Automation of hybridization and capture based next generation sequencing library preparation requires reduction of on-deck bead binding and heated wash temperatures.

Capture-based library preparation for next generation sequencing (NGS) offers a balance between sequencing depth and bioinformatics cost of analysis. Liquid handling automation enhances the reliability of the library preparation process by reducing sample-to-sample variation and substantially enhances throughput, particularly when it can be employed in a 'walk-away' fashion with limited hands-on interaction. This requires complex series of mixing and heating steps like those utilized in capture chemistries to happen on the liquid handler. While developing liquid handling automation for Integrated DNA Technologies (IDT) xGen Exome, Illumina TruSight Oncology 500, and Personal Genome Diagnostics (PGDx) elio Plasma Resolve chemistries on the PerkinElmer Sciclone liquid handler, we found that applying the capture temperatures recommended for manual library preparation results in low yield on automation. To restore the final library yield, we reduced bead binding and/or heated wash temperatures of the Peltier heaters on the liquid handlers by about 10°C. Since this applied across three unique capture-based chemistries, we consider this a generalizable principle of automating capture on the Sciclone. We hypothesize that this is driven by the very different thermodynamic environments represented by a sealed plate on a thermal cycler and a plate with a lid on a Peltier heater. This phenomenon should be considered when automating NGS library preparation on PerkinElmer Sciclone instruments.

Applying Standard Clinical Chemistry Assay Validation to Droplet Digital PCR Quantitative Liquid Biopsy Testing.

BACKGROUND: Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS: We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS: For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R 2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS: Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

Prognostic association of plasma cell-free DNA-based androgen receptor amplification and circulating tumor cells in pre-chemotherapy metastatic castration-resistant prostate cancer patients.

BACKGROUND: The prognostic significance of plasma cell-free DNA (cfDNA) androgen receptor amplification (ARamp) in metastatic castration-resistant prostate cancer (mCRPC) compared with circulating tumor cell (CTC) counts is not known. METHODS: As part of correlative aims of a prospective study in mCRPC, concurrent and serial collections of plasma and CTCs were performed. Specimen collections were performed at baseline after progression on androgen deprivation therapy and then 12 weeks later. QuantStudio3D digital PCR system was used to determine plasma cfDNA AR copy number variations and Cell search assay for enumerating CTC counts. Association of baseline cfDNA ARamp status/CTC counts with overall survival (OS) (primary goal) was evaluated using Kaplan-Meier method and log-rank test (p ≤ 0.05 for significance) and receiver operator curves (ROCs) for ARamp status and CTC counts ≥5. A multivariate analysis was performed using Cox regression models that included ARamp, CTC counts, and other clinical factors. RESULTS: ARamp was detected in 19/70 patients at baseline. At the time of analysis, 28/70 patients had died (median follow-up 806 days; interquartile range: 535-966). ARamp was associated with poor OS (2-year OS of 35% in ARamp vs. 71% in non-ARamp; log-rank p value ≤0.0001). Baseline CTC counts ≥5 (vs. <5) was also associated with poor survival (2-year OS of 44 vs. 74%; log-rank p = 0.001). ROC analysis demonstrated area under the curve of 0.66 for ARamp-based prognosis and 0.68 for CTC count-based prognosis (p = 0.84 for difference). The best two variables included for multivariable analysis were ARamp and CTC counts ≥5; however, the two-factor model was not significantly better than using ARamp alone for predicting survival (hazard ratio = 5.25; p = 0.0002). CONCLUSIONS: CTCs and plasma cfDNA ARamp were observed to have equal prognostic value in mCRPC. Larger cohorts that incorporate molecular and clinical factors are needed to further refine prognosis in CRPC.

Circulating tumor cells are associated with poor overall survival in patients with cholangiocarcinoma.

