Kevin D. Staton

Quantification of nanoscale nuclear refractive index changes during the cell cycle

Intrigued by our recent finding that the nuclear refractive index is significantly increased in malignant cells and histologically normal cells in clinical histology specimens derived from cancer patients, we sought to identify potential biological mechanisms underlying the observed phenomena. The cell cycle is an ordered series of events that describes the intervals of cell growth, DNA replication, and mitosis that precede cell division. Since abnormal cell cycles and increased proliferation are characteristic of many human cancer cells, we hypothesized that the observed increase in nuclear refractive index could be related to an abundance or accumulation of cells derived from cancer patients at a specific point or phase(s) of the cell cycle. Here we show that changes in nuclear refractive index of fixed cells are seen as synchronized populations of cells that proceed through the cell cycle, and that increased nuclear refractive index is strongly correlated with increased DNA content. We therefore propose that an abundance of cells undergoing DNA replication and mitosis may explain the increase in nuclear refractive index observed in both malignant and histologically normal cells from cancer patients. Our findings suggest that nuclear refractive index may be a novel physical parameter for early cancer detection and risk stratification.

Investigation of depth-resolved nanoscale structural changes in regulated cell proliferation and chromatin decondensation

We present depth-resolved spatial-domain low-coherence quantitative phase microscopy, a simple approach that utilizes coherence gating to construct a depth-resolved structural feature vector quantifying sub-resolution axial structural changes at different optical depths within the sample. We show that this feature vector is independent of sample thickness variation, and identifies nanoscale structural changes in clinically prepared samples. We present numerical simulations and experimental validation to demonstrate the feasibility of the approach. We also perform experiments using unstained cells to investigate the nanoscale structural changes in regulated cell proliferation through cell cycle and chromatin decondensation induced by histone acetylation.

Spectral encoding of spatial frequency approach for characterization of nanoscale structures

An approach to acquire axial structural information at nanoscale is demonstrated. It is based on spectral encoding of spatial frequency principle to reconstruct the structural information about the axial profile of the three-dimensional (3D) spatial frequency for each image point. This approach overcomes the fundamental limitations of current optical techniques and provides nanoscale accuracy and sensitivity in characterizing axial structures. Numerical simulation and experimental results are presented.

Assessment of Nuclear Nanomorphology Marker to Improve the Detection of Malignancy From Bile Duct Biopsy Specimens

OBJECTIVES The accurate diagnosis of malignancy from small bile duct biopsy specimens is often challenging. This proof-of-concept study assessed the feasibility of a novel optical technology, spatial-domain low-coherence quantitative phase microscopy (SL-QPM), that assesses nanoscale structural alterations in epithelial nuclei for improving the diagnosis of malignancy in bile duct biopsy specimens. METHODS The SL-QPM analysis was performed on standard histology specimens of bile duct biopsy specimens from 45 patients. We analyzed normal cells with benign follow-up, histologically normal cells with pancreaticobiliary malignancy, and malignant epithelial cells. RESULTS The SL-QPM-derived nuclear nanomorphology marker can not only distinguish benign and malignant epithelial cells but can also detect features of malignancy in those cells normal by light microscopy with a discriminatory accuracy of 0.90. When combining pathology with SL-QPM, the sensitivity is improved to 88.5% from 65.4% of conventional pathology, while maintaining 100% specificity. CONCLUSIONS SL-QPM-derived nuclear nanomorphology markers represent a novel approach for detecting malignancy from histologically normal-appearing epithelial cells, with potential as an adjunctive test in patients with negative or inconclusive pathologic diagnosis on bile duct biopsy specimens.

Nuclear Nano-architecture Markers of Gastric Cardia and Upper Squamous Esophagus Detect Esophageal Cancer “Field Effect”

Background: Barretts esophagus (BE) affects up to 12 million Americans and confers an increased risk for development of esophageal adenocarcinoma (EAC). EAC is often fatal unless detected early. Given the high prevalence, high cost of surveillance and relatively low risk of most affected individuals, identification of high-risk patients for additional scrutiny, regular surveillance, or ablative therapy is crucial. The exploration of “field effect” by probing uninvolved esophageal mucosa to predict the risk of EAC has the potential as an improved surveillance and prevention strategy. In this study, we evaluate the ability of nuclear nano-architecture markers from normal squamous esophagus and gastric cardia to detect the “field effect” of esophageal dysplasia and EAC, and their response to endoscopic therapy. Methods: Patients with normal esophagus, gastroesophageal reflux, BE and EAC were eligible for enrollment. We performed endoscopic cytology brushings of the gastric cardia, ~1-2 cm below the gastroesophageal junction, and of the normal squamous esophageal mucosa at ~20 cm from the incisors and standard cytology slides were made using Thinprep method. Optical analysis was performed on the cell nuclei of cytologically normal-appearing epithelial cells. Results: The study cohort consisted of 128 patients. The nuclear nano-architecture markers detected the presence of esophageal dysplasia and EAC with statistical significance. The field effect does not exhibit a spatial dependence. These markers reverted toward normal in response to endoscopic therapy. Conclusions: Optical analysis of gastric cardia and upper squamous esophagus represents a potentially viable method to improve risk stratification and ease of surveillance of patients with Barretts esophagus and to monitor the efficacy of ablative therapy.

