Combined EsophaCap cytology and MUC2 immunohistochemistry for screening of intestinal metaplasia, dysplasia and carcinoma.

Purpose: The incidence of esophageal adenocarcinoma (EAC) has increased by 700% in Western countries over the last 30 years. Although clinical guidelines call for endoscopic surveillance for EAC among high-risk populations, fewer than 5% of new EAC patients are under surveillance at the time of diagnosis. We studied the accuracy of combined cytopathology and MUC2 immunohistochemistry (IHC) for screening of Intestinal Metaplasia (IM), dysplasia and EAC, using specimens collected from the EsophaCap swallowable encapsulated cytology sponge from Canada and United States. Patients and methods: By comparing the EsophaCap cytological diagnosis with concurrent endoscopic biopsies performed on the same patients in 28 cases, we first built up the cytology diagnostic categories and criteria. Based on these criteria, 136 cases were evaluated by both cytology and MUC2 IHC with blinded to patient biopsy diagnosis. Results: We first set up categories and criteria for cytological diagnosis of EscophaCap samples. Based on these, we divided our evaluated cytological samples into two groups: non-IM group and IM or dysplasia or adenocarcinoma group. Using the biopsy as our gold standard to screen IM, dysplasia and EAC by combined cytology and MUC2 IHC, the sensitivity and specificity were 68% and 91%, respectively, which is in the range of clinically useful cytological screening tests such as the cervical Pap smear. Conclusions: Combined EsophaCap cytology and MUC2 IHC could be a good screening test for IM and Beyond.

Corrigendum to "Considerations and quality controls when analyzing cell-free tumor DNA" [Biomol. Detect. Quantif. 17 (2019) 100078].

[This corrects the article DOI: 10.1016/j.bdq.2018.12.003.].

Detection of Circulating Tumor DNA in Plasma: A Potential Biomarker for Esophageal Adenocarcinoma.

BACKGROUND: Recent literature has demonstrated the potential of "liquid biopsy" and detection of circulating tumor (ct)DNA as a cancer biomarker. However, to date there is a lack of data specific to esophageal adenocarcinoma (EAC). This study was conducted to determine how detection and quantification of ctDNA changes with disease burden in patients with EAC and evaluate its potential as a biomarker in this population. METHODS: Blood samples were obtained from patients with stage I to IV EAC. Longitudinal blood samples were collected from a subset of patients. Imaging studies and pathology reports were reviewed to determine disease course. Tumor samples were sequenced to identify mutations. Mutations in plasma DNA were detected using custom, barcoded, patient-specific sequencing libraries. Mutations in plasma were quantified, and associations with disease stage and response to therapy were explored. RESULTS: Plasma samples from a final cohort of 38 patients were evaluated. Baseline plasma samples were ctDNA positive for 18 patients (47%) overall, with tumor allele frequencies ranging from 0.05% to 5.30%. Detection frequency of ctDNA and quantity of ctDNA increased with stage. Data from longitudinal samples indicate that ctDNA levels correlate with and precede evidence of response to therapy or recurrence. CONCLUSIONS: ctDNA can be detected in plasma of EAC patients and correlates with disease burden. Detection of ctDNA in early-stage EAC is challenging and may limit diagnostic applications. However, our data demonstrate the potential of ctDNA as a dynamic biomarker to monitor treatment response and disease recurrence in patients with EAC.

The Molecular Biologic Basis of Esophageal and Gastric Cancers.

Esophageal cancer and gastric cancer are leading causes of cancer-related mortality worldwide. In this article, the authors discuss the molecular biology of esophageal and gastric cancer with a focus on esophageal adenocarcinoma. They review data from The Cancer Genome Atlas project and advances in the molecular stratification and classification of esophageal carcinoma and gastric cancer. They also summarize advances in microRNA, molecular staging, gene expression profiling, tumor microenvironment, and detection of circulating tumor DNA. Finally, the authors summarize some of the implications of understanding the molecular basis of esophageal cancer and future directions in the management of esophageal cancer.

Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements.

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.

Considerations and quality controls when analyzing cell-free tumor DNA.

Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.

Impact of Polymerase Fidelity on Background Error Rates in Next-Generation Sequencing with Unique Molecular Identifiers/Barcodes.

