The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing.

In our previous study, we detected the effects of centrifugal forces on plasma RNA quantification by quantitative reverse transcription PCR. The aims of this study were to perform targeted mRNA sequencing and data analysis in healthy donors' plasma prepared by two centrifugation protocols and to investigate the effects of centrifugal forces on plasma mRNA quality and quantity. Targeted mRNA sequencing was performed using a custom panel with 108 colorectal cancer-related genes in 18 healthy donors' plasma that prepared by (1) 3,500 g for 10 min at 4°C and (2) 1,600 g for 10 min at 4°C followed by 16,000 g for 10 min at 4°C. Results showed that plasma ribosomal RNA was detected in 16/18 (88.9%) 3,500 g and 6/18 (33.3%) 1,600 g followed by 16,000 g centrifuged plasma. For targeted sequencing, 75/108 (69.4%) and 86/108 (79.6%) genes were detected in 3,500 and 1,600 g followed by 16,000 g, respectively, while 16/108 (14.8%) genes were not detected in both centrifugations. Detailed analysis showed that 2 of 108 (1.85%) genes showed lower expressions in 3,500 g than in 1,600 g followed by 16,000 g. The median expressions of genes in 3,500 g were positively correlated with the expressions in 1,600 g followed by 16,000 g (R2 = 0.9471, P < 0.0001, Spearman rank correlation). Meanwhile, plasma samples were not distinctively clustered based on centrifugal forces according to hierarchical clustering. Targeted mRNA sequencing and subsequent data analysis were performed in this study to investigate the effects of two different centrifugal forces that are commonly used in plasma collection. Our targeted sequencing results help to understand the centrifugal force effects on plasma mRNA, and these findings show that the centrifugation protocol for plasma mRNA research using targeted sequencing can be standardized which facilitates multicenter studies for comparison and quality assurance in the future.

Menstrual Blood Human Papillomavirus DNA and TAP1 Gene Polymorphisms as Potential Biomarkers for Screening and Monitoring of Cervical Squamous Intraepithelial Lesion.

Background: Human papillomavirus (HPV) is a known causative factor in the etiology of cervical cancer. Methods: HPV DNA genotyping was performed in menstrual blood (MB) collected in napkins from patients with cervical intraepithelial neoplasia (CIN), HPV infection and sexually active apparently normal subjects. In the same patient cohort, MB TAP1 I333V and TAP1 D637G gene polymorphisms were examined. Results: The sensitivity, specificity, and positive and negative predictive values of HPV DNA in the detection of CIN or HPV infection were 83% (223 of 268), 98% (131 of 134), 99% (223 of 226), and 74% (131 of 176), respectively. Moreover, HPV DNA was found in 24% (28/118) patients who had loop electrosurgical excision procedure treatment and 0% (0/76) HPV infected or CIN1 patient with proven recovery. On the other hand, the risk of developing high-grade CIN was significantly reduced for AG and GG genotypes compared with AA genotype and for carriers with a G allele compared with those with an A allele for both polymorphisms. Conclusions: MB HPV DNA is a potential noninvasive marker for screening and monitoring of squamous intraepithelial lesion. Together with TAP1 I333V and TAP1 D637G gene polymorphisms, the combined test may be useful for stratifying high-risk patients for better follow-up strategies.

Implementation and new insights in molecular diagnostics for HIV infection.

INTRODUCTION: Acquired immunodeficiency syndrome (AIDS) is a kind of acquired disease that breaks down the immune system. Human immunodeficiency virus (HIV) is the causative agent of AIDS. By the end of 2016, there were 36.7 million people living with HIV worldwide. Early diagnosis can alert infected individuals to risk behaviors in order to control HIV transmission. Infected individuals are also benefited from proper treatment and management upon early diagnosis. Thanks to the public awareness of the disease, the annual increase of new HIV infections has been slowly declining over the past decades. The advent of molecular diagnostics has allowed early detection and better management of HIV infected patients. Areas covered: In this review, the authors summarized and discussed the current and future technologies in molecular diagnosis as well as the biomarkers developed for HIV infection. Expert Commentary: A simple and rapid detection of viral load is important for patients and doctors to monitor HIV progression and antiretroviral treatment efficiency. In the near future, it is expected that new technologies such as digital PCR and CRISPR-based technology will play more important role in HIV detection and patient management.