Nanoscale nuclear architecture for cancer diagnosis by spatial-domain low-coherence quantitative phase microscopy

Alterations in nuclear architecture are the hallmark diagnostic characteristic of cancer cells. In this work, we show that the nuclear architectural characteristics quantified by spatial-domain low-coherence quantitative phase microscopy (SL-QPM), is more sensitive for the identification of cancer cells than conventional cytopathology. We demonstrated the importance of nuclear architectural characteristics in both an animal model of intestinal carcinogenesis - APC/Min mouse model and human cytology specimens with colorectal cancer by identifying cancer from cytologically noncancerous appearing cells. The determination of nanoscale nuclear architecture using this simple and practical optical instrument is a significant advance towards cancer diagnosis.

Nuclear Refractive Index Properties of Non-Dysplastic Metaplastic Cells to Detect the Presence of Esophageal High-Grade Dysplasia and Adenocarcinoma From Barrett's Esophagus

BACKGROUND: Radiofrequency ablation (RFA) is an endoscopic ablation modality used to treat Barretts esophagus (BE) with the goal of eliminating dysplasia and metaplasia. Factors associated with stricture formation or incomplete eradication of intestinal metaplasia (EIM) are poorly understood. AIM: To determine the factors associated with stricture formation or incomplete EIM. METHODS: This was a retrospective study of all patients treated with RFA for BE at a tertiary care referral center between June 2006 and November 2010. Pertinent information was extracted from medical records, including: demographics, history of BE (pre-ablation histology, duration of pre-treatment dysplasia), medication and substance use, indicators of GERD activity (symptoms, presence of erosive esophagitis), upper endoscopy findings (Prague criteria, hiatus hernia), ablation outcomes (elimination of metaplasia and dysplasia), and complications (perforation, stricture, bleeding, and hospitalization). Outcomes related to RFA were described for all patients as well as stratified by pre-ablation histology. Comparative analysis of patients with and without stricture and complete and incomplete elimination of dysplasia were performed with non-parametric tests (Fishers exact test for categorical data, Wilcoxon rank-sum test for continuous data) to determine associated factors. RESULTS: Among 113 patients who received RFA for BE (22 low-grade dysplasia, 77 high-grade dysplasia, 14 intramucosal carcinoma), 83 (73.5%) completed treatment with 95.2% complete elimination of dysplasia and 85.5% complete EIM. Of the remaining 30 subjects, 22 had ongoing treatment, 6 were lost to follow up, one had treatment delay for antireflux surgery and 1 opted for esophagectomy. Nine patients (8.0%) experienced a treatment-related complication, including8 strictures and 1 post-procedure hemorrhage. Stricture formation was associated with receiving endoscopic mucosal resection (75.0% vs. 36.2%, p=0.05) and number of EMR sessions (mean 1.4 vs. 0.4, p=0.007). A trend toward stricture formation existed with number of focal RFA treatments (mean 3.3 vs. 2.2, p=0.09), active NSAID use (75.0% vs. 44.8%, p=0.14) and prior peptic stricture (25.0% vs. 7.6%, p=0.15). Incomplete EIM was associated with ongoing GERD symptoms (75.0% vs. 33.8%, p=0.01) while increased Prague M length had a trend toward association (mean 6.6 vs. 4.5cm, p=0.11). CONCLUSIONS: RFA at a tertiary referral center is both safe (8.0% with complications, mostly benign strictures) and efficacious (95.2% elimination of dysplasia, 85.5% elimination of intestinal metaplasia). Previous EMR is associated with stricture formation while ongoing GERD symptoms are associated with incomplete elimination of intestinal metaplasia. These findings should be considered in planning treatment protocols for patients with BE.

Assessment of Nuclear Nano-morphology Markers for Predicting Cancer Progression Risk in Patients with Pre-cancerous Breast Lesions

We evaluate whether nano-morphology markers from cell nuclei derived from spatial-domain low-coherence quantitative phase microscopy from patients with precancerous breast lesios can identify high-risk patients for cancer progression.

