Improved risk stratification of nasopharyngeal cancer by targeted sequencing of Epstein-Barr virus DNA in post-treatment plasma.

BACKGROUND: Quantitative measurement of plasma Epstein-Barr virus (EBV) DNA by real-time PCR at the end of primary treatment is a robust prognostic marker for nasopharyngeal carcinoma (NPC) patients. However, up to 40% of patients who would later develop disease recurrence had undetectable post-treatment plasma EBV DNA. Targeted sequencing for the entire EBV genome potentially allows a more comprehensive and unbiased detection of plasma EBV DNA and enables the use of other parameters such as fragment size as biomarkers. Hence, we explored if plasma EBV DNA sequencing might allow more accurate prognostication of NPC patients. PATIENTS AND METHODS: Plasma samples collected from 769 patients with stage IIB-IVB NPC at 6-8 weeks after radiotherapy were analysed using targeted sequencing for EBV DNA. RESULTS: The sensitivities of the PCR-based analysis, at a cut-off of any detectable levels of plasma EBV DNA, for prediction of local and distant recurrences were 42.3% and 85.3%, respectively. The sequencing-based analysis (involving quantitation and size profiling) achieved better performance for both local and distant recurrences than PCR. Using a cut-off of the proportion of plasma EBV DNA deduced by sequencing at 0.01%, the sensitivities of the sequencing-based analysis for local and distant recurrences were 88.5% and 97.1%, with the resultant negative predictive values of 99.1% and 99.4%, respectively. Among patients with undetectable EBV DNA on quantitative PCR, sequencing could further define a subgroup that enjoyed superior survival outcomes based on the proportion of plasma EBV DNA, with a 5-year progression-free survival (PFS) approaching 90%. On multivariate analysis, sequencing-based quantitative level of plasma EBV DNA was the independent prognostic factor with the highest hazard ratio for prediction of overall survival and PFS. CONCLUSION: NPC prognostication using post-treatment plasma EBV DNA could be enhanced through sequencing.

What do we need to obtain high quality circulating tumor DNA (ctDNA) for routine diagnostic test in oncology? - Considerations on pre-analytical aspects by the IFCC workgroup cfDNA.

The analysis of circulating cell free DNA is an important tool for the analysis of tumor resistance, tumor heterogeneity, detection of minimal residual disease and detection of allograft rejection in kidney or heart transplant patients. The proper use of this technique is important, and starts with considering pre-analytic aspects. The current paper addresses some important technical considerations to ensure the proper and harmonized use of cfDNA techniques.

Integrating postradiotherapy plasma Epstein-Barr virus DNA and TNM stage for risk stratification of nasopharyngeal carcinoma to adjuvant therapy.

BACKGROUND: After curative radiotherapy (RT) or chemoradiation (CRT), there is no validated tool to accurately identify patients for adjuvant therapy in nasopharyngeal carcinoma (NPC). Post-RT circulating plasma Epstein-Barr virus (EBV) DNA can detect minimal residual disease and is associated with recurrence and survival independent of TNM (tumor-lymph node-metastasis) stage. We aimed to develop and validate a risk model for stratification of NPC patients after completion of RT/CRT to observation or adjuvant therapy. PATIENTS AND METHODS: The prospective multicenter 0502 EBV DNA screening cohort (Hong Kong NPC Study Group 0502 trial) enrolled from 2006 to 2015 (n = 745) was used for model development. For internal validation, we pooled independent patient cohorts from prospective clinical studies enrolled from 1997 to 2006 (n = 340). For external validation, we used retrospective cohort of NPC patients treated at Sun Yat-sen University Cancer Center from 2009 to 2012 (n = 837). Eligible patients had histologically confirmed NPC of Union for International Cancer Control (UICC) 7th Edition stage II-IVB who completed curative RT/CRT with or without neoadjuvant chemotherapy, had post-RT EBV DNA tested within 120 days after RT and received no adjuvant therapy. The primary end point was overall survival (OS). We used recursive-partitioning analysis (RPA) to classify patients into groups of low, intermediate, and high risk of death. RESULTS: Combining post-RT EBV DNA level (0, 1-49, 50-499, and ≥500 copies/ml) and TNM stage (II, III, IVAB), RPA model classified patients into low-, intermediate-, and high-risk groups with 5-year OS of 89.4%, 78.5% and 37.2%, respectively. The RPA low-risk group had comparable OS to TNM stage II (5-year OS 88.5%) but identified more patients (64.8% versus stage II 28.1%) that could potentially be spared adjuvant therapy toxicity. The RPA model (c-index 0.712) showed better risk discrimination than either the TNM stage (0.604) or post-RT EBV DNA alone (0.675) with improved calibration and consistence. These results were validated in both internal and external cohorts. CONCLUSION: Combining post-RT EBV DNA and TNM stage improved risk stratification in NPC.