UNLABELLED: Circulating tumor cells (CTCs) in blood are associated with poor survival of patients with breast, prostate, or colon cancer. We hypothesized that CTCs are associated with poor survival of patients with cholangiocarcinoma (CCA). Eighty-eight patients with CCA were prospectively enrolled at Mayo Clinic Rochester between June 2010 and September 2014. The CellSearch system by Veridex was used for detection of CTCs in peripheral blood. Associations between CTC, patient and tumor characteristics, and survival were examined using the Cox's proportional hazards model. Fifteen patients (17%) were positive for CTC ≥2 and 8 patients (9%) for CTC ≥5. CTCs were associated with tumor extent. CTC ≥2 (hazard ratio [HR]: 2.5; 95% confidence interval [CI]: 1.1-5.4; P = 0.02) and CTC ≥5 (HR, 4.1; 95% CI: 1.4-10.8; P = 0.01) were both independent predictors of survival. In subgroup analyses, CTC ≥2 (HR, 8.2; 95% CI: 1.8-57.5; P < 0.01) and CTC ≥5 (HR, 7.7; 95% CI: 1.4-42.9; P = 0.02) were both associated with shorter survival among patients with metastasis. There was a trend toward association of CTC ≥5 with shorter survival in patients with nonmetastatic CCA (HR, 4.3; 95% CI: 1.0-13.8; P = 0.06). CTC ≥2 (HR, 10.5; 95% CI: 2.2-40.1; P < 0.01) and CTC ≥5 (HR, 10.2; 95% CI: 1.5-42.3; P = 0.02) were both associated with shorter survival among patients with perihilar/distal CCA. CTC ≥5 was associated with shorter survival of patients with intrahepatic CCA (HR, 4.2; 95% CI: 1.1-14.1; P = 0.04). CONCLUSION: CTCs were associated with more-aggressive tumor characteristics and independently associated with survival in patients with CCA. Assessment of CTCs may be useful for identifying CCA patients at risk of early mortality.

An Optimized Set of Fluorescence In Situ Hybridization Probes for Detection of Pancreatobiliary Tract Cancer in Cytology Brush Samples.

BACKGROUND & AIMS: Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis. METHODS: We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH. RESULTS: The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of brush samples, pancreatobiliary FISH identified samples from patients with malignancy with a significantly higher level of sensitivity (64.7%) than the UroVysion probes (45.9%) (P < .001) or routine cytology analysis (18.8%) (P < .001), but similar specificity (92.9%, 90.8%, and 100.0% respectively). Factors significantly associated with detection of carcinoma, in adjusted analyses, included detection of polysomy by pancreatobiliary FISH (P < .001), a mass by cross-sectional imaging (P < .001), cancer cells by routine cytology (overall P = .003), as well as absence of primary sclerosing cholangitis (P = .011). CONCLUSIONS: We identified a set of FISH probes that detects cancer cells in pancreatobiliary brush samples from patients with and without primary sclerosing cholangitis with higher levels of sensitivity than UroVysion probes. Cytologic brushing test results and clinical features were independently associated with detection of cancer and might be used to identify patients with pancreatobiliary cancers.

Endoscopic ultrasound fine-needle aspiration cytology mutation profiling using targeted next-generation sequencing: personalized care for rectal cancer.

OBJECTIVES: In an era of precision medicine, our aim was to determine the frequency and theranostic potential of mutations identified in malignant lymph nodes (LNs) sampled by endoscopic ultrasound fine-needle aspiration (EUS FNA) of patients with rectal cancer by targeted next-generation sequencing (NGS). METHODS: The NGS Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA) and MiSeq (Illumina, San Diego, CA) sequencers were used to sequence and assess for 2,800 or more possible mutations in 50 established cancer-associated genes. RESULTS: Among 102 patients, 89% had 194 pathogenic alterations identified in 19 genes. The identification of KRAS, NRAS, or BRAF mutations suggests that 42% are likely nonresponders to anti-epidermal growth factor receptor therapy. Among KRAS, NRAS, or BRAF wild-type patients, alterations in eight genes linked to alternative therapies were identified in 44%. CONCLUSIONS: Our data demonstrate the successful ability to apply a single multiplex test to allow multigene mutation detection from malignant LN cytology specimen DNA collected by EUS FNA.

Frequency of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathway pathogenic alterations in EUS-FNA sampled malignant lymph nodes in rectal cancer with theranostic potential.

BACKGROUND: Targeted next-generation sequencing has the potential to stratify a tumor by molecular subtype and aid the development of a biomarker profile for prognostic risk stratification and theranostic potential. OBJECTIVE: To assess the frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens. DESIGN: Multigene molecular profiling of archived malignant EUS-FNA lymph node cytology specimens using the Ion Ampliseq Cancer Hotspot Panel v2, which targets at least 2855 possible mutations within 50 cancer-associated genes. SETTING: Single tertiary referral center. PATIENTS: Sporadic, treatment naive, locally advanced primary rectal cancer by EUS-FNA (n = 76) who subsequently completed neoadjuvant therapy with on-site oncologic surgery. MAIN OUTCOME MEASUREMENTS: The frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens by the mitogen-activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling pathways, by KRAS or NRAS wild-type lymph node status, by extramesenteric lymph node status, and by a complete pathologic response status. RESULTS: Eleven patients (14.5%) were 50-gene panel wild-type. Sixty-five patients had 139 pathogenic alterations (2 [1-3] per patient) in 13 of 50 evaluated genes. The following represent a spectrum of identified alterations: TP53 (n = 52; 68.4%), APC (n = 36; 47.4%), KRAS (n = 22; 28.9%), FBXW7 (n = 8; 10.5%), NRAS (n = 6; 7.9%), PIK3CA (n = 4; 5.3%), SMAD4 (n = 3; 3.9%), and BRAF (n = 3; 3.9%). Pathogenic alterations were identified in the MAPK and PI3K signaling pathways in 41% and 5% of patients, respectively. LIMITATIONS: Findings were limited to a 50 cancer-associated gene analysis. CONCLUSIONS: Molecular EUS lymph node assessments using cancer "hotspot" panels can identify pathogenic alteration frequency and distribution and have theranostic potential for individualized patient care.

