associated with LUAD metastasis (P < 0.01). Our study suggests that CH25H may function as a prognostic and risk stratification biomarker for LUAD.

Response to trametinib in a nonsmall cell lung cancer patient with osimertinib resistance harboring GNAS R201C and R201H mutations: a case report.

Osimertinib, an orally administered third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is widely approved for the first-line and second-line treatment of advanced non-small-cell lung cancer (NSCLC) with EGFR mutations. However, the rapid development of osimertinib resistance renders the unsustainable treatment benefit. Patients with EGFR -mutated NSCLC who develop osimertinib resistance, especially those acquiring relatively rare and 'off-target' resistance mutations, still lack effective therapeutic options for postosimertinib therapy. Herein, we reported a 73-year-old woman diagnosed with T1N3M1 lung adenocarcinoma harboring EGFR L858R mutation, who acquired two GNAS mutations (R201C and R201H) and lost the EGFR L858R mutation after progression on icotinib and osimertinib. The patient was subsequently treated with trametinib and there was no obvious tumor increase. Our study revealed that GNAS R201 can confer the osimertinib resistance in EGFR -positive NSCLC, and present the first report of the prevalence of GNAS R201C and R201H mutants in NSCLC which response to trametinib treatment. Our case suggests that trametinib could be a treatment option in NSCLC patients harboring GNAS -activating mutations.

FAT1 and MSH2 Are Predictive Prognostic Markers for Chinese Osteosarcoma Patients Following Chemotherapeutic Treatment.

Osteosarcoma is characterized by diverse genetic mutations, including single-nucleotide variants (SNVs), which can complicate clinical outcomes of the treatment. This study identified key mutations or polymorphisms in genes that correlate with osteosarcoma prognoses. A total of 110 patients with osteosarcoma were assigned to "good" or "poor" cohorts depending on their 5-year disease-free survival (DFS) after surgery and chemotherapeutic treatment. We performed next-generation sequencing analysis of tumor tissues for prognosis-associated SNVs in 315 tumorigenesis-related genes, followed by modeling of clinical outcomes for these patients using random forest classification via a support vector machine (SVM). Data from the Chinese Millionome Database were used to compare SNV frequency in osteosarcoma patients and healthy people. SVM screening identified 17 nonsynonymous SNVs located in 15 genes, of which rs17224367 and rs3733406 (located in MSH2 and FAT1, respectively) were strongly correlated with osteosarcoma prognosis. These results were verified in a 26-patient validation cohort, confirming that these SNVs could be used to predict prognosis. These results demonstrated that two SNVs located in MSH2 and FAT1 are associated with prognosis of osteosarcoma patients. © 2022 American Society for Bone and Mineral Research (ASBMR).

Rapid discrimination between tuberculosis and sarcoidosis using next-generation sequencing.

OBJECTIVES: Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (MTB), has similar clinical, radiological, and histopathological characteristics to sarcoidosis (SA). Accurately distinguishing SA from TB remains a clinical challenge. METHODS: A total of 44 TB patients and 47 SA patients who were clinically diagnosed using chest radiography, pathological examination, routine smear microscopy, and microbial culture were enrolled in this study. The MTB genome was captured and sequenced directly from tissue specimens obtained upon operation or biopsy, and the feasibility of next-generation sequencing (NGS) for the MTB genome in the differential diagnosis of TB from SA was evaluated. RESULTS: Using a depth >10× and coverage >15% of the sequencing data, TB patients were identified via the NGS approach directly using operation or biopsy specimens without clinical pretreatment. The sensitivity, specificity, and concordance of the NGS method were 81.8% (36/44), 95.7% (45/47), and 89.0% (81/91), respectively (kappa = 0.78, 95% confidence interval 0.65-0.91; P<0.001). CONCLUSIONS: This study established an improved NGS strategy for rapidly distinguishing patients with TB from those with SA and has potential clinical benefits.

Bronchial Washing Fluid Versus Plasma and Bronchoscopy Biopsy Samples for Detecting Epidermal Growth Factor Receptor Mutation Status in Lung Cancer.