Early Detection of Cancer: Evaluation of MR Imaging Grading Systems in Patients with Suspected Nasopharyngeal Carcinoma.

BACKGROUND AND PURPOSE: We evaluated modifications to our contrast-enhanced MR imaging grading system for symptomatic patients with suspected nasopharyngeal carcinoma, aimed at improving discrimination of early-stage cancer and benign hyperplasia. We evaluated a second non-contrast-enhanced MR imaging grading system for asymptomatic patients from nasopharyngeal carcinoma plasma screening programs. MATERIALS AND METHODS: Dedicated nasopharyngeal MR imaging before (plain scan system) and after intravenous contrast administration (current and modified systems) was reviewed in patients from a nasopharyngeal carcinoma-endemic region, comprising 383 patients with suspected disease without nasopharyngeal carcinoma and 383 patients with nasopharyngeal carcinoma. The modified and plain scan systems refined primary tumor criteria, added a nodal assessment, and expanded the system from 4 to 5 grades. The overall combined sensitivity and specificity of the 3 systems were compared using the extended McNemar test (a χ2 value [Formula: see text]> 5.99 indicates significance). RESULTS: The current, modified, and plain scan MR imaging systems yielded sensitivities of 99.74%, 97.91%, and 97.65%, respectively, and specificities of 63.45%, 89.56% and 86.42%, respectively. The modified system yielded significantly better performance than the current ([Formula: see text] = 122) and plain scan ([Formula: see text] = 6.1) systems. The percentages of patients with nasopharyngeal carcinoma in grades 1-2, grade 3, and grades 4-5 for the modified and plain scan MR imaging systems were 0.42% and 0.44%; 6.31% and 6.96%; and 90.36% and 87.79%, respectively. No additional cancers were detected after contrast administration in cases of a plain scan graded 1-2. CONCLUSIONS: We propose a modified MR imaging grading system that improves diagnostic performance for nasopharyngeal carcinoma detection. Contrast was not valuable for low MR imaging grades, and the plain scan shows potential for use in screening programs.

Complementary roles of MRI and endoscopic examination in the early detection of nasopharyngeal carcinoma.

BACKGROUND: Early-stage nasopharyngeal carcinoma (NPC) evades detection when the primary tumor is hidden from view on endoscopic examination. Therefore, in a prospective study of subjects being screened for NPC using plasma Epstein-Barr virus (EBV) DNA, we conducted a study to investigate whether magnetic resonance imaging (MRI) could detect endoscopically occult NPC. PATIENTS AND METHODS: Participants with persistently positive EBV DNA underwent endoscopic examination and biopsy when suspicious for NPC, followed by MRI blinded to the endoscopic findings. Participants with a negative endoscopic examination and positive MRI were recalled for biopsy or surveillance. Diagnostic performance was assessed by calculating sensitivity, specificity and accuracy, based on the histologic confirmation of NPC in the initial study or in a follow-up period of at least two years. RESULTS: Endoscopic examination and MRI were performed on 275 participants, 34 had NPC, 2 had other cancers and 239 without cancer were followed-up for a median of 36 months (24-60 months). Sensitivity, specificity and accuracy were 76.5%, 97.5% and 94.9%, respectively, for endoscopic examination and 91.2%, 97.5% and 96.7%, respectively, for MRI. NPC was detected only by endoscopic examination in 1/34 (2.9%) participants (a participant with stage I disease), and only by MRI in 6/34 (17.6%) participants (stage I = 4, II = 1, III = 1), two of whom had stage I disease and follow-up showing slow growth on MRI but no change on endoscopic examination for 36 months. CONCLUSION: MRI has a complementary role to play in NPC detection and can enable the earlier detection of endoscopically occult NPC.