Jagged 2 silencing inhibits motility and invasiveness of colorectal cancer cell lines.

Although the Notch pathway has been reported to be activated in colorectal cancer (CRC), limited information is available regarding the expression and role of its ligand, Jagged 2 (JAG2), in CRC. Using immunohistochemistry, the present study demonstrated that JAG2 protein expression may be detected in up to 95% of CRC cases and is 3-fold upregulated in tumor cells compared to surrounding normal tissues. This finding suggests that JAG2 may have a role in the tumorigenicity of CRC. To further investigate the cellular functions of JAG2 expression in CRC, two different small interfering RNAs (siRNAs) were used to downregulate JAG2 expression in CRC cell lines (HCT116, DLD-1 and HT-29). The results indicated that JAG2 knockdown inhibits the motility and invasiveness of CRC cell lines without significantly affecting cell proliferation. These findings implicate JAG2 in promoting aggressiveness of CRC, and lay the foundation for its future development as a therapeutic target for the treatment of CRC.

TP53-induced glycolysis and apoptosis regulator promotes proliferation and invasiveness of nasopharyngeal carcinoma cells.

The TP53-induced glycolysis and apoptosis regulator (TIGAR) is the protein product of the p53 target gene, C12orf5. TIGAR blocks glycolysis and promotes cellular metabolism via the pentose phosphate pathway; it promotes the production of cellular nicotinamide adenine dinucleotide phosphate (NADPH), which leads to enhanced scavenging of intracellular reactive oxygen species, and inhibition of oxidative stress-induced apoptosis in normal cells. Our previous study identified a novel nucleoside analog that inhibited cellular growth and induced apoptosis in nasopharyngeal carcinoma (NPC) cell lines via downregulation of TIGAR expression. Furthermore, the growth inhibitory effects of c-Met tyrosine kinase inhibitors were ameliorated by the overexpression of TIGAR in the NPC cell lines. These results indicate a significant role for TIGAR expression in the survival of NPCs. The present study aimed to further define the function of TIGAR expression in NPC cells. In total, 36 formalin-fixed, paraffin-embedded NPC tissue samples were obtained for the immunohistochemical determination of TIGAR expression. The effects of TIGAR expression on cell proliferation, NADPH production and cellular invasiveness were also assessed in NPC cell lines. Overall, TIGAR was overexpressed in 27/36 (75%) of the NPC tissues compared with the adjacent non-cancer epithelial cells. Similarly, TIGAR overexpression was also observed in a panel of six NPC cell lines compared with normal NP460 hTert and Het1A cell lines. TIGAR overexpression led to increased cellular growth, NADPH production and invasiveness of the NPC cell lines, whereas a knockdown of TIGAR expression resulted in significant inhibition of cellular growth and invasiveness. The expression of the two mesenchymal markers, fibronectin and vimentin, was increased by TIGAR overexpression, but reduced following TIGAR-knockdown. The present study revealed that TIGAR overexpression led to increased cellular growth, NADPH production and invasiveness, and the maintenance of a mesenchymal phenotype, in NPC tissues.

Clinical significance of frizzled homolog 3 protein in colorectal cancer patients.

