The contribution of hereditary cancer-related germline mutations to lung cancer susceptibility.

BACKGROUND: Germline variations may contribute to lung cancer susceptibility besides environmental factors. The influence of germline mutations on lung cancer susceptibility and their correlation with somatic mutations has not been systematically investigated. METHODS: In this study, germline mutations from 1,026 non-small cell lung cancer (NSCLC) patients were analyzed with a 58-gene next-generation sequencing (NGS) panel containing known hereditary cancer-related genes, and were categorized based on American College of Medical Genetics and Genomics (ACMG) guidelines in pathogenicity, and the corresponding somatic mutations were analyzed using a 605-gene NGS panel containing known cancer-related genes. RESULTS: Plausible genetic susceptibility was found in 4.7% of lung cancer patients, in which 14 patients with pathogenic mutations (P group) and 34 patients with likely-pathogenic mutations (LP group) were identified. The ratio of the first degree relatives with lung cancer history of the P groups was significantly higher than the Non-P group (P=0.009). The ratio of lung cancer patients with history of other cancers was higher in P (P=0.0007) or LP (P=0.017) group than the Non-P group. Pathogenic mutations fell most commonly in BRCA2, followed by CHEK2 and ATM. Likely-pathogenic mutations fell most commonly in NTRK1 and EXT2, followed by BRIP1 and PALB2. These genes are involved in DNA repair, cell cycle regulation and tumor suppression. By comparing the germline mutation frequency from this study with that from the whole population or East Asian population (gnomAD database), we found that the overall odds ratio (OR) for P or LP group was 17.93 and 15.86, respectively, when compared with the whole population, and was 2.88 and 3.80, respectively, when compared with the East Asian population, suggesting the germline mutations of the P and LP groups were risk factors for lung cancer. Somatic mutation analysis revealed no significant difference in tumor mutation burden (TMB) among the groups, although a trend of lower TMB in the pathogenic group was found. The SNV/INDEL mutation frequency of TP53 in the P group was significantly lower than the other two groups, and the copy number variation (CNV) mutation frequency of PIK3CA and MET was significantly higher than the Non-P group. Pathway enrichment analysis found no significant difference in aberrant pathways among the three groups. CONCLUSIONS: A proportion of 4.7% of patients carrying germline variants may be potentially linked to increased susceptibility to lung cancer. Patients with pathogenic germline mutations exhibited stronger family history and higher lung cancer risk.

Comprehensive analysis of POLE and POLD1 Gene Variations identifies cancer patients potentially benefit from immunotherapy in Chinese population.

POLE/POLD1 gene variants have been suggested as potential markers for immunotherapy due to their significant association with the tumor mutational burden (TMB), an effective indicator for response prediction in immunotherapy. However, the correlation of POLE/POLD1 variants with MSI, MMR, TMB, MMR-related and key driver gene mutations needs to be defined to support patient recruitment and therapeutic effect assessment in immunotherapy. 1,392 Chinese cancer patients were recruited, and the correlation of POLE/POLD1 variants with existing immunotherapeutic markers and cancer pathways was investigated. A next-generation sequencing panel including 605 cancer-related genes was used for variant sequencing. It was found that the frequency of POLE variants was not statistically different from that in COSMIC database, while the frequency of POLD1 variants was significantly higher in lung cancer. c.857 C > G and c.2091dupC were potential high frequency variants in Chinese cancer patients. Patients carrying POLE damaging variants were significantly younger than POLE/POLD1 WT patients. Patients carrying POLE/POLD1 damaging variants exhibited significantly higher TMB and frequency of MMR gene variants than POLE/POLD1 WT patients. Patients with POLE damaging variants also exhibited significantly higher frequency of driver gene variants than POLE/POLD1 WT patients. Further analysis showed that POLE damaging variants may affect the cancer development through MMR, TGFß and RTK/RAS/RAF signaling pathways, and POLD1 through MMR pathways. In conclusion, this study identified key characteristics and regions of POLE/POLD1 genes that correlates with TMB, MMR gene mutations and key driver gene mutations, and provided theoretical and practical basis for patient selection based on POLE/POLD1 gene status in immunotherapy.

Double-Stranded DNA in Exosomes of Malignant Pleural Effusions as a Novel DNA Source for EGFR Mutation Detection in Lung Adenocarcinoma.