Plasma Epstein-Barr viral DNA load at midpoint of radiotherapy course predicts outcome in advanced-stage nasopharyngeal carcinoma.

BACKGROUND: To test the hypothesis that prognostication of treatment outcome is feasible by biomarker response at midcourse of chemoradiotherapy (CRT)/radiotherapy (RT), with respect to the plasma load of Epstein-Barr viral (EBV) DNA in nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS: One hundred seven patients with stage IIB-IV NPC were prospectively studied. Plasma EBV DNA load was measured by quantitative PCR before therapy (pre-DNA), at completion of 4 weeks of CRT/RT (mid-DNA), and within 3 months of completion of therapy (post-DNA). The end points are post-DNA load, a recognized surrogate of survival, and clinical outcome. RESULTS: Ninety-three percent of patients had detectable EBV DNA before therapy (median load = 972 copies/ml). EBV DNA became undetectable in 55 (51%) patients at the end of week 4 of therapy. Detectable mid-DNA was associated with worse clinical outcome (median follow-up time, 6.2 years), for distant failure [hazard ratio (HR) 12.02, 95% confidence interval (CI) 2.78-51.93; P < 0.0001], progression-free survival (PFS; HR 4.05, 95% CI 1.89-8.67, P < 0.0001), and overall survival (OS; HR 3.29, 95% CI 1.37-7.90, P = 0.0077). Seventy-four percent of all failures were associated with detectable mid-DNA, whereas 34% of all failures were associated with detectable post-DNA. Stratification by tumor stage (IIB, III, IV) has no significant prognostic effect. CONCLUSIONS: Unfavorable EBV DNA response at midcourse of RT/CRT is an adverse prognosticator for treatment outcome, is linked to majority of all failures, and discriminates outcome better than tumor stage. The data could provide a basis for trial design that addresses alteration of therapy intensity during the latter phase of CRT, and adjuvant therapy. Validation studies are awaited.

Cancer genome scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing.

BACKGROUND: Tumor-derived DNA can be found in the plasma of cancer patients. In this study, we explored the use of shotgun massively parallel sequencing (MPS) of plasma DNA from cancer patients to scan a cancer genome noninvasively. METHODS: Four hepatocellular carcinoma patients and a patient with synchronous breast and ovarian cancers were recruited. DNA was extracted from the tumor tissues, and the preoperative and postoperative plasma samples of these patients were analyzed with shotgun MPS. RESULTS: We achieved the genomewide profiling of copy number aberrations and point mutations in the plasma of the cancer patients. By detecting and quantifying the genomewide aggregated allelic loss and point mutations, we determined the fractional concentrations of tumor-derived DNA in plasma and correlated these values with tumor size and surgical treatment. We also demonstrated the potential utility of this approach for the analysis of complex oncologic scenarios by studying the patient with 2 synchronous cancers. Through the use of multiregional sequencing of tumoral tissues and shotgun sequencing of plasma DNA, we have shown that plasma DNA sequencing is a valuable approach for studying tumoral heterogeneity. CONCLUSIONS: Shotgun DNA sequencing of plasma is a potentially powerful tool for cancer detection, monitoring, and research.

Detection of circulating fetal nucleic acids: a review of methods and applications.

The discovery of cell-free circulating fetal nucleic acids in maternal plasma has opened up new possibilities in non-invasive prenatal diagnosis. The rapid advancement of this field in the past decade is catalysed by the discovery of new classes of fetal nucleic acid markers and technological developments in nucleic acid detection and amplification. In this review, some of the more significant recent developments in this field will be discussed, including the detection of single molecule, chromosomal aneuploidies, single nucleotide variations and placental microRNAs in maternal plasma.

Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art.

Fetal nucleic acids in maternal plasma have opened up new possibilities for noninvasive prenatal diagnosis of chromosomal aneuploidies. One approach is based on the measurement of the allelic ratio of single nucleotide polymorphisms in the coding region of placental mRNA. Another approach is through the analysis of DNA fragments with different patterns of DNA methylation between fetal and maternal DNA. One other alternative is to enrich the fractional concentration of fetal DNA in maternal plasma using physical or chemical methods. Finally, the development of more precise digital polymerase chain reaction-based methods for fetal nucleic acid analysis might further catalyse the developments in this area. It is hoped that plasma-based molecular prenatal diagnosis might ultimately make prenatal testing safer for pregnant women and their fetuses.