Frizzled homolog 3 receptor was up-regulated in several gastrointestinal cancers such as esophageal and gastric cancers. Moreover, frizzled homolog 3 has recently reported to be expressed in colorectal adenoma specimens. In the present study, we investigated the clinical significance of frizzled homolog 3 protein in colorectal cancer patients. Using immunocytochemical staining, frizzled homolog 3 expression was examined in 186 colorectal cancer specimens, 79 colorectal adenoma specimens, 133 colorectal polyp specimens, 127 colorectal cancer specimens with lymph node and/or distant metastasis, 310 specimens of various non-colorectal cancer metastatic carcinomas and 40 specimens with simultaneous occurrence of colorectal cancer, colorectal adenoma and colorectal polyp. Statistical analysis was used to correlate frizzled homolog 3 protein expression to the clinicohistopathological factors, recurrence/metastasis and survival after follow-up for 42 months in colorectal cancer patients. Frizzled homolog 3 protein was expressed in 100% colorectal cancer specimens, 89% colorectal adenoma specimens, 75% colorectal polyp specimens and 69% normal colorectal epithelial tissues. Moreover, frizzled homolog 3 immunocytochemical scores were highly correlated with colorectal cancer progression. Furthermore, frizzled homolog 3 was expressed in a comparatively lower percentage of metastatic hepatocellular carcinoma and metastatic renal clear cell carcinoma with focal and very weak staining than other metastatic tumor types. On the other hand, the frizzled homolog 3 immunocytochemical scores of colorectal adenomas with synchronous colorectal carcinomas were significantly higher than those of pure colorectal adenomas. Statistical analysis showed that frizzled homolog 3 immunocytochemical scores were associated with Dukes stage and lymph node status. Finally, stratified groups of colorectal cancer patients had significant differences in their recurrence/metastasis and survival. In conclusion, the present large-scale study has clearly showed that frizzled homolog 3 protein can generate clinically important information for colorectal cancer patients.

Advanced technologies for studying circulating tumor cells at the protein level.

Metastasis is the main cause of cancer death. As the tumor progresses, cells from the primary tumor site are shed into the bloodstream as circulating tumor cells (CTCs). Eventually, these cells colonize other organs and form distant metastases. It is therefore imperative that we gain a better understanding of the biological characteristics of CTCs for development of novel treatment modalities to minimize metastasis-associated cancer deaths. In recent years, rapid developments in technologies for the study of CTCs have taken place. We now have a variety of tools for the isolation and examination of CTCs which were not available before. This review introduces some commonly used protein markers in CTC investigations and summarizes a few advanced technologies which have been successfully applied for studying CTC biology at the protein level.

Human papillomavirus DNA detection in menstrual blood from patients with cervical intraepithelial neoplasia and condyloma acuminatum.

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases.

STAT3 activation contributes directly to Epstein-Barr virus-mediated invasiveness of nasopharyngeal cancer cells in vitro.

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)-associated head and neck cancer prevalent in Asia. Although with reasons not fully understood, the intrinsic invasiveness of NPC is believed to be EBV-linked. Recently, EBV was found to induce STAT3 activation. Constitutive STAT3 activation correlated with advanced clinical staging in NPC. We hypothesized that STAT3 activation by EBV directly contributes to the intrinsic invasiveness of NPC cells. Phospho-STAT3-Tyr705 was detected in high percentage of NPC tumors (7/10 cases). Using a paired NPC cell line model, HONE-1 and the EBV-infected counterpart, HONE-1-EBV, we found that HONE-1-EBV expressed a higher level of phospho-STAT3-Tyr705 and was approximately 11-fold more invasive than HONE-1. In HONE-1-EBV, STAT3 siRNA targeting inhibited both spontaneous and serum-induced invasion, as well as cell growth. Conversely, activation of STAT3 (by expressing an activated STAT3 mutant, namely STAT3C) in the parental HONE-1, mimicking EBV-induced STAT3 activation, significantly enhanced its invasiveness and proliferation, which was accompanied by increased expression of markers of mesenchymal status, proliferation and anti-apoptosis. Our results demonstrated that EBV-induced STAT3 activation is responsible for NPC cell proliferation and invasion. This was further confirmed by a small molecule inhibitor of JAK/STAT3, JSI-124. JSI-124 inhibited STAT3 activation in HONE-1-EBV, with subsequent growth inhibition, induction of PARP cleavage, abrogation of anchorage-independent growth and invasion. We found that EBV-independent activation of STAT3 by a growth factor, EGF, also contributed to NPC invasion. In conclusion, EBV-induced STAT3 activation directly contributes to the intrinsic invasiveness of NPC cells and STAT3 targeting may be beneficial in treating aggressive NPC.

Advanced proteomic technologies for cancer biomarker discovery.