Use of optical biomarkers from nondysplastic metaplastic cells on the detection of high-grade dysplasia and adenocarcinoma from Barrett’s esophagus.

14 Background: Patients with Barretts esophagus (BE), defined as presence of intestinal metaplasia (IM) in the esophagus, require surveillance due to an increased risk of developing esophageal adenocarcinoma (EAC). The biggest challenge in the current surveillance methodology (i.e., Seattle protocol) is random sampling with the inability to identify those patients with non-dysplastic BE on surveillance who have occult high grade dysplasia (HGD) or will eventually progress to HGD or EAC. We propose a novel approach based on the concept of field effect to detect HGD or EAC through the analysis of non-dysplastic IM, thus identifying a high risk BE population. Our group uses a unique microscope - spatial-domain low-coherence quantitative phase microscopy (SL-QPM) to detect changes in nuclear structure as small as 0.9 nm, a scale 1000 times smaller than what conventional microscopy detects. We hypothesize that the SL-QPM-derived optical biomarkers of non-dysplastic IM would distinguish BE patients with EAC/HGD from those without neoplasia. METHODS We performed a retrospective study of 60 BE patients who underwent Seattle protocol biopsies: 33 BE patients with IM only and 27 BE patients with HGD or EAC (21 HGD, 6 EAC). H&E stained slides with non-dysplastic IM on review by an expert pathologist were used. The distance between the selected IM biopsy and HGD/EAC was 1 to 4 cm. Forty to 60 columnar cells from each case were analyzed. RESULTS We identified three optical biomarkers (nuclear optical path length, intra-nuclear uniformity, entropy) that can distinguish non-dysplastic BE from patients with HGD and EAC with statistical significance (P < 0.01). A prediction model combining all three optical biomarkers can distinguish BE patients with HGD/EAC from those with IM only at 89% sensitivity and 76% specificity (accuracy = 0.87). CONCLUSIONS The accurate assessment of nanoscale optical biomarkers by SL-QPM is a promising approach for detecting dysplastic/neoplastic Barretts epithelium from non-dysplastic IM. This approach could potentially simplify BE surveillance by identifying a subset of high-risk BE patients that warrant intensive surveillance.

Su1930 Nuclear Nano-Morphology Markers From Rectal Tissue for the Surveillance of Colorectal Cancer in Patients With Ulcerative Colitis

Background: Inflammatory bowel disease (IBD), including Crohns disease (CD) and ulcerative colitis (UC), is considered to result from the interaction of environmental factors, including intestinal microbiota, with host immune mechanisms in genetically susceptible individuals. Association studies in human and mice indicate that no single organisms are causal for IBD, but instead reflect a broad dysbiosis, suggesting that distinct organisms sharing similar traits may be operative. Owing to the complexity and low inter-individual overlap of the intestinal microbiota, no biologically and ecologically integrated microbial signature of this dysbiosis has been identified in IBD. Results: We performed a study of endoscopic lavage samples from different anatomical sites in 64 subjects (32 normal (NM), 16 CD and 16 UC patients in remission). The V4 region of the 16S ribosomal RNA gene of 190 total mucosal lavage samples was sequenced on an Illumina HiSeq 2000. The microbial composition of lavage specimens was more similar to mucosal biopsies versus fecal compartment (phylum-level Pearson similarity analysis with published datasets), suggesting that mucosal lavage sampling is a good reflection of mucosal-associated microbiota. Phylogenetic diversity was reduced in IBD patients compared to NM subjects. While expected from prior studies of active disease, this is the first demonstration that such differences persist in clinically quiescent phases of disease. A subset of IBD-enriched microbial communities was identified by PCoA of the unweighted UniFrac distance matrix. Network-based analysis of microbial co-occurrence patterns identified 8 functional microbial communities (FMCs). Only one FMC was related to any of the previously reported fecal enterotypes. Bioinformatic validation with two independent mucosal (biopsy) datasets showed that these FMCs are a significantly preserved characteristic of the mucosal surface ecology. Two FMCs were positively and negatively associated with IBD phenotype, and with ATG16L1 and NOD2 risk alleles. Conclusions: We have introduced mucosal lavage as a new sampling method to study host-microbial interaction, and module analysis to identify integrated mucosal ecology parameters for microbial community structure. Our results demonstrate the feasibility of detecting subtle variation between NM and IBD microbiota using relatively short Illumina reads. The identification of FMCs indicated that the intestinal microbiota can be defined as a set of reproducible microbial communities, and that a subset of these are selectively augmented or depleted in IBD patient samples, and with related disease-associated genotypes. Such disease-associated communities provide a new tool to define disease states as microbial ecologic units, and potentially novel integrated units to monitor deleterious microbial states and for therapeutic targeting.