Development of cancer biomarker assays from dna in various bodily fluids

Abstract DNA from cancer cells can enter surrounding fluid after apoptosis or necrosis. This DNA can be identified by sampling and sequencing the fluid looking for the mutations that gave rise to the cancer, referred to as a liquid biopsy. We have developed several liquid biopsies for the purpose of diagnosis and treatment selection through the use of targeted PCR and high throughput sequencing. We used the SafeSeqS methodology, a molecular barcoding technology previously developed in this lab, to further reduce sequencing errors to aid us in finding these rare mutations. We developed assays that identified genetic mutations from saliva, blood plasma, and pancreatic cysts. We investigating head and neck squamous cell carcinoma (HNSCC) from the oral cavity, oropharynx, hypopharynx, and larynx in 93 patients using a combination of saliva and plasma. We first identified either the E7 gene of HPV 16 or a primary mutation in the tumor and then tried to detect their presence in saliva and plasma. We collected saliva from every patient, while we only had plasma samples from 43 patients. We found that saliva performed best in oral cavity cancers detecting 100% of those patients, while plasma performed similarly across all sites detecting 87% of HNSCC patients. We detected mutations in 96% of patients when both saliva and plasma were available. Pancreatic cyst fluid was used to aid clinicians in the classification of pancreatic cysts. We created an 8 gene panel to look for mutations as well as additional tests for loss of heterozygosity and aneuploidy. We used DNA from fluid captured through endoscopic aspiration as well as from surgically resected cysts. When combined with the typical clinical features we were able to accurately predict which cysts needed surgery with a sensitivity and specificity of 89% and 69% respectively. Each of these projects began as an attempt to aid clinicians in dealing with these various diseases. Our results illustrate that liquid biopsies can be developed into an effective tool for the fight against cancer

Unrestricted somatic stem cells (USSC) from human umbilical cord blood display uncommitted epigenetic signatures of the major stem cell pluripotency genes

AbstractUnrestricted somatic stem cells (USSC) from human cord blood display a broad differentiation potential for ectodermal, mesodermal, and endodermal cell types. The molecular basis for these stem cell properties is unclear and unlike embryonic stem cells (ESC) none of the major stem cell factors OCT4, SOX2, and NANOG exhibits significant expression in USSC. Here, we report that these key stem cell genes hold an epigenetic state in between that of an ESC and a terminally differentiated cell type. DNA methylation analysis exhibits partial demethylation of the regulatory region of OCT4 and a demethylated state of the NANOG and SOX2 promoter/enhancer regions. Further genome-wide DNA methylation profiling identified a partially demethylated state of the telomerase gene hTERT. Moreover, none of the pluripotency factors exhibited a repressive histone signature. Notably, SOX2 exhibits a bivalent histone signature consisting of the opposing histone marks dimeH3K4 and trimeH3K27, which is typically found on genes that are "poised" for transcription. Consequently, ectopic expression of OCT4 in USSC led to rapid induction of expression of its known target gene SOX2. Our data suggest that incomplete epigenetic repression and a "poised" epigenetic status of pluripotency genes preserves the USSC potential to be able to react adequately to distinct differentiation and reprogramming cues

A methodology for exploring biomarker – phenotype associations: application to flow cytometry data and systemic sclerosis clinical manifestations

BACKGROUND: This work seeks to develop a methodology for identifying reliable biomarkers of disease activity, progression and outcome through the identification of significant associations between high-throughput flow cytometry (FC) data and interstitial lung disease (ILD) - a systemic sclerosis (SSc, or scleroderma) clinical phenotype which is the leading cause of morbidity and mortality in SSc. A specific aim of the work involves developing a clinically useful screening tool that could yield accurate assessments of disease state such as the risk or presence of SSc-ILD, the activity of lung involvement and the likelihood to respond to therapeutic intervention. Ultimately this instrument could facilitate a refined stratification of SSc patients into clinically relevant subsets at the time of diagnosis and subsequently during the course of the disease and thus help in preventing bad outcomes from disease progression or unnecessary treatment side effects. The methods utilized in the work involve: (1) clinical and peripheral blood flow cytometry data (Immune Response In Scleroderma, IRIS) from consented patients followed at the Johns Hopkins Scleroderma Center. (2) machine learning (Conditional Random Forests - CRF) coupled with Gene Set Enrichment Analysis (GSEA) to identify subsets of FC variables that are highly effective in classifying ILD patients; and (3) stochastic simulation to design, train and validate ILD risk screening tools. RESULTS: Our hybrid analysis approach (CRF-GSEA) proved successful in predicting SSc patient ILD status with a high degree of success (>82 % correct classification in validation; 79 patients in the training data set, 40 patients in the validation data set). CONCLUSIONS: IRIS flow cytometry data provides useful information in assessing the ILD status of SSc patients. Our new approach combining Conditional Random Forests and Gene Set Enrichment Analysis was successful in identifying a subset of flow cytometry variables to create a screening tool that proved effective in correctly identifying ILD patients in the training and validation data sets. From a somewhat broader perspective, the identification of subsets of flow cytometry variables that exhibit coordinated movement (i.e., multi-variable up or down regulation) may lead to insights into possible effector pathways and thereby improve the state of knowledge of systemic sclerosis pathogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12859-015-0722-x) contains supplementary material, which is available to authorized users