Detection of circulating tumor cells in high-risk endometrial cancer.

AIM: To evaluate the presence of circulating tumor cells (CTCs) in patients with high-risk endometrial cancer (EC). PATIENTS AND METHODS: We prospectively included 28 patients with a preoperative diagnosis of grade 3 EC undergoing surgery from June 2010 to December 2011. Their preoperative blood samples were tested for the presence of CTCs using an immunomagnetic and immunofluorescence assay technique. RESULTS: Overall, 2 out of 28 patients (7%) were positive for CTCs. The presence of positive CTCs was significantly associated with myometrial invasion (MI) (33% vs. 0% for MI>50% vs. ≤50%; p=0.04) and lymph node positivity (40% vs. 0% for positive vs. negative nodes; p=0.03). Only patients with endometrioid histology had positive CTCs (29% in endometrioid vs. 0% in nonendometrioid; p=0.06). CONCLUSION: The presence of positive CTCs was associated with deep MI and lymph node positivity. The absence of CTCs in patients with type II histology suggests the need to find other markers in this sub-group of patients.

Somatic STK11 and concomitant STK11/KRAS mutational frequency in stage IV lung adenocarcinoma adrenal metastases.

Somatic serine/threonine kinase 11 (STK11) also known as liver kinase B1 (LKB1) is a tumor suppressor gene and ranks as the third most frequently mutated gene in lung adenocarcinoma. However, current molecular testing guidelines recommend evaluating for epidermal growth factor receptor mutations and ALK fusions to guide therapy in all patients with advanced stage adenocarcinoma, regardless of gender, race, or smoking history. Identifying alternative "driver" mutations and using actionable targeted pharmacotherapy is a key approach to providing effective individualized medical care. The analytical sensitivity and parallel multigene approach of targeted next-generation sequencing is an attractive methodology for use for cytology specimens. The presented lung adenocarcinoma study revealed that STK11 mutations alone and concomitant KRAS/STK11 mutations were identified in 18.2% and 4.5% of solitary adrenal metastases, respectively. Molecular profiling of epidermal growth factor receptor tyrosine kinase inhibitor resistant tumors may help to identify patients who would most benefit from alternative single or dual pathway inhibition potentially leading to a revision in current molecular testing guidelines.

Kinase genotype analysis of gastric gastrointestinal stromal tumor cytology samples using targeted next-generation sequencing.

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy.

Biliary dysplasia in primary sclerosing cholangitis harbors cytogenetic abnormalities similar to cholangiocarcinoma.

Grading criteria for biliary dysplasia associated with primary sclerosing cholangitis (PSC) have been recently described. Although a dysplasia to cholangiocarcinoma (CCA) sequence is implied, supportive data are lacking. Seventeen liver explants with biliary dysplasia from patients with PSC were selected. Formalin-fixed, paraffin-embedded blocks from each patient were evaluated to identify areas of normal/reactive biliary epithelium, intestinal metaplasia, low-grade dysplasia, high-grade dysplasia, and CCA. Areas of interest were assessed for chromosomal alteration with fluorescence in situ hybridization using probes directed to locus 9p21 and centromeres 3, 7, and 17. The cutoffs for calling probe copy number abnormalities for polysomy, single locus gain, and homozygous 9p21 loss were established by receiver operating characteristic curve analysis. Of 4 areas of intestinal metaplasia, 19 low-grade dysplasias, 19 high-grade dysplasias, and 5 CCAs, 0%, 11%, 58%, and 40% displayed polysomy and 0%, 0%, 16%, and 40% exhibited homozygous 9p21 loss as the most severe abnormality, respectively. Patients with prior or current CCA were more likely to display polysomy in dysplasia than patients without CCA (70% versus 14%; P = .05); however, high-grade dysplasia was proportionally more common in the CCA-associated dysplasia group. Polysomy and homozygous 9p21 loss are detected in biliary dysplasia and CCA. These findings support a dysplasia-carcinoma sequence in PSC patients and suggest that fluorescence in situ hybridization analysis could help refine the grading of biliary dysplasia in these patients.