Proteomic technologies have experienced major improvements in recent years. Such advances have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis, prognosis and treatment monitoring of cancer patients. This review will focus on four state-of-the-art proteomic technologies, namely 2D difference gel electrophoresis, MALDI imaging mass spectrometry, electron transfer dissociation mass spectrometry and reverse-phase protein array. The major advancements these techniques have brought about and examples of their applications in cancer biomarker discovery will be presented in this review, so that readers can appreciate the immense progress in proteomic technologies from 1997 to 2008. Finally, a summary will be presented that discusses current hurdles faced by proteomic researchers, such as the wide dynamic range of protein abundance, standardization of protocols and validation of cancer biomarkers, and a 5-year view of potential solutions to such problems will be provided.

Clinical significance of cytokeratin 20-positive circulating tumor cells detected by a refined immunomagnetic enrichment assay in colorectal cancer patients.

PURPOSE: Current immunomagnetic enrichment method can only detect general epithelial antigens of circulating tumor cells (CTC). Further characterization of the CTCs to provide specific information on the tumor type is not possible. We attempted to overcome this drawback by developing the methodology for using a gastrointestinal-specific anti-cytokeratin (CK) 20 antibody to detect CTCs in colorectal cancer patients' blood. EXPERIMENTAL DESIGN: The protocol was validated using a colorectal cancer SW480 cell line. The clinical significance of findings in colorectal cancer was investigated by detecting CK20-positive CTCs (pCTC) in patients with colorectal cancer, other common cancers, colorectal adenoma, benign colorectal diseases, and normal subjects. Moreover, the malignant nature of CK20 pCTCs was examined by comparing chromosome 17 aberration patterns with those from the corresponding primary tumors. RESULTS: The assay successfully showed CK20-positive SW480 cells. When applied in patient samples, the detection rates were 62% (132 colorectal cancer patients; median number = 11 CTCs), 0% (120 patients with other common cancers), 6% (50 colorectal adenoma patients), 0% (120 patients with benign colorectal diseases), and 0% (40 normal subjects). Furthermore, statistical analysis showed that CK20 pCTC numbers were associated with tumor-node-metastasis stage and lymph node status. Using the median CK20 pCTC numbers as the cutoff points, stratified groups of colorectal cancer patients had significant differences in their recurrence, metastasis, and survival. Finally, chromosome 17 aneusomy in 90% of colorectal cancer patients with CK20 pCTCs matched with those from the primary tumors. CONCLUSIONS: Detection of CK20 pCTCs using the new protocol could generate clinically important information for colorectal cancer patients.

Identification of 5-fluorouracil response proteins in colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry.

Colorectal cancer (CRC) is the second most prevalent cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) is a standard chemotherapeutic drug to treat CRC. However, the response rate is less than 20% and patients who have responded to 5-FU may become resistant. Therefore there is an urgent need to examine the 5-FU response proteins so that patients with no response to 5-FU can change to other treatment strategies promptly. In this study, the proteomic expression profile in a CRC cell line SW480 before and after 5-FU treatment was examined using 2-dimensional electrophoresis technology. Fourteen proteins with differential expression were identified using mass spectrometry and 7 of them were validated using immunocytochemical (ICC) staining. Protein identification indicated that cyclophilin A, cytokeratin 19 (CK19), cytokeratin 8 (CK8), ras-related nuclear protein, heat shock protein 27 (hsp27) and peroxiredoxin 6 (Prx 6) were upregulated whereas heat shock protein 60 (hsp60), cytokeratin 18 (CK18), cytokeratin 9 (CK9), carbamoylphosphate synthetase I, alpha-enolase, heat shock protein 70 (hsp70), nm23 and beta-actin were down-regulated. Seven of the 14 proteins detected were validated by ICC staining, which showed that the expression of hsp27, Prx 6 and hsp70 correlated with that from proteomics profiling. Our results suggest that hsp27, Prx 6 and hsp70 are potential 5-FU response proteins and they may represent potential targets for further evaluation in other 5-FU-sensitive and -resistant CRC cell lines.