Background: Exosomes are cell-derived vesicles and bear a specific set of nucleic acids including DNA (exoDNA). Thus, this study is to explore whether exoDNA in malignant pleural effusions (MPEs) could be a novel DNA source for mutation detection of epidermal growth factor receptor (EGFR). Methods: In this study, 52 lung adenocarcinoma patients were enrolled, and EGFR mutation status was detected with tumor tissues as well as cell blocks and exosomes in MPEs. The sensitivity, specificity and consistency of EGFR detection using exosomes were evaluated, compared with gene detection using tumor tissues and cell blocks. And the clinical response of patients who were detected as EGFR mutation in exosomes and treated with EGFR tyrosine kinase inhibitor (EGFR-TKI) was explored. Results: Gene detection using exosomes showed sensitivity of 100%, specificity of 96.55% and coincidence rate of 98.08% (Kappa = 0.961, P < 0.001), compared with detection using tumor tissues and cell blocks. After EGFR-TKI treatment, patients detected as EGFR mutation by exosomes showed efficacy rate of 83% and disease control rate of 100%. And patients who were detected as wild type in tumor tissues or cell blocks but EGFR mutation in exosomes turned up as PR or SD. Conclusions: These results demonstrated that exoDNA in MPEs could be used as a DNA source for EGFR detection in lung adenocarcinoma.

The mSHOX2 is capable of assessing the therapeutic effect and predicting the prognosis of stage IV lung cancer.

BACKGROUND: Instant monitoring of the therapeutic effect of systematic therapy in late-stage lung cancer is crucial for response assessment and strategy adjustment. Previous study found that specific plasma methylation markers may be applied to therapeutic effect assessment. In order to investigate the performance of plasma mSHOX2 in assessing the therapeutic effect and predicting the prognosis of stage IV lung cancer, we performed the study focusing on patients underwent chemotherapy or tyrosine kinase inhibitor (TKI)-based targeted therapy. METHODS: Blood samples from 163 subjects, including 30 stage I, 29 stage II, 26 stage III and 68 stage IV lung cancer patients, were recruited in this study. Quantitative relationship between primary tumor size and the plasma mSHOX2 level was established. Blood samples before therapy and two cycles after therapy were obtained from 68 stage IV patients, and the mSHOX2 level was quantified as ΔΔCt. RESULTS: Sharp decrease of plasma mSHOX2 level was seen in patients with partial response (PR) while not in those with stable disease (SD). The plasma mSHOX2 level change reflected the degree of response and correlated with the maximal diameter of primary tumors in linear relationship. The mSHOX2 levels before and two cycles after therapy were predictors of the overall survival, while the mSHOX2 level change or the tumor size change were not predictors of the overall survival. Furthermore, univariable and multivariable Cox regression revealed that mSHOX2 level before therapy was the only independent predictor of the overall survival with a hazard ratio of 1.414. CONCLUSIONS: mSHOX2 is effective for therapeutic effect assessment and prognosis prediction of stage IV lung cancer patients underwent systematic therapy.

The quantitative profiling of blood mSEPT9 determines the detection performance on colorectal tumors.

AIM: To investigate the quantitative relationship between the positive detection rate (PDR) in colorectal tumor detection and the mSEPT9 level. MATERIALS & METHODS: The level of blood mSEPT9 in various colorectal diseases was quantified by the Epi proColon 2.0 assay. ΔΔCt values were calculated representing the mSEPT9 level. A total of 1347 subjects were recruited in this quantitative study. RESULTS: PDR or sensitivity was positively correlated with the progression of colorectal tumors and the mSEPT9 level in an exponential relationship. The mSEPT9 level of CRC exhibited a distinct pattern of distribution. Strong correlation was found between mSEPT9 level and PDR or sensitivity in various tumor differentiation, pathological types or metastasis. CONCLUSION: The quantitative profiling of blood mSEPT9 determines the detection performance on colorectal tumors.

The blood mSEPT9 is capable of assessing the surgical therapeutic effect and the prognosis of colorectal cancer.

AIM: To investigate the performance of methylated SEPT9 (mSEPT9) in assessing the surgical therapeutic effect and prognosis of colorectal cancer (CRC). METHODS: Blood samples before surgery and 1 and 7 days after surgery were obtained from 120 CRC patients, and mSEPT9 and carcinoembryonic antigen (CEA) assays were performed. RESULTS: The mean plasma mSEPT9 level showed 57.6-times and 131.1-times decrease 1 day and 7 days after surgery, respectively. In contrast, mean CEA levels showed 2.51-and 2.70-times decrease 1 and 7 days after surgery. 86.7% of patients can be assessed by mSEPT9 while 44.2% can be assessed by CEA. Positive mSEPT9 detection before surgery correlated with higher risk of death after surgery. CONCLUSION: mSEPT9 is effective for CRC postsurgical assessment and prognosis prediction.

The performance of the SEPT9 gene methylation assay and a comparison with other CRC screening tests: A meta-analysis.