Lung cancer adrenal gland metastasis: Optimal fine-needle aspirate and touch preparation smear cellularity characteristics for successful theranostic next-generation sequencing.

BACKGROUND: Multigene molecular testing to guide personalized therapy for oncology patients is of increasing clinical relevance. Molecular testing of fine-needle aspiration samples is underused, but when acquired with minimally invasive techniques could become the standard of care to obtain theranostic specimens. The aims of the current study were to identify key cytology specimen selection criteria suitable for next-generation sequencing (NGS) and to determine the prevalence and spectrum of pathogenic alterations in a cohort of patients with AJCC stage IV lung cancer. METHODS: A total of 70 adrenal gland cytology specimens with direct smears were screened to identify 56 patients with a single slide containing at least 300 total cells and 20% tumor nuclei. After DNA extraction, the NGS protocols were used to simultaneously detect mutations in >2800 exonic regions in 50 key cancer genes. RESULTS: A total of 28 specimens produced acceptable NGS results. Specimens with a combined critical cell mass (>5000 viable cells) and a DNA concentration >5 ng/µL resulted in a 95% chance of successful sequencing. A total of 37 pathogenic alterations were identified in 10 genes and in 25 patients (85%). A pathogenic alteration (≥1) linked to available or developing targeted therapies was revealed in 50% of cases. CONCLUSIONS: The data from the current study demonstrate that theranostic NGS can be applied to adrenal gland metastasis using routine cytologic smear specimens. The characteristics of such smears could be evaluated during onsite adequacy assessment by cytopathology professionals. This model greatly augments the opportunity for customized genotype-directed therapy from minimally invasive techniques.

Comparison of methods to detect neoplasia in patients undergoing endoscopic ultrasound-guided fine-needle aspiration.

BACKGROUND & AIMS: Digital image analysis (DIA) and fluorescence in situ hybridization (FISH) can be used to evaluate biliary strictures with greater accuracy than conventional cytology (CC). We performed a prospective evaluation of the accuracy of CC, compared with that of DIA and FISH, in detection of malignancy in patients undergoing endoscopic ultrasonography (EUS) fine-needle aspiration (FNA). METHODS: We collected a minimum of 6 FNA samples from each of 250 patients during EUS. CC or DIA and FISH analyses were performed on every other specimen (from every other FNA pass); patients were randomly assigned to the first test performed. CC slides were reviewed by gastrointestinal cytopathologists who were blinded to all data. Findings from cytohistologic analysis, after a minimum 24-month follow-up period, were used as the standard (n = 202; median age, 65 years). RESULTS: Aspirates were collected from lymph nodes (n = 111), pancreas (n = 61), gastrointestinal lumen wall (n = 9), periluminal mass (n = 4), liver (n = 8), and miscellaneous sites (n = 9). Matched samples provided a mean of 3.2 passes for CC and 1.6 passes for DIA and FISH. The data indicate a potential lack of utility for DIA. The combination of CC and FISH detected malignancy with 11% greater sensitivity than CC alone (P = .0002), but specificity was reduced from 100% to 96%. CONCLUSIONS: FISH analysis identifies neoplastic lesions with significantly greater sensitivity than CC in patients with diverse pathologies who underwent EUS with FNA, despite limited tissue sampling for FISH analysis.

Fluorescence in situ hybridization mapping of esophagectomy specimens from patients with Barrett's esophagus with high-grade dysplasia or adenocarcinoma.

