The recommendation of re-classification of variants of uncertain significance (VUS) in adult genetic disorders patients.

Since variants of uncertain significance (VUS) reported in genetic testing cannot be acted upon clinically, this classification may delay or prohibit precise diagnosis and genetic counseling in adult genetic disorders patients. Large-scale analyses about qualitatively distinct lines of evidence used for VUS can make them re-classification more accurately. We analyzed 458 Chinese adult patients WES data, within 15 pathogenic evidence PS1, PS2, PM1, PM6 and PP4 were not used for VUS pathogenic classification, meanwhile the PP3, BP4, PP2 were used much more frequently. The PM2_Supporting was used most widely for all reported variants. There were also 31 null variants (nonsense, frameshift, canonical ±1 or 2 splice sites) which were probably the disease-causing variants of the patients were classified as VUS. By analyzed the evidence used for all VUS we recommend that appropriate genetic counseling, reliable releasing of in-house data, allele frequency comparison between case and control, expanded verification in patient family, co-segregation analysis and functional assays were urgent need to gather more evidence to reclassify VUS. We also found adult patients with nervous system disease were reported the most phenotype-associated VUS and the lower the phenotypic specificity, the more reported VUS. This result emphasized the importance of pretest genetic counseling which would make less reporting of VUS. Our result revealed the characteristics of the pathogenic classification evidence used for VUS in adult genetic disorders patients for the first time, recommend a rules-based process to evaluate the pathogenicity of VUS which could provide a strong basis for accurately evaluating the pathogenicity and clinical grade information of VUS. Meanwhile, we further expanded the genetic spectrum and improve the diagnostic rate of adult genetic disorders.

Intronic position +9 and -9 are potentially splicing sites boundary from intronic variants analysis of whole exome sequencing data.

Whole exome sequencing (WES) can also detect some intronic variants, which may affect splicing and gene expression, but how to use these intronic variants, and the characteristics about them has not been reported. This study aims to reveal the characteristics of intronic variant in WES data, to further improve the clinical diagnostic value of WES. A total of 269 WES data was analyzed, 688,778 raw variants were called, among these 367,469 intronic variants were in intronic regions flanking exons which was upstream/downstream region of the exon (default is 200 bps). Contrary to expectation, the number of intronic variants with quality control (QC) passed was the lowest at the +2 and -2 positions but not at the +1 and -1 positions. The plausible explanation was that the former had the worst effect on trans-splicing, whereas the latter did not completely abolish splicing. And surprisingly, the number of intronic variants that passed QC was the highest at the +9 and -9 positions, indicating a potential splicing site boundary. The proportion of variants which could not pass QC filtering (false variants) in the intronic regions flanking exons generally accord with "S"-shaped curve. At +5 and -5 positions, the number of variants predicted damaging by software was most. This was also the position at which many pathogenic variants had been reported in recent years. Our study revealed the characteristics of intronic variant in WES data for the first time, we found the +9 and -9 positions might be a potentially splicing sites boundary and +5 and -5 positions were potentially important sites affecting splicing or gene expression, the +2 and -2 positions seem more important splicing site than +1 and -1 positions, and we found variants in intronic regions flanking exons over ± 50 bps may be unreliable. This result can help researchers find more useful variants and demonstrate that WES data is valuable for intronic variants analysis.

Postoperative circulating tumor DNA testing based on tumor naïve strategy after liver metastasis surgery in colorectal cancer patients.

Objective: There is still a lack of highly sensitive methods for monitoring recurrence of colorectal cancer patients after liver metastasis surgery. The aim of this study was to evaluate the prognostic value of tumor-naive ctDNA detection after resection of colorectal liver metastases (CRLM). Methods: Patients with resectable CRLM were prospectively enrolled. Based on the tumor-naive strategy, NGS panels containing 15 colorectal cancer hotspot mutated genes were used to detect ctDNA 3-6 weeks after surgery. Results: A total of 67 patients were included in the study, and the positive rate of postoperative ctDNA was 77.6% (52/67). Patients with positive ctDNA had a significantly higher risk of recurrence after surgery (HR 3.596, 95% CI 1.479 to 8.744, P = 0.005), and a higher proportion relapsed within 3 months after surgery (46.7% vs 3.8%). The C-index of postoperative ctDNA in predicting recurrence was higher than that of CRS and postoperative CEA. The nomogram combining CRS and postoperative ctDNA can improve the accuracy of recurrence prediction. Conclusion: Tumor-naive ctDNA detection can detect molecular residual lesions in patients with colorectal cancer after liver metastasis, and its prognostic value is superior to conventional clinical factors.

Germline mutations in homologous recombination repair genes among Chinese pancreatic ductal adenocarcinoma patients detected using next-generation sequencing.

BACKGROUND: Genetic testing for pancreatic ductal adenocarcinoma (PDAC) patients is in constant development. However, the status of homologous recombination repair (HRR) genes in unselected Chinese PDAC has not been fully explored. This study aims to characterize the profile of germline mutations in HRR genes in Chinese PDAC patients. METHODS: A cohort of 256 PDAC patients were enrolled at Zhongshan Hospital Fudan University between 2019 and 2021. Germline DNA was analyzed by next-generation sequencing using a multigene panel of the 21 HRR genes. RESULTS: The germline pathogenic (P)/likely pathogenic (LP) variant rates were 7.0% (18/256) in unselected patients with pancreatic cancer. Among them, 1.6% (4/256) were identified as harboring BRCA2 variants, and 5.5% (14/256) patients carried non-BRCA variants. Variants were detected in eight non-BRCA genes, including ATM (4, 1.6%), PALB2 (4, 1.6%), ATR (1, 0.4%), BRIP1 (1, 0.4%), CHEK2 (1, 0.4%), MRE11 (1, 0.4%), PTEN (1, 0.4%), and STK11 (1, 0.4%). ATM, BRCA2, and PALB2 were the most prevalent variant genes. If only BRCA1/2 was tested, 5.5% of P/LP variants would have been lost. Further, we found that the landscape of P/LP HRR variants in various population cohorts was quite different. However, no significant difference was found in clinical characteristics between germline HRR P/LP carriers and non-carriers. In our study, one case carrying a germline PALB2 variant showed a long-time response to platinum-based chemotherapy and PARP inhibitor. CONCLUSION: This study comprehensively depicts the prevalence and characteristics of germline HRR mutations in unselected Chinese PDAC patients. Our findings show the clinical utility of a multigene panel may increase the detection of P/LP HRR carriers.

Development of Multiplex Drop-Off Digital PCR Assays for Hotspot Mutation Detection of KRAS, NRAS, BRAF, and PIK3CA in the Plasma of Colorectal Cancer Patients.

The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. The analytical performance of the assays was assessed using synthetic oligonucleotides, which were further validated on plasma cell-free DNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cell-free DNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring.

Role of blood mSEPT9 in evaluating tumor burden and disease monitoring in colorectal cancer patients.

PURPOSE: This study aimed to investigate the correlation between mSEPT9 and tumor burden as well as the role of mSEPT9 in monitoring colorectal cancer (CRC) patients. METHODS: A total of 309 patients were recruited and received mSEPT9 detection in this retrospective study. Clinicopathologic characteristics were collected, including age, gender, differentiation, gene mutation, stage, and tumor markers. The correlation between mSEPT9 and clinical tumor burden was analyzed. A relative mSEPT9 value was determined using the ΔΔCt method. RESULTS: The overall positivity rate of mSEPT9 was 39.8% in CRC patients. mSEPT9 status was significantly associated with disease status and tumor markers (CEA and CA19-9). The mSEPT9 positivity rates were 15.6%, 50.0%, 64.4%, and 70.0% for P0M0, P1M0, P0M1, and P1M1 patients, respectively (p < 0.001). Among 137 CRC patients who received mSEPT9 assay before surgery, the pre-operation mSEPT9 positivity rate increased significantly from stage I to stage IV (Stage I vs. II vs. III vs. IV 25% vs. 59.1% vs. 57.1% vs. 70.0%, respectively). Consecutive blood samples were obtained from 26 patients during therapy. The patients with increased mSEPT9 levels showed a higher progression rate. CONCLUSIONS: mSEPT9 was a biomarker reflecting tumor burden, and serial detections of mSEPT9 could be a promising strategy for disease monitoring in CRC patients.

Cross-platform comparison of next-generation sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for detecting KRAS/NRAS/BRAF/PIK3CA mutations in cfDNA from metastatic colorectal cancer patients.

BACKGROUND: Examining tumor KRAS/NRAS/BRAF/PIK3CA status in metastatic colorectal cancer (mCRC) is essential for treatment selection and prognosis evaluation. Cell-free DNA (cfDNA) in plasma is a feasible source for tumor gene analysis. METHODS: In this study, we recruited mCRC patients and analyzed their KRAS/NRAS/BRAF/PIK3CA status in cfDNA using two platforms, next-generation sequencing (NGS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The performance between the two platforms and the concordance rate between cfDNA and tissue were analyzed. The relationship between cfDNA-related variables and clinical variables was also assessed. Tumor mutations in cfDNA from patients receiving continuous treatments were monitored in the follow-ups. RESULTS: Next-generation sequencing and MALDI-TOF had similar specificity (100.0% vs. 99.3%) and negative predictive value (99.9% vs. 99.4%), whereas NGS had higher sensitivity (97.1% vs. 85.3% of MALDI-TOF) and positive predictive value (100% vs. 82.9% of MALDI-TOF). The overall concordance rate of NGS and MALDI-TOF was 98.6%. For the reportable types of mutations in both cfDNA and tissue, the concordance rate was 96.1%. Among 28 tissue-positive patients, the allele frequencies of tumor mutations in cfDNA were higher in patients with primary tumor burden (p = 0.0141). Both CEA and CA 19-9 were positively correlated with cfDNA concentration (r = 0.3278 and r = 0.3992). The allele frequencies of tumor mutations changed with disease progression. CONCLUSIONS: Next-generation sequencing showed slightly better performance in detecting cfDNA mutations and was more suitable for clinical practice. cfDNA-related variables reflected the tumor status and showed a promising potential in monitoring disease progression.

Chemotherapy-associated clonal hematopoiesis mutations should be taken seriously in plasma cell-free DNA KRAS/NRAS/BRAF genotyping for metastatic colorectal cancer.

BACKGROUND: Genotyping of plasma cell-free DNA (cfDNA) is an increasingly important method to assess the tumor mutation status in colorectal cancer (CRC) patients. Clonal hematopoiesis (CH) releases non-tumor somatic mutations into blood, causing false positive results in cfDNA-based tumor genotyping. It is still not clear if CH should be examined in all CRC patients undergoing cfDNA analysis. METHODS: We analyzed cfDNA KRAS, NRAS and BRAF genotypes in 236 metastatic CRC patients, who had matched tissue genotyping results, by next-generation sequencing using plasma cfDNA. The cfDNA-only mutations with allele frequencies (AFs) < 5% were highly suspicious for being CH-derived mutations. The origins of cfDNA mutations were confirmed by droplet digital polymerase chain reaction (ddPCR) using paired peripheral blood cells (PBCs) and CH-derived mutations were finally determined. One patient with a CH-derived mutation was followed up and the subpopulation of blood cells, in which CH was present, was investigated. RESULTS: Three CH-derived mutations, KRAS Q61H, KRAS G12D and KRAS G12V, were identified in the patient cohort. All three patients harboring corresponding CH-derived mutations had a prior chemotherapy history. The CH-derived KRAS G12V mutation in a patient was found only present in lymphocytes and persisting under treatment. For all cfDNA mutations, the CH-derived ones were clustered in the patients with < 5% mutation AF and prior chemotherapy. CONCLUSION: The prevalence of CH in CRC patients was limited, and prior chemotherapy was a contributing factor of CH. It is recommended for patients with < 5% mutation AF and prior chemotherapy to have genotyping analysis of their PBCs following plasma cfDNA genotyping.

Role of circulating free DNA in evaluating clinical tumor burden and predicting survival in Chinese metastatic colorectal cancer patients.

BACKGROUND: The aim of this study was to explore the utility of circulating free DNA (cfDNA) in the evaluation of clinical tumor burden and survival in Chinese patients with metastatic colorectal cancer (mCRC) and to preliminarily summarize some metastatic characteristics associated with mutational status. METHODS: A panel covering a total of 197 hotspot mutations of KRAS, NRAS, BRAF and PIK3CA was used to evaluate the mutational status in plasma by next-generation sequencing (NGS) technology in 126 patients with mCRC. An amplification-refractory mutation system (ARMS) was used to analyze genomic DNA from matched tissue samples. Clinical markers including carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), carbohydrate antigen 125 (CA125), neuron-specific enolase (NSE) and lactate dehydrogenase (LDH) in serum and the sum of all tumor diameters on CT or PET/CT were collected to indicate clinical tumor burden. The correlations between cfDNA and clinical tumor burden were analyzed using Pearson correlation and linear regression models. The median progression-free survival (PFS) and 1-year overall survival (OS) rates were calculated by Kaplan-Meier (K-M) survival analysis. RESULTS: Of the 126 enrolled patients, patients who were tested positive for mutations in plasma accounted for 45.2% (57/126). Mutations in KRAS, NRAS, BRAF and PIK3CA were detected in 37.3% (47/126), 1.6% (2/126), 3.2% (4/126) and 13.5% (17/126) of patients, respectively. The overall concordance rate of mutational status between plasma and matched tissues was 78.6% (99/126). Sixteen patients had mutations in plasma that were not detected in tissue, including some rare hotspot mutations. The cfDNA concentration was significantly correlated with the levels of clinical markers, especially CEA (P < 0.0001, Pearson r = 0.81), LDH (P < 0.0001, Pearson r = 0.84) and the sum of tumor diameters (P < 0.0001, Pearson r = 0.80). Patients with a high cfDNA concentration (> 17.91 ng/ml) had shorter median progression-free survival (6.6 versus 11.7 months, P < 0.0001) and lower 1-year overall survival rate (56% versus 94%, P < 0.0001) than those with a low cfDNA concentration (≤17.91 ng/ml). The most common metastatic site was the liver (77.8%), followed by the lymph nodes (62.7%), lung (40.5%), peritoneum (14.3%) and bone (10.3%), in all patients. There was no significant difference in metastasis between different mutational statuses. CONCLUSION: Analyzing mutations in plasma could provide a more comprehensive overview of the mutational landscape than analyzing mutations in tissue. The cfDNA concentration could be a quantitative biomarker of tumor burden and could predict survival in Chinese patients with mCRC.

Elevated soluble programmed death-ligand 1 levels indicate immunosuppression and poor prognosis in hepatocellular carcinoma patients undergoing transcatheter arterial chemoembolization.

AIMS: The present study aimed to determine the prognostic significance of soluble Programmed Death-ligand 1 (sPD-L1) in hepatocellular carcinoma (HCC) patients undergoing transcatheter arterial chemoembolization (TACE). METHODS: We treated 114 HCC patients with TACE from 2012 to 2013 and determined their sPD-L1 levels by enzyme-linked immunosorbent assay. We evaluated prognosis according to mRESIST criteria and analyzed prognostic values by Cox regression and Kaplan-Meier analysis. We further evaluated correlations between sPD-L1 level and inflammatory status, as well as immunosuppressive environment. RESULTS: sPD-L1 levels were significantly increased in patients who developed HCC progression (P = 0.002) and death (P < 0.001). Patients with higher pre-treatment sPD-L1 levels had a significantly shorter time to progression (10.50 vs. 18.25 months, P = 0.001) and decreased overall survival (16.50 vs. 28.50 months, P = 0.003). Importantly, sPD-L1 levels positively correlated with SII (r = 0.284, P = 0.002), sIL-2R (r = 0.239, P = 0.010), IL-10 (r = 0.283, P = 0.002), HBV-DNA loads (r = 0.229, P = 0.014), and CRP (r = 0.237, P = 0.011). Moreover, high sPD-L1 levels had increased numbers of Treg cells (FOXP3+; P = 0.026), Macrophage cells (CD68+; P = 0.014), and M2-Macrophage cells (CD163+; P = 0.026) CONCLUSIONS: sPD-L1 level is a prognostic indicator of poor outcomes after TACE. High sPD-L1 might reflect increased immune activation in an immunosuppressive environment that hindered anti-tumor response activity.

Sphere-forming culture enriches liver cancer stem cells and reveals Stearoyl-CoA desaturase 1 as a potential therapeutic target.

BACKGROUNDS: The role of sphere-forming culture in enriching subpopulations with stem-cell properties in hepatocellular carcinoma (HCC) is unclear. The present study investigates its value in enriching cancer stem cells (CSCs) subpopulations and the mechanism by which HCC CSCs are maintained. METHODS: HCC cell lines and fresh primary tumor cells were cultured in serum-free and ultra-low attachment conditions to allow formation of HCC spheres. In vitro and in vivo experiments were performed to evaluate CSC characteristics. Expression levels of CSC-related genes were assessed by qRT-PCR and the correlation between sphere formation and clinical characteristics was investigated. Finally, gene expression profiling was performed to explore the molecular mechanism underlying HCC CSC maintenance. RESULTS: We found that both cell lines and primary tumor cells formed spheres. HCC spheres possessed the capacity for self-renewal, proliferation, drug resistance, and contained different subpopulations of CSCs. Of interest, 500 sphere-forming Huh7 cells or 200 primary tumor cells could generate tumors in immunodeficient animals. Sphere formation correlated with size, multiple tumors, satellite lesions, and advanced stage. Further investigation identified that the PPARα-SCD1 axis plays an important role in maintenance of the CSC properties of HCC sphere cells by promoting nuclear accumulation of ß-Catenin. Inhibition of SCD1 interfered with sphere formation, down-regulated expression of CSC-related markers, and reduced ß-Catenin nuclear accumulation. CONCLUSIONS: Sphere-forming culture can effectively enrich subpopulations with stem-cell properties, which are maintained through activation of the PPARα-SCD1 axis. Therefore, we suggest that targeting the SCD1-related CSC machinery might provide a novel insight into HCC treatment.

Analytical and clinical validation of a novel amplicon-based NGS assay for the evaluation of circulating tumor DNA in metastatic colorectal cancer patients.

Background Evaluating the tumor RAS/BRAF status is important for treatment selection and prognosis assessment in metastatic colorectal cancer (mCRC) patients. Correction of artifacts from library preparation and sequencing is essential for accurately analyzing circulating tumor DNA (ctDNA) mutations. Here, we assessed the analytical and clinical performance of a novel amplicon-based next-generation sequencing (NGS) assay, Firefly™, which employs a concatemer-based error correction strategy. Methods Firefly assay targeting KRAS/NRAS/BRAF/PIK3CA was evaluated using cell-free DNA (cfDNA) reference standards and cfDNA samples from 184 mCRC patients. Plasma results were compared to the mutation status determined by ARMS-based PCR from matched tissue. Samples with a mutation abundance below the limit of detection (LOD) were retested again by droplet digital polymerase chain reaction (ddPCR) or NGS. Results The Firefly assay demonstrated superior sensitivity and specificity with a 98.89% detection rate at an allele frequency (AF) of 0.2% for 20 ng cfDNA. Generally, 40.76% and 48.37% of the patients were reported to be positive by NGS of plasma cfDNA and ARMS of FFPE tissue, respectively. The concordance rate between the two platforms was 80.11%. In the pre-treatment cohort, the concordance rate between plasma and tissue was 93.33%, based on the 17 common exons that Firefly™ and ARMS genotyped, and the positive percent agreement (PPA) and negative percent agreement (NPA) for KRAS/NRAS/BRAF/PIK3CA were 100% and 99.60%, respectively. Conclusions Total plasma cfDNA detected by Firefly offers a viable complement for mutation profiling in CRC patients, given the high agreement with matched tumor samples. Together, these data demonstrate that Firefly could be routinely applied for clinical applications in mCRC patients.

CD73 promotes hepatocellular carcinoma progression and metastasis via activating PI3K/AKT signaling by inducing Rap1-mediated membrane localization of P110ß and predicts poor prognosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. METHODS: CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110ß and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. RESULTS: In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110ß to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. CONCLUSIONS: CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.

Application of Serum Annexin A3 in Diagnosis, Outcome Prediction and Therapeutic Response Evaluation for Patients with Hepatocellular Carcinoma.

PURPOSE: Annexin A3 (ANXA3) could induce progression of hepatocellular carcinoma (HCC) via promoting stem cell traits of CD133-positive cells. Moreover, serum ANXA3 showed preliminary diagnostic potential, however further validation was required. Meanwhile, the prognostic value of ANXA3 remained elusive. The present study aimed to validate diagnostic performance and further systematically investigate the prognostic value of serum ANXA3. METHODS: Serum ANXA3 of 368 HCC patients was determined by enzyme-linked immunosorbent assay (ELISA); 295 of these patients underwent resection and 73 underwent transcatheter arterial chemoembolization (TACE). Diagnostic performance of ANXA3 was evaluated by receiver operating characteristic (ROC) analysis, and the prognostic value was evaluated by Cox regression and Kaplan-Meier analysis. To evaluate the relationship between serum ANXA3 and circulating CD133 mRNA-positive tumor cells (CD133mRNA+ CTCs), real-time polymerase chain reaction was conducted in 69 patients who underwent resection. RESULTS: Serum ANXA3 provided greater diagnostic performance than α-fetoprotein (area under the curve [AUC] 0.869 vs. 0.782), especially in early diagnosis (AUC 0.852 vs. 0.757) and discriminating HCC from patients at risk (0.832 vs. 0.736). Pretreatment ANXA3 was an independent predictor of tumor recurrence (hazard ratio [HR] 1.87, 95% confidence interval [CI] 1.26-2.76, p = 0.002)/progression (HR 1.88, 95% CI 1.04-3.43, p = 0.038) and survival (resectable: HR 2.26, 95% CI 1.44-3.56, p = 0.001; unresectable: HR 2.08, 95% CI 1.10-4.05, p = 0.025), and retained its performance in low-recurrence-risk subgroups. Specifically, dynamic changes of ANXA3-positive status was associated with worse prognosis. ANXA3 was positively correlated with CD133mRNA+ CTCs (r = 0.601, p < 0.001). In patients with detectable CD133mRNA+ CTC, high ANXA3 was positively associated with a higher risk of recurrence and shorter overall survival. CONCLUSIONS: Serum ANXA3 shows promise as a biomarker for diagnosis, outcome prediction, and therapeutic response evaluation in patients with HCC.

Circulating Tumor Cells with Stem-Like Phenotypes for Diagnosis, Prognosis, and Therapeutic Response Evaluation in Hepatocellular Carcinoma.

Background: In the present study, we assessed the clinical value of circulating tumor cells (CTC) with stem-like phenotypes for diagnosis, prognosis, and surveillance in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) by an optimized qPCR-based detection platform.Methods: Differing subsets of CTCs were investigated, and a multimarker diagnostic CTC panel was constructed in a multicenter patient study with independent validation (total n = 1,006), including healthy individuals and patients with chronic hepatitis B infection (CHB), liver cirrhosis (LC), benign hepatic lesion (BHL), and HBV-related HCC, with area under the receiver operating characteristic curve (AUC-ROC) reflecting diagnostic accuracy. The role of the CTC panel in treatment response surveillance and its prognostic significance were further investigated.Results: The AUC of the CTC panel was 0.88 in the training set [sensitivity = 72.5%, specificity = 95.0%, positive predictive value (PPV) = 92.4, negative predictive value (NPV) = 77.8] and 0.93 in the validation set (sensitivity = 82.1%, specificity = 94.2%, PPV = 89.9, NPV = 89.3). This panel performed equally well in detecting early-stage and α-fetoprotein-negative HCC, as well as differentiating HCC from CHB, LC, and BHL. The CTC load was decreased significantly after tumor resection, and patients with persistently high CTC load showed a propensity of tumor recurrence after surgery. The prognostic significance of the CTC panel in predicting tumor recurrence was further confirmed [training: HR = 2.692; 95% confidence interval (CI), 1.617-4.483; P < 0.001; and validation: HR = 3.127; 95% CI, 1.360-7.190; P = 0.007].Conclusions: Our CTC panel showed high sensitivity and specificity in HCC diagnosis and could be a real-time parameter for risk prediction and treatment monitoring, enabling early decision-making to tailor effective antitumor strategies. Clin Cancer Res; 24(9); 2203-13. ©2018 AACR.

Serum IgG4:IgG Ratio Predicts Recurrence of Patients with Hepatocellular Carcinoma after Curative Resection.

Aim: IgG4 is associated with a Th1-to-Th2 switch, which plays a vital role in metastasis, in patients with malignances; thus, we aimed to investigate its clinical significance in predicting hepatocellular carcinoma (HCC) recurrence in the present study. Methods: The correlation between serum IgG4:IgG ratio and recurrence was analyzed in a cohort of 195 patients undergoing curative resection in 2012. Another 100 patients were analyzed in a prospective independent cohort during 2012-2013 to validate the value of serum IgG4. Serum IgG4 and total IgG concentrations were measured with an automatic immune analyzer and the optimal cutoff value for serum IgG4 levels was determined by X-tile software. Results: Our data revealed that serum IgG4:IgG were significantly elevated in patients with tumor recurrence (P<0.05). A cutoff IgG:IgG4 ratio of 0.08 was set to stratify HCC patients into high (>0.08) and low (≤0.08) groups. High serum IgG4:IgG ratio correlated with significantly shorter time-to-recurrence (median 11.85 months vs. 39.20, P=0.005). Univariate and multivariate analyses demonstrated that serum IgG4:IgG ratio is an independent indicator of tumor recurrence and this retained its clinical significance even in conventional low-recurrence-risk subgroups, including patients with low α-fetoprotein and early-stage diseases. Conclusion: Our results demonstrated that elevated serum IgG4:IgG ratio is associated with poor clinical outcomes in HCC patients and therefore, and can serve as a novel prognostic predictor for HCC patients undergoing resection. Analyzing serum IgG4 would be useful to tailor individualized therapies for patients.

Extraction of crude polysaccharides from Duchesnea indica (Andrews) Focke: optimization by response surface methodology.

A full set of optimization procedure was applied to the extraction of anti-viral polysaccharides from Duchesnea indica (Andrews) Focke. By Plackett-Burman factorial design, three parameters (extraction time, extraction temperature, and ratio of water to raw material) were identified as significant to the extraction yield. However, no significant parameters had been identified for antiviral activity. A three-level-three-factor Box-Behnken factorial design was then employed to further optimize the extraction condition. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis and also examined using appropriate statistical methods. This led to the construction of a response surface indicating the optimal values for each parameter and response studied. Concerning the extraction yield, an extraction at 98.51 ºC for 6.16 h with a ratio of water to raw material of 30.94 mL/g was found to be optimal. Under the optimized conditions, the experimental yield was 6.430 ± 0.078%, which was well matched with the predicted yield of 6.509%.

Clinical significance of EpCAM mRNA-positive circulating tumor cells in hepatocellular carcinoma by an optimized negative enrichment and qRT-PCR-based platform.

PURPOSE: This study aimed to construct a novel platform for the detection of circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC) and to investigate the clinical significance of epithelial cell adhesion molecule mRNA-positive (EpCAM(mRNA+)) CTCs using this platform. EXPERIMENTAL DESIGN: An optimized platform for CTC detection was constructed by evaluating different negative enrichment, mRNA isolation, and cDNA synthesis procedures and compared with the CellSearch system. A total of 299 patients with HCC were recruited into this prospective study; of these, 157 who received curative resection, 76 who received transcatheter arterial chemoembolization (TACE), and 66 who received radiotherapy were tested using our platform. The diagnostic value of EpCAM(mRNA+) CTCs was investigated in 122 patients with HCC who underwent resection and 120 control subjects. RESULTS: The optimized negative enrichment and quantitative real-time PCR (qRT-PCR)-based CTC detection platform had high sensitivity, specificity, and reproducibility and a low sample volume requirement. This platform showed a potential diagnostic value in patients with HCC and exhibited 76.7% consistency with the CellSearch system (r = 0.54, P < 0.050). Pretreatment CTC level showed prognostic significance in patients with HCC treated with resection, TACE, and radiotherapy (all P < 0.050). Most of the patients showed a decrease in CTC levels after treatment that reflected tumor response. In contrast, patients with an increased CTC level showed disease progression after treatment. CONCLUSIONS: We established an optimized platform based on negative enrichment and qRT-PCR for highly sensitive, specific, and reproducible CTC detection. This platform might be clinically useful in auxiliary diagnosis, treatment response assessment, and early decision-making to tailor the most effective antitumor strategies.

Unlabeled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in pancreatic adenocarcinoma tissues.

BACKGROUND: The aim of our study was to establish an unlabeled-probe high-resolution melting (HRM) approach to the detection of Kirsten RAS (KRAS) codon 12 and 13 mutations in pancreatic adenocarcinoma (PA) tissues as a novel and effective diagnostic technique. METHODS: We tested the sensitivity and specificity of this genotyping approach in cell lines with known KRAS mutations using 166 bp amplicons and 37 bp wild-type probe to detect KRAS codon 12 and 13 mutations. We screened 49 PA tissues to be subsequently sequenced to confirm the mutations. Simultaneously, we tested the specimens using Sanger sequencing and then used target-DNA cloning and sequencing for verification. RESULTS: It was found that unlabeled-probe HRM was reliable in detecting 3% of mutant cell lines DNA diluted with that of the wild-type, whereas Sanger sequencing could only discriminate 20% mutant cell ratios. In detecting 49 specimens, the former was capable of detecting 23 mutations (46.9%); and the latter could observe 15 (30.6%). For further verification, T-A DNA cloning and sequencing was applied to the differences, with the results matching those of the unlabeled-probe HRM. CONCLUSIONS: It was concluded that the unlabeled-probe HRM approach can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in diagnosing and treating PA.