Liquid biopsy and detection of tumor-associated mutations in cell-free circulating DNA often requires the ability to identify single nucleotide variants at allele frequencies below 0.1%. Standard sequencing protocols cannot achieve this level of sensitivity due to background noise from DNA damage and polymerase induced errors. Addition of unique molecular identifiers allows identification and removal of errors responsible for this background noise. Theoretically, high fidelity enzymes will also reduce error rates in barcoded NGS but this has not been thoroughly explored. We evaluated the impact of polymerase fidelity on the magnitude of error reduction at different steps of barcoded NGS library construction. We find that barcoding itself displays largest impact on error reduction, even with low fidelity polymerases. Use of high fidelity polymerases in the barcoding step of library construction further suppresses error in barcoded NGS, and allows detection of variant alleles below 0.1% allele frequency. However, the improvement in error correction is modest and is not directly proportional to polymerase fidelity. Depending on the specific application, other polymerase characteristics such as multiplexing capacity, PCR efficiency, buffer requirements and ability to amplify targets with high GC content may outweigh the relatively small additional decrease in error afforded by ultra-high fidelity polymerases.

Plasma circulating tumor DNA as a potential tool for disease monitoring in head and neck cancer.

BACKGROUND: Recommendations for perioperative therapy in head and neck cancer are not explicit and recurrence occurs frequently. Circulating tumor DNA is an emerging cancer biomarker, but has not been extensively explored for detection of recurrence in head and neck cancer. METHODS: Patients diagnosed with head and neck squamous cell carcinoma were recruited into the study protocol. Tumors were sequenced to identify patient-specific mutations. Mutations were then identified in plasma circulating tumor DNA from pre-treatment blood samples and longitudinally during standard follow-up. Circulating tumor DNA status during follow-up was correlated to disease recurrence. RESULTS: Samples were taken from eight patients. Tumor mutations were verified in seven patients. Baseline circulating tumor DNA was positive in six patients. Recurrence occurred in four patients, two of whom had detectable circulating tumor DNA prior to recurrence. CONCLUSION: Circulating tumor DNA is a potential tool for disease and recurrence monitoring following curative therapy in head and neck cancer, allowing for better prognostication, and/or modification of treatment strategies.

miRNA profiling of primary lung and head and neck squamous cell carcinomas: Addressing a diagnostic dilemma.

OBJECTIVE: To determine whether microRNA (miRNA) profiling of primary lung and head and neck squamous cell carcinomas could be useful to identify a specific miRNA signature that can be used to further discriminate between primary lung squamous carcinomas and metastatic lesions in patients with a history of head and neck squamous cell cancer. METHODS: Specimens of resected primary head and neck and lung squamous cell carcinomas were obtained from formalin-fixed, paraffin-embedded blocks. Paraffin blocks were sectioned and deparaffinized, and total RNA was isolated and profiled. Quantitative polymerase chain reaction was performed to verify array results. RESULTS: Twelve head and neck and 16 lung squamous cell carcinoma samples met quality control metrics and were included for analysis. Forty-eight miRNAs were differentially expressed (P < .05) between the 2 groups. Of these, 30 were also significantly associated (q < .25) with tumor type in 2 independent sets of primary head and neck and lung squamous carcinomas profiled by The Cancer Genome Atlas consortium, including miR-34a and miR-10a. The ratio of miR-10a and miR-10b was especially predictive of primary cancer site in all 3 data sets, with area under the (receiver operating characteristics) curve values ranging from 0.922 to 0.982. Quantitative polymerase chain reaction confirmed the association of miR-34a expression and the miR-10:miR-10b ratio with tumor type. CONCLUSIONS: MicroRNA expression may be useful for discriminating between head and neck and lung squamous cell carcinomas, including miR-34a and the miR-10a:miR-10b ratio. This differentiation has clinical importance because it could help determine the appropriate therapeutic approach.

Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing.

Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

Bile salt receptor TGR5 is highly expressed in esophageal adenocarcinoma and precancerous lesions with significantly worse overall survival and gender differences.