The potential of circulating tumor DNA (ctDNA) to reshape the design of clinical trials testing adjuvant therapy in patients with early stage cancers

BACKGROUND: The conventional approach to testing the benefit of adjuvant therapies in patients (pts) with relatively favorable prognoses is to follow a large number of pts for long periods of time, hoping that mature outcome data will document an improved outcome compared to control pts. We reasoned that the design of such trials could be improved if pts with minimal residual disease could be identified a priori through the presence of ctDNA, and the effects of adjuvant therapy then assessed through serial ctDNA assays. METHODS: We carried out a prospective trial in 231 pts with Stage II colon cancer. Serial plasma samples were collected every 3 months starting 4-10 weeks after surgery. Somatic mutations in pts' tumors were identified via sequencing of 15 genes commonly mutated in colon cancer. We then designed personalized assays to quantify ctDNA in plasma samples. Adjuvant chemotherapy was administered at clinician discretion, blinded to ctDNA analysis. RESULTS: Somatic mutations were identified in 230 (99.6%) of tumors. Matching ctDNA was detected in the immediate post-operative period in 14 of 178 (8%) pts not treated with chemotherapy, 11 of whom had recurred (79%) at a median follow-up of 27 months. In contrast, recurrence occurred in only 16 (10%) of the 164 pts with negative ctDNA not treated with chemotherapy (HR 15.66, log-rank P<0.0001). ctDNA was detected in the immediate post-operative period in 6 of 52 pts who went on to receive chemotherapy. The ctDNA status turned from positive to negative during adjuvant treatment phase in all 6 pts (100%) but became positive again following completion of chemotherapy in 2 pts, both of whom have recurred. In patients with serial samples available, the median lead-time between ctDNA detection and radiologic-recurrence was 167 days. CONCLUSIONS: Detection of ctDNA in pts with resected stage II colon cancer provides direct evidence of residual disease. As well as defining pts at very high risk of later radiologic-recurrence, serial ctDNA analysis may provide an early readout of adjuvant treatment effect. Including ctDNA analyses would increase the efficiency of clinical trials testing the benefit of adjuvant treatment

Feeding ecology of the deep-sea lanternshark Etmopterus pusillus (Elasmobranchii: Etmopteridae) in the northeast Atlantic

This study provides the first description of the feeding ecology of the smooth lanternshark Etmopterus pusillus based on stomach contents of specimens caught as bycatch in the Algarve (southern Portugal) with bottom trawling and bottom longline. The diet of E. pusillus consists mainly of fish (dry weight (% W)=87.1%; frequency of occurrence (%FO)=28.6%; number (%N)=30.3%), crustaceans (%W=7.7%; %FO=36.7%; %N=3.4%) and cephalopods (%W=4.7%; %FO=11.3%; %N=11.1%). The diet did not vary between sexes. Ontogenic changes were detected: crustaceans decreased in importance as the sharks increased in size and fish became dominant in the diet of adults. Combining two fishing methods provided broad information on the diet of E. pusillus, as bottom trawling caught smaller specimens and longlines caught larger individuals. E. pusillus feeds mainly on non-commercial species, and therefore does not compete directly with commercial fisheries. Finally, E. pusillus feeds in various parts of the water column and thus it can access a wide range of prey; however, this also means that it can be caught by both gears, making it more vulnerable in terms of conservation.info:eu-repo/semantics/publishedVersio

TERT promoter mutations occur early in urothelial neoplasia and are biomarkers of early disease and disease recurrence in urine

Activating mutations occur in the promoter of the telomerase reverse transcriptase (TERT) gene in 66% of muscle-invasive urothelial carcinomas. To explore their role in bladder cancer development and to assess their utility as urine markers for early detection, we sequenced the TERT promoter in 76 well-characterized papillary and flat noninvasive urothelial carcinomas, including 28 pTa low-grade transitional cell carcinomas (TCC), 31 pTa high-grade TCCs, and 17 pTis carcinoma in situ lesions. We also evaluated the sequence of the TERT promoter in a separate series of 14 early bladder neoplasms and matched follow-up urine samples to determine whether urine TERT status was an indicator of disease recurrence. A high rate of TERT promoter mutation was observed in both papillary and flat lesions, as well as in low- and high-grade noninvasive urothelial neoplasms (mean: 74%). In addition, among patients whose tumors harbored TERT promoter mutations, the same mutations were present in follow-up urines in seven of eight patients that recurred but in none of the six patients that did not recur (P < 0.001). TERT promoter mutations occur in both papillary and flat lesions, are the most frequent genetic alterations identified to date in noninvasive precursor lesions of the bladder, are detectable in urine, and seem to be strongly associated with bladder cancer recurrence. These provocative results suggest that TERT promoter mutations may offer a useful urinary biomarker for both early detection and monitoring of bladder neoplasia. ©2013 AACR