The SEPT9 gene methylation assay is the first FDA-approved blood assay for colorectal cancer (CRC) screening. Fecal immunochemical test (FIT), FIT-DNA test and CEA assay are also in vitro diagnostic (IVD) tests used in CRC screening. This meta-analysis aims to review the SEPT9 assay performance and compare it with other IVD CRC screening tests. By searching the Ovid MEDLINE, EMBASE, CBMdisc and CJFD database, 25 out of 180 studies were identified to report the SEPT9 assay performance. 2613 CRC cases and 6030 controls were included, and sensitivity and specificity were used to evaluate its performance at various algorithms. 1/3 algorithm exhibited the best sensitivity while 2/3 and 1/1 algorithm exhibited the best balance between sensitivity and specificity. The performance of the blood SEPT9 assay is superior to that of the serum protein markers and the FIT test in symptomatic population, while appeared to be less potent than FIT and FIT-DNA tests in asymptomatic population. In conclusion, 1/3 algorithm is recommended for CRC screening, and 2/3 or 1/1 algorithms are suitable for early detection for diagnostic purpose. The SEPT9 assay exhibited better performance in symptomatic population than in asymptomatic population.

The SEPT9 gene methylation assay is capable of detecting colorectal adenoma in opportunistic screening.

AIM: This study validated the detection of colorectal adenoma in opportunistic screening using the SEPT9 gene methylation assay. MATERIALS & METHODS: Plasma samples including 85 colorectal cancers, 364 adenomas, 216 hyperplastic polyps, 372 other gastrointestinal diseases and 324 normal subjects, were obtained and tested using the Epi proColon 2.0 CE assay. RESULTS & CONCLUSION: The SEPT9 assay detected 38.7% of all types of adenoma, including 27.8% of serrated adenoma, 28.7% of tubular adenoma, 53.7% of tubulovillous adenoma and 83.3% of villous adenoma. It also detected 27.5% of nonadvanced adenoma (NAA), 47.0% of advanced adenoma (AA) without high-grade dysplasia and 62.5% of AA with high-grade dysplasia. The average adenoma detection rate was 31.8% (95% CI: 28.3-35.4%) with the Boston Bowel Preparation Scale score at 7.6 ± 1.2 (mean ± SD). Our study provided strong evidence for the application of the SEPT9 assay in AA detection in opportunistic screening.

KRAS and BRAF mutations in serum exosomes from patients with colorectal cancer in a Chinese population.

The efficacy of epidermal growth factor receptor- targeted therapy is significantly associated with Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-raf serine/threonine kinase proto-oncogene (BRAF) mutation in patients with colorectal cancer (CRC), for which the standard gene testing is currently performed using tumor tissue DNA. The aim of the present study was to compare the presence of KRAS and BRAF mutations in the serum exosome and primary tumor tissue from patients with CRC. Genomic DNA were extracted from the tumor tissues of 35 patients with histologically-confirmed CRC and exosomal mRNA were obtained from peripheral blood, which were collected from the corresponding patients prior to surgery. Three mutations in the KRAS gene (codons 12, 13 and 61) and a mutation in the BRAF gene (codon 600) were detected using a polymerase chain reaction-based sequencing method and their presence were compared between tumor tissues and the matched serum exosomes. The KRAS mutation rates in tumor tissues and the matched serum exosomes were 57.6 and 42.4%, respectively, which was not significantly different (P=0.063). The detection rate of the BRAF mutation was 24.2 and 18.2% in tumor tissues and the matched serum exosomes, respectively, and there was no significant difference (P=0.500). The patients with CRC that had a KRAS mutation of codon 12 in exon 2 in their tumor tissues and serum exosomes were significantly older compared with those without this mutation (tumor tissue, P=0.002; serum exosome, P=0.022). The sensitivity of KRAS and BRAF mutation detection using exosomal mRNA was 73.7 and 75%, respectively. The specificity of the detected mutations exhibited an efficiency of 100%, and the total consistency rate was 94.9 and 93.9% for KRAS and BRAF mutations, respectively. These results suggested that serum exosomal mRNA may be used as a novel source for the rapid and non-invasive genotyping of patients with CRC.

Effectiveness of circulating tumor DNA for detection of KRAS gene mutations in colorectal cancer patients: a meta-analysis.