The progression of intestinal metaplasia to esophageal adenocarcinoma in patients with Barrett's esophagus is partly driven by chromosomal alterations that activate oncogenes and inactivate tumor suppressor genes. The goal of this study was to determine how alterations of 4 frequently affected genes correlate with the range of histopathologic lesions observed in resected esophagi of patients with Barrett's esophagus. Fluorescence in situ hybridization was used to assess 83 tissue sections from 10 Barrett's esophagus esophagogastrectomy specimens for chromosomal alterations of 8q24 (MYC), 9p21 (CDKN2A; alias P16), 17q12 (ERBB2), and 20q13.2 (ZNF217). Histologic lesions assessed included gastric metaplasia (n = 8), intestinal metaplasia (n = 43), low-grade dysplasia (n = 28), high-grade dysplasia (n = 25), and adenocarcinoma (n = 16). Histologic maps showing the correlation between fluorescence in situ hybridization abnormalities and corresponding histology were created for all patients. Chromosomal abnormalities included 9p21 loss, single locus gain, and polysomy. A greater number of chromosomal alterations were detected as the severity of histologic diagnosis increased from intestinal metaplasia to adenocarcinoma. All patients had alterations involving the CDKN2A gene. CDKN2A loss was the only abnormality detected in 20 (47%) of 43 areas of intestinal metaplasia. Polysomy, the most common abnormality in dysplastic epithelium and adenocarcinoma, was observed in 16 (57%) of 28 low-grade dysplasia, 22 (88%) of 25 high-grade dysplasia, and 16 (100%) of 16 adenocarcinoma. The findings of this study improve our understanding of the role that chromosomal instability and alterations of tumor suppressor genes such as CDKN2A and oncogenes such as ERBB2 play in the progression of intestinal metaplasia to adenocarcinoma in patients with Barrett's esophagus.

Comparison of circulating endothelial cell/platelet count ratio to aspartate transaminase/platelet ratio index for identifying patients with cirrhosis.

BACKGROUND/OBJECTIVES: Circulating endothelial cells (CECs) are indicative of vascular injury and correlate with severity of vascular diseases. A pilot study showed that the ratio of CEC to platelet count (CEC/PC) was effective in predicting cirrhosis. Therefore, we evaluated CEC/PC in a larger cohort of patients, correlated it with cirrhosis, and compared its operating characteristics with previously described biomarker for cirrhosis, the AST/platelet ratio index (APRI). METHODS: Fifty-three patients with cirrhosis, 20 matched healthy controls, and 9 patients with noncirrhotic liver disease were recruited. Peripheral blood sample was collected and analyzed to enumerate nucleated CEC CD146+, CD105+, CD45- using a commercial assay. RESULTS: Median CEC counts were significantly higher in patients with cirrhosis (62 cells/4 mL, interquartile range [IQR]: 43.5-121) as compared with controls (31 cells/4 mL, IQR: 22.2-40). The CEC/PC was also significantly elevated in cirrhotics (0.69, IQR: 0.39-1.48) compared with controls (0.12, IQR: 0.09-0.20) and noncirrhotics (0.21, IQR: 0.08-0.43). Receiver operator characteristic (ROC) analysis revealed that CEC cutoff value of ≥37 cells/4 mL showed sensitivity of 81% and specificity of 75% for differentiating cirrhosis from controls (area under the curve [AUC]: 0.80; 95% confidence interval [CI] 0.67-0.91). The CEC/PC ratio cutoff value of ≥0.23 showed sensitivity of 91% and specificity of 82% (AUC: 0.92; 95% CI 0.83-0.99). The APRI cutoff value of ≥0.4 showed sensitivity of 94% and specificity of 85% for differentiating cirrhosis from control patients (AUC: 0.96; 95% CI 0.90-1.0). A product of CEC and APRI, termed CAPRI (CEC-APRI), effectively distinguished patients with cirrhosis from controls; with cutoff value of ≥12.7, showing higher sensitivity of 98% and specificity of 85% (AUC: 0.98; 95% CI 0.96-1.0). CONCLUSION: The CEC/PC ratio is significantly elevated in patients with cirrhosis and demonstrates comparable operating characteristics to previously described APRI. Furthermore, CAPRI, compiled as product of CEC to APRI showed outstanding ability to distinguish patients with cirrhosis from controls, although larger studies are necessary for validation.

Assessment of fluorescence in situ hybridization and hybrid capture 2 analyses of cervical cytology specimens diagnosed as low grade squamous intraepithelial lesion for the detection of high grade cervical intraepithelial neoplasia.

OBJECTIVE: To assess Hybrid Capture 2 (HC2) and fluorescence in situ hybridization (FISH) for the detection of cervical intraepithelial neoplasia 2 or worse (CIN 2+) in patients with a cytologic diagnosis of low grade squamous intraepithelial lesion (LSIL). STUDY DESIGN: Residual samples from 115 LSIL-diagnosed cervical cytology specimens were evaluated by high-risk human papillomavirus (HR-HPV) HC2 testing and FISH using biotin-labeled probes to HR-HPV and chromosomal probes to 3q26 (TERC) and 8q24 (CMYC). A cervical biopsy diagnosis of CIN 2+ was considered as evidence of high grade disease. RESULTS: The positive and negative predictive values of HC2 and FISH for detecting patients with CIN 2+ were 32% vs. 37% and 100% vs. 93%, respectively. The sensitivities of HC2 and FISH for CIN 2+ were not significantly different (100% vs