Bile acid reflux in the esophagus plays an important role in the carcinogenesis of esophageal adenocarcinoma (EAC). The G-protein coupled bile acid receptor (TGR5) has been associated with the development of gastrointestinal cancer. However, little is known regarding the role of TGR5 in esophageal carcinoma and precancerous lesions. We analyzed genomic DNA from 116 EACs for copy number aberrations via Affymetrix SNP6.0 microarrays. The TGR5 gene locus was amplified in 12.7% (14/116) of the EACs. The TGR5 protein expression was also assessed using immunohistochemistry from tissue microarrays, including Barrett's esophagus (BE), low-(LGD) and high-grade dysplasia (HGD), columnar cell metaplasia (CM), squamous epithelium (SE), EAC and squamous cell carcinoma. The TGR5 protein was highly expressed in 71% of EAC (75/106), 100% of HGD (11/11), 72% of LGD (13/18), 66% of BE (23/35), 84% of CM (52/62), and 36% of SE (30/83). The patients with high expression of TGR5 exhibited significantly worse overall survival compared to the patients with nonhigh expression. TGR5 high expression was significantly increased in the males compared to the females in all cases with an odds ratio of 1.9 times. The vitamin D receptor (VDR) was significantly correlated with TGR5 expression. Our findings indicated that TGR5 may play an important role in the development and prognosis of EAC through a bile acid ligand. Gender differences in TGR5 and VDR expression may explain why males have a higher incidence of EAC compared to females.

Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR.

Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications.

Bile Salts at Low pH Cause Dilation of Intercellular Spaces in In Vitro Stratified Primary Esophageal Cells, Possibly by Modulating Wnt Signaling.

BACKGROUND: The presence of dilated intercellular spaces in the stratified squamous lining of the esophagus is the pathognomonic feature of reflux esophagitis secondary to gastroesophageal reflux disease (GERD). In addition to stomach acid, bile salts are major constituents of gastroesophageal refluxate. The aim of our study was to determine the effect of bile salts cocktail at different pHs on epithelial junctions in an in vitro transwell model of stratified esophageal squamous epithelium. DISCUSSION: Human telomerase reverse transcriptase (hTERT) immortalized primary esophageal EPC1 cells were grown on polyester transwell surfaces in calcium-enriched media. The cells exhibited gradual stratification into an 11-layered squamous epithelium over 7 days, together with epithelial barrier function as indicated by increased transepithelial electrical resistance (TEER). This stratified epithelium demonstrated well-formed tight junctions, adherens junctions, and desmosomes as visualized by immunofluorescence and electron microscopy. When exposed to short pulses of bile salts at pH 5, but not either condition alone, there was loss of stratification and decrease in TEER, concomitant with disruption of adherens junctions, tight junctions, and desmosomes, leading to the appearance of dilated intercellular spaces. At the cellular level, bile salts at pH 5 activated the Wnt pathway (indicated by increased ß-catenin Ser552 phosphorylation). CONCLUSION: In conclusion, in our in vitro transwell model bile salts at pH 5, but not bile salts or media at pH 5 alone, modulate Wnt signaling, disrupt different junctional complexes, and cause increased permeability of stratified squamous esophageal epithelium. These changes approximate the appearance of dilated intercellular space similar to that found in GERD patients.

Properties of targeted preamplification in DNA and cDNA quantification.

OBJECTIVE: Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). METHODS: To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. RESULT: The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. CONCLUSION: On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

Vitamin D receptor is highly expressed in precancerous lesions and esophageal adenocarcinoma with significant sex difference.

Bile acid reflux into the esophagus is important in the development of esophageal adenocarcinoma (EAC). Recently, vitamin D receptor (VDR) was recognized as a bile acid receptor as well as a vitamin receptor. Expression of VDR is reported to influence the development of various types of cancer, such as those of the breast, liver, and colon. However, little is known about the role of VDR in esophageal neoplasms. We investigated the clinicopathological role of VDR in esophageal tumors. We analyzed genomic DNA from 116 EACs for copy number aberrations. The VDR locus was amplified in 7% of EACs. Expression of the VDR protein was also detected by immunohistochemistry from tissue microarrays created from tissues of Barrett esophagus (BE), low-grade (LGD) and high-grade dysplasia (HGD), columnar cell metaplasia (CCM), squamous epithelium (SE), EAC, and esophageal squamous cell carcinoma (ESCC). The protein was highly expressed in 88% of CCM (58/66), 95% of BE (35/37), 100% of the 19 LGD, 94% of HGD (15/16), and 79% of EAC (86/109), but expression in SE and ESCC was rare. Female patients with EAC and CCM were significantly less likely to have high VDR expression than male patients. The overall survival rate was significantly different for patients with tumors exhibiting VDR amplification versus nonamplification. Our findings suggest that VDR plays a role in the early development of EAC through a bile acid ligand. The sex difference in VDR expression may help to explain why men have a high incidence of EAC.