BACKGROUND: Bronchial washing fluid (BWF) is a common specimen collected during bronchoscopy and has been suggested to contain both tumor cells and cell-free DNA. However, there is no consensus on the feasibility of BWF in epidermal growth factor receptor (EGFR) genetic analysis because of the limited sample size and varying results in previous studies. This study compared the feasibility, sensitivity, and specificity of detecting EGFR mutation using BWF, bronchoscopy biopsy, and plasma samples in patients with lung cancer (LC). MATERIALS AND METHODS: A total of 144 patients (110 with LC and 34 without LC) were enrolled in the study. During diagnostic bronchoscopy for suspected LC lesions, bronchial washing with saline was performed directly or through a guide sheath. BWF was collected as well as paired bronchoscopy biopsy and plasma samples, and EGFR mutation testing was performed via highly sensitive blocker polymerase chain reaction. The EGFR mutation status of histologic samples was set as the standard reference. RESULTS: Compared with the histologic samples, the sensitivity, specificity, and concordance rate of EGFR mutation detected in BWF samples were 92.5%, 100%, and 97.9%, respectively. Moreover, BWF showed a higher sensitivity in EGFR mutation testing than both plasma (100% [8/8] vs. 62.5% [5/8], p = 0.095) and bronchoscopy biopsy samples (92.5% [37/40] vs. 77.5% [31/40], p = 0.012) and identified EGFR mutations in 6 cases whose biopsy failed to establish an LC diagnosis. The diameter of the target lesion and its contact degree with BWF were positive predictive factors for EGFR testing results. CONCLUSIONS: BWF yields a high sensitivity in EGFR mutation testing, having high concordance with histologic samples, and presenting the benefit of rapid EGFR mutation detection in LC patients.

Distinguishing Rectal Cancer from Colon Cancer Based on the Support Vector Machine Method and RNA-sequencing Data.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. Several studies have indicated that rectal cancer is significantly different from colon cancer in terms of treatment, prognosis, and metastasis. Recently, the differential mRNA expression of colon cancer and rectal cancer has received a great deal of attention. The current study aimed to identify significant differences between colon cancer and rectal cancer based on RNA sequencing (RNA-seq) data via support vector machines (SVM). Here, 393 CRC samples from the The Cancer Genome Atlas (TCGA) database were investigated, including 298 patients with colon cancer and 95 with rectal cancer. Following the random forest (RF) analysis of the mRNA expression data, 96 genes such as HOXB13, PRAC, and BCLAF1 were identified and utilized to build the SVM classification model with the Leave-One-Out Cross-validation (LOOCV) algorithm. In the training (n=196) and the validation cohorts (n=197), the accuracy (82.1 % and 82.2 %, respectively) and the AUC (0.87 and 0.91, respectively) indicated that the established optimal SVM classification model distinguished colon cancer from rectal cancer reasonably. However, additional experiments are required to validate the predicted gene expression levels and functions.

Comprehensive Determination of Mycobacterium tuberculosis and Nontuberculous Mycobacteria From Targeted Capture Sequencing.

Infection of Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) challenges effective pulmonary infectious disease control. Current phenotypic and molecular assays could not comprehensively and accurately diagnose MTB, NTM, and drug resistance. Next-generation sequencing allows an "all-in-one" approach providing results on expected drug susceptibility testing (DST) and the genotype of NTM strains. In this study, targeted capture sequencing was used to analyze the genetic backgrounds of 4 MTB strains and 32 NTM pathogenic strains in 30 clinical samples, including 14 sputum specimens and 16 bronchoalveolar lavage fluid samples. Through comparing with other TB diagnostic tests, we proved that targeted capture sequencing could be used as a highly sensitive (91.3%) and accurate (83.3%) method to diagnose TB, as well as MGIT 960. Also, we identified 7 NTM strains in 11 patients; among them, seven patients were MTB/NTM co-affected, which indicated that it was a meaningful tool for the diagnosis and treatment of NTM infection diseases in clinic. However, based on a drug-resistant mutation library (1,325 drug resistance loci), only 9 drug resistance strains and 22 drug resistance loci were discovered, having considerable discordance with the drug-resistant results of MGIT 960. Our finding indicated that targeted capture sequencing approach was applicable for the comprehensive and accurate diagnosis of MTB and NTM. However, from data presented here, the DST results identified by next-generation sequencing (NGS) showed a relatively low consistency with MGIT 960, especially in sputum samples. Further work should be done to explore the reasons for low drug-resistance detection rate of NGS.

Multiplexed molecular profiling of lung cancer with malignant pleural effusion using next generation sequencing in Chinese patients.

Lung cancer is the most common type of cancer and the leading cause of cancer-associated death worldwide. Malignant pleural effusion (MPE), which is observed in ~50% of advanced non-small cell lung cancer (NSCLC) cases, and most frequently in lung adenocarcinoma, is a common complication of stage III-IV NSCLC, and it can be used to predict a poor prognosis. In the present study, multiple oncogene mutations were detected, including 17 genes closely associated with initiation of advanced lung cancer, in 108 MPE samples using next generation sequencing (NGS). The NGS data of the present study had broader coverage, deeper sequencing depth and higher capture efficiency compared with NGS findings of previous studies on MPE. In the present study, using NGS, it was demonstrated that 93 patients (86%) harbored EGFR mutations and 62 patients possessed mutations in EGFR exons 18-21, which are targets of available treatment agents. EGFR L858R and exon 19 indel mutations were the most frequently observed alterations, with frequencies of 31 and 25%, respectively. In 1 patient, an EGFR amplification was identified and 6 patients possessed a T790M mutation. ALK + EML4 gene fusions were identified in 6 patients, a ROS1 + CD74 gene fusion was detected in 1 patient and 10 patients possessed a BIM (also known as BCL2L11) 2,903-bp intron deletion. In 4 patients, significant KRAS mutations (G12D, G12S, G13C and A146T) were observed, which are associated with resistance to afatinib, icotinib, erlotinib and gefitinib. There were 83 patients with ERBB2 mutations, but only two of these mutations were targets of available treatments. The results of the present study indicate that MPE is a reliable specimen for NGS based detection of somatic mutations.