Circulating tumour-derived nucleic acids in cancer patients: potential applications as tumour markers.

Tumour-associated changes have been observed in the circulating nucleic acids of cancer patients and have been proposed to be useful for the detection and monitoring of cancers. In this review, different approaches for detecting tumour-associated nucleic acids in the circulation and their potential applications as tumour markers are discussed.

Evidence of circulating donor genetic material in bone allotransplantation.

Musculoskeletal allotransplantion is the most common form of human tissue transplantation. Unlike solid organ transplants, bone allotransplants undergo rigorous processing and are considered non-viable tissue. In this study, we propose that donor genetic material may exist in circulation after bone allotransplantation. Fifty-one female patients who received bone allotransplants from male donors were assessed. Blood plasma samples were analyzed using real-time quantitative polymerase chain reaction (PCR) with dual labeled fluorogenic probes for the presence of the SRY gene on the Y chromosome. Of the total 51 patients, the SRY sequence was detected in 6 patients. Five were positive at day 1 postoperatively and negative thereafter, with the remaining patient positive at 3 months post-transplantation. Our results document, for the first time, the presence of donor DNA in the circulation of recipients after bone allotransplantation. Our findings suggest a potential new investigative tool to assess the postoperative status of bone allotransplants.

Recent developments in fetal nucleic acids in maternal plasma: implications to noninvasive prenatal fetal blood group genotyping.

The discovery of circulating cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Fetal DNA in maternal plasma has been used for the noninvasive prenatal determination of the RhD status of fetuses carried by RhD-negative pregnant women. In such analysis, the possible need of an internal control for the presence of detectable amounts of fetal DNA in a particular maternal plasma sample has been actively discussed. Recently, the development of a robust method for discriminating single nucleotide differences in plasma DNA using single allele base extension reaction (SABER) followed by matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has opened up the possibilities of using a panel of single nucleotide polymorphisms as such a positive control. A second approach is the recent successful development of fetal epigenetic markers which can be developed into universal fetal DNA markers. These developments hold promise to allow the eventual widespread utilization of maternal plasma DNA analysis for the noninvasive prenatal diagnosis of blood group mismatches between the mother and fetus.

Nuclear beta-catenin and Ki-67 expression in choriocarcinoma and its pre-malignant form.

OBJECTIVE: To study the expression of nuclear beta-catenin and Ki-67 in patients with normal gestation products (NGP), complete hydatidiform moles (CHM), and choriocarcinoma to elucidate their roles in carcinogenesis and their interrelations. METHODS: Expression of nuclear beta-catenin and Ki-67 was studied by immunohistochemistry using paraffin embedded blocks. Sixty NGP, 60 CHM, and 10 choriocarcinomas were analysed. In addition, approximately 400 trophoblasts each in 40 NGP, 40 CHM, and 10 choriocarcinomas from the same batch of samples were microdissected for quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) to compare beta-catenin mRNA concentration among them. RESULTS: In the chorionic villi of NGP, beta-catenin was consistently expressed in the nuclei of cytotrophoblasts but not syncytiotrophoblasts. Nuclear beta-catenin expression was comparatively reduced in CHM trophoblasts and was absent in choriocarcinoma. By contrast, Ki-67 expression was increased from cytotrophoblasts but not in syncytiotrophoblasts in the chorionic villi of NGP to CHM trophoblasts and choriocarcinoma. Using Q-RT-PCR, beta-catenin mRNA was detected in 10 NGP, 13 CHM, and three choriocarcinoma specimens, with median copy numbers of 43,230, 18,229, and 17,334 per 400 trophoblasts, respectively. A housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was detected as a control in the NGP, CHM, and choriocarcinoma specimens, with median copy numbers of 51,300, 54,270, and 97,150 per 400 trophoblasts, respectively. Thus median beta-catenin mRNA values after normalisation were 0.85 in NGP (n = 10), 0.31 in CHM (n = 13), and 0.16 in choriocarcinoma (n = 3). CONCLUSIONS: Decreased nuclear beta-catenin expression and increased Ki-67 expression may be involved in choriocarcinoma carcinogenesis. The findings also suggest that nuclear beta-catenin may play a role in trophoblast differentiation during normal placental development.