Cyclin E involved in early stage carcinogenesis of esophageal adenocarcinoma by SNP DNA microarray and immunohistochemical studies.

BACKGROUND: Cyclin E is a cell cycle regulator which is critical for driving G1/S transition. Abnormal levels of cyclin E have been found in many cancers. However, the level changes of cyclin E in esophageal adenocarcinoma and its precancerous lesion have not been well studied. Here, we focus on the gene amplification and expression of cyclin E in these lesions, and aim to ascertain the relationship with clinicopathological characteristics. METHODS: Genomic DNA was analyzed from 116 esophageal adenocarcinoma and 26 precancerous lesion patients using Affymetrix SNP 6.0 arrays. The protein overexpression of cyclin E was also detected using immunohistochemistry from tissue microarrays containing esophageal adenocarcinoma and precancerous lesions. Patient survival and other clinical data were collected and analyzed. The intensity and percentage of the cyclin E expressing cells in tissue microarrays were scored by two pathologists. Fisher exact tests and Kaplan-Meier methods were used to analyze data. RESULTS: By genomic analysis, cyclin E was amplified in 19.0% of the EAC samples. By immunohistochemistry, high expression of cyclin E was observed in 2.3% of squamous mucosa tissues, 3.7% in columnar cell metaplasia, 5.8% in Barrett's esophagus, 19.0% in low grade dysplasia, 35.7% in high grade dysplasia, and 16.7% in esophageal adenocarcinoma. The differences in cyclin E high expression between neoplastic groups and non-dysplasia groups are statistically significant (p < 0.05). The prognosis for patients with high cyclin E expression appeared slightly better than for those with low cyclin E expression although this was not statistically significant (p = 0.13). CONCLUSIONS: The expression of cyclin E significantly increases from non-dysplasia esophageal lesion to low and high grade dysplasia, suggesting that cyclin E plays an important role in the early stage of carcinogenesis. Importantly, cyclin E is also amplified and highly expressed in a subset of esophageal adenocarcinoma patients, but this increase is not associated with worse prognosis.

Comparison of cancer-associated genetic abnormalities in columnar-lined esophagus tissues with and without goblet cells.

OBJECTIVE: To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells. BACKGROUND: Barrett's esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barrett's esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC. METHODS: Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing. RESULTS: Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples. CONCLUSIONS: This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.

Bile acid at low pH reduces squamous differentiation and activates EGFR signaling in esophageal squamous cells in 3-D culture.

BACKGROUND: Barrett's esophagus is a preneoplastic metaplasia in which the normal squamous epithelium of the esophagus changes to an intestinal, columnar phenotype due to long-term gastro-esophageal reflux. The major components of this reflux are bile and stomach acid. Previous in vitro studies on the effect of bile and acid on esophageal cells have predominantly relied on transformed esophageal squamous cells or cancer cells grown in monolayer culture. DISCUSSION: In this study, we expanded our previous work using an immortalized primary esophageal squamous cell line (EPC1). We demonstrate that EPC1 cells form a multi-layer, stratified epithelium when grown on polyester transwell filters in media supplemented with calcium. When exposed to short pulses of bile and pH 5, but not either condition alone, EPC1 cells demonstrate a reduction in stratification layers and reduced expression of squamous epithelium-specific genes. Bile at pH 5 also causes activation of epidermal growth factor receptor and down-stream pathways. Blocking epidermal growth factor receptor activation partially attenuates the effects of bile acid and pH 5. These results suggest that bile at low pH, but not bile or low pH alone, promotes loss of differentiation status of stratified squamous esophageal epithelium in vitro, possibly by initiating a mucosal repair response through epidermal growth factor activation.