Next-generation sequencing in thymic epithelial tumors uncovered novel genomic aberration sites and strong correlation between TMB and MSH6 single nucleotide variations.

Thymic epithelial tumors (TET) including thymomas and thymic carcinomas are rare, but they are common primary tumors in the anterior mediastinum. The etiology and tumorigenesis of TET remain unclear. To better understand the novel aberrations of this rare tumor and provide more significant mutation sites for targeted therapy, we performed next-generation sequencing detection on 55 patients with TET. Our results showed that most genes in 12 core pathways harbored aberrations of indeterminate potential. In 4 genes (ARID1A, KMT2C, TGFBR2 and MAP3K1), the indel frequency was above 90%. Dozens of genes, including TGFBR2, KMT2C, PRKDC, ATR, CHD2, SDHA, KDM5A, CHEK1, MSH6 and POLE, possessed frameshift indel with different frequencies in different hotspot sites, which could be the new targets of therapy for TET. For the first time, we revealed a strong correlation between the tumor mutational burden and single nucleotide variations, but not frameshift, on DNA mismatch repair gene MSH6 in TET.

Detection of EGFR mutations using target capture sequencing in plasma of patients with non-small-cell lung cancer.

PURPOSE : Circulating tumour DNA (ctDNA) is a promising biomarker for detection of non-invasive epidermal growth factor receptor ( EGFR) mutations in patients with non-small-cell lung cancer (NSCLC). However, the existing methods have limitations in sensitivity or in availability. The aim was to evaluate the accuracy of capture target sequencing for detecting EGFR mutations in ctDNA. METHODS : A total of 79 patients with NSCLC and available plasma and matched tissue specimens were enrolled. Through capture target sequencing, mutations were searched in over 20 000 reads obtained from each exon region. Parameters corresponding to the limit of detection and limit of quantification were used as the thresholds for mutation detection. To evaluate the accuracy, detection of EGFR mutations in matched tissue samples was performed by target capture sequencing and the amplification refractory mutation system (ARMS). RESULTS:   : EGFR mutations were discovered in 32.9 % (26/79) of the patients with NSCLC, the overall rate of consistency for the 79 paired plasma and tissue samples was 86.1 % (68/79). The sensitivity and specificity of detecting EGFR mutations in the plasma were 72.7 % and 95.7 %. In terms of the EGFR mutations identified by ARMS, the overall consistency was 78.5 % (62/79) in three groups. Of 21 patients with EGFR sensitive mutation defined by next generation sequencing in ctDNA, 20 (95.2%) showed long-term disease control with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) treatment; the median progression-free survival was 10.8 months (95% CI 9.1 to 16.8). CONCLUSIONS: Target capture sequencing of ctDNA can be used for genotyping of EGFR in patients with NSCLC, which may enable a direct recommendation for EGFR TKI on the basis of positive results with plasma DNA.

Clinical test on circulating tumor cells in peripheral blood of lung cancer patients, based on novel immunomagnetic beads.

This paper aims to establish a novel and highly sensitive method to detect circulating tumor cells (CTCs) in the peripheral blood of patients with lung cancer. This therefore enables the discovery of invisible micrometastasis in the early stage of lung cancer, leading to better prognostic assessments of lung cancer and detection of the post-operative tumor recurrence and metastasis, treatment options, and evaluation of curative effects. In this research study, various lung cancer cells were mixed with adult blood samples to simulate blood samples of tumor patients. With novel test methods, CTCs in peripheral blood of lung cancer patients were calculated, after the reaction between the cells obtained from the mix and EpCAM (epithelial cell adhesion molecule) antibodies which were marked by immunomagnetic beads. The results showed that 18 out of 42 (42.9%) lung cancer patients had a positive CTCs, which increased with tumor enlargement or metastasis. CTCs were not detected in a total of 20 blood samples from healthy volunteers. This indicated that the technology of novel immunomagnetic bead-enrichment could effectively separate and identify CTCs in peripheral blood of lung cancer patients, which is of great clinical value for prognostic assessments and treatment guidance of lung cancer.