Nuclear beta-catenin expression is rare and its potential association with short survival in colorectal signet-ring cell carcinoma.

Colorectal signet-ring cell carcinoma (SRCC) is a rare cancer and the prognosis is usually very poor. The biologic pathways involved in its oncogenesis are unknown. beta-catenin, a key target in the Wnt-signaling pathway, is recognized to play an important role in the carcinogenesis in conventional colorectal carcinoma. This study explores the involvement of Wnt-signaling molecules beta-catenin and cyclin D1, cell cycle regulators cyclin D3, proliferative index Ki-67, apoptotic index, and angiogenic indicator CD31 in 20 colorectal SRCC paraffin-embedded specimens. Results showed that there were 2 specimens with nuclear beta-catenin and higher expression of cyclin D1 than the remaining 18 specimens. Surprisingly, those 2 patients had a much shorter survival of 6 months than the remaining 15 patients, who had around 24 months. Moreover, all colorectal SRCC specimens had an overexpression of cyclin D1, cyclin D3, and Ki-67, as well as much more angiogenesis and apoptosis than adjacent normal epithelial tissues. The authors make the preliminary comment that nuclear beta-catenin is a rare phenomenon in colorectal SRCC, but the involvement of it may indicate a worse prognosis with shorter survival than colorectal SRCC without nuclear beta-catenin expression. Besides, overexpression of cyclin D1, cyclin D3, Ki-67, and increased angiogenesis and apoptosis may play a vital role in promoting colorectal SRCC development.

Circulating nucleic acid analysis: diagnostic applications for acute pathologies.

Much research interest has been shown in recent years for the development of molecular diagnostic strategies based on the analysis of DNA/RNA molecules that are present in the plasma/serum of human subjects. Reported applications include the diagnosis, prognostication or monitoring of malignancies and pregnancy-associated complications. While researchers have speculated that cell death is a potential mechanism that leads to the release of DNA/RNA into the circulation, studies have demonstrated that indeed increased amounts of plasma DNA and RNA could be detected in patients sustaining acute traumatic injuries. The degree of plasma DNA elevation correlated with the severity of injury. Similarly, plasma DNA concentrations have been shown to correlate with indices of prognostic significance in patients with acute stroke. It is expected that new diagnostic markers based on plasma RNA detection could be developed for the evaluation of acute pathologies.

Quantitation of circulating DNA in the serum of breast cancer patients by real-time PCR.

The purpose of this study was to quantify the level of serum DNA in different groups of primary breast cancer patients and in healthy controls using real-time quantitative PCR in order to determine whether such measurements have diagnostic or prognostic value. A total of 96 serum samples of patients with primary breast cancer before surgery (with positive or negative lymph nodes and with high or low relapse-free survival) as well as 24 healthy controls were analysed. DNA concentrations in the serum of the patients differed significantly from the concentration of serum DNA in the controls (medians were 221 and 63 ng x ml(-1), respectively, P<0.001 M-W test). However, no statistically significant difference was observed between the patient groups (P=0.87, M-W test). The serum DNA levels were elevated independently of the size of primary tumour or lymph node metastases. The overall survival of patients with serum DNA concentrations >221 ng x ml(-1) was better than patients with serum DNA concentration

Genomic characterisation of the severe acute respiratory syndrome coronavirus of Amoy Gardens outbreak in Hong Kong.

Severe acute respiratory syndrome (SARS) is a global health concern. In Hong Kong, two major outbreaks, one hospital based and the other in the Amoy Gardens apartments, were identified. The frequency of diarrhoea, admission to intensive care, and mortality differed significantly between the two outbreaks. We did genomic sequencing for viral isolates from five Amoy Gardens patients. The virus sequence was identical in four of these five patients. The sequence data from one hospital case and the four identical community cases had only three nucleotide differences. Alterations in the SARS coronavirus genome are unlikely to have caused the distinctive clinical features of the Amoy Gardens patients, and these results highlight the importance of non-viral genomic factors in this outbreak.