Circulating tumor DNA (ctDNA) can be identified in the peripheral blood of patients and harbors the genomic alterations found in tumor tissues, which provides a noninvasive approach for detection of gene mutations. We conducted this meta-analysis to investigate whether ctDNA can be used for monitoring KRAS gene mutations in colorectal cancer (CRC) patients. Medline, Embase, Cochrane Library and Web of Science were searched for the included eligible studies in English, and data were extracted for statistical analysis according to the numbers of true-positive (TP), true-negative (TN), false-positive (FP) and false-negative (FN) cases. Sensitivity, specificity and diagnostic odds ratio (DOR) were calculated, and the area under the receiver operating characteristic curve (AUROC) was used to evaluate the diagnostic performance. After independent searching and reviewing, 21 studies involving 1,812 cancer patients were analyzed. The overall sensitivity, specificity and DOR were 0.67 (95% confidence interval [CI] =0.55-0.78), 0.96 (95% CI =0.93-0.98) and 53.95 (95% CI =26.24-110.92), respectively. The AUROC was 0.95 (95% CI =0.92-0.96), which indicated the high diagnostic accuracy of ctDNA. After stratified analysis, we found the higher diagnostic accuracy in subgroup of patients detected in blood sample of plasma. The ctDNA may be an ideal source for detection of KRAS gene mutations in CRC patients with high specificity and diagnostic value.

Colony-stimulating factor 2 enhances the developmental competence of yak (Poephagus grunniens) preimplantation embryos by modulating the expression of heat shock protein 70 kDa 1A.

Colony-stimulating factor 2 (CSF2) is known to promote the development and survival of rodents and ruminants preimplantation embryos; however, the effect of CSF2 on yak embryos has not been reported. The objective of this study was to investigate the effects of CSF2 on the developmental competence of yak embryos cultured in vitro in modified synthetic oviduct fluid (mSOF) medium and on the expression pattern of heat shock protein 70 kDa 1A (HSPA1A). In each experiment, cumulus-oocyte complexes (COCs) were matured in vitro and fertilized with frozen-thawed semen. Zygotes were treated with varying concentrations of CSF2 (0, 10, 50, 100 ng/mL) until day 8 after fertilization. Embryo development was calculated as the percentage of oocytes that formed embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst stages. The total cell numbers (TCN) per blastocyst and their allocation to the inner cell mass (ICM) and trophectoderm (TE) lineages were determined using differential CDX2 staining. The expression of HSPA1A was examined by quantitative real-time PCR (qRT-PCR) and immunochemistry to determine the mRNA and protein levels. The results showed that treatment with 50 ng/mL CSF2 significantly (P < 0.05) increased the rate of blastocyst formation (19.01% versus 9.93%) and the TCN per blastocyst (96.94 versus 81.41) compared to the control group. However, no significant differences were observed in the other stages of development. qRT-PCR analysis confirmed that treatment with 50 ng/mL CSF2 significantly (P < 0.05) inhibited the expression of HSPA1A mRNA in blastocysts cultured in vitro relative to the control group, but there were no significant differences between the other treatment groups. Immunocytochemical analysis confirmed that HSPA1A protein accumulation was gradually reduced in yak blastocysts cultured in 0, 10, 100 or 50 ng/mL CSF2, however, no significant differences were observed between the 10 and 100 ng/mL treatments (P > 0.05). In conclusion, these findings demonstrate that CSF2 inhibits the expression of HSPA1A to facilitate yak blastocyst formation and increase cell numbers.

The performance of the mSEPT9 assay is influenced by algorithm, cancer stage and age, but not sex and cancer location.

PURPOSE: This study aims to examine the influence of algorithm and subject-related factors, including cancer stage, age, sex, and cancer location, on the performance of the SEPT9 gene methylation test, an assay approved by the US FDA for colorectal cancer (CRC) screening. METHODS: A total of 1225 subjects were recruited in this opportunistic screening study, including 388 CRC patients, 139 subjects with adenoma, 108 subjects with hyperplastic polyps, and 590 subjects with no evidence of disease (NED). Epi proColon 2.0 CE assay was used to examine the blood level of SEPT9 gene methylation. RESULTS: It was found that tests using 1/3 algorithm exhibited higher detection rate than those using the 2/3 algorithm for CRC, adenoma, hyperplastic polyps, while the false positive rate in subjects with NED was also higher with 1/3 algorithm. The positive detection rate (PDR) of the assay for stage 0 and I CRC were lower than later stages (Stage II, III and IV). Interestingly, the normal subjects above 60 years old exhibited significantly higher PDR than subjects from younger groups, while no significant change in PDR was observed among age groups in CRC patients. Furthermore, no difference in the PDR for CRC was found between male and female, and the PDR for CRC at various colorectal locations were essentially identical. CONCLUSIONS: Algorithm, cancer stage and age are factors affecting the detection rate of the SEPT9 assay, while sex and cancer location appeared to have no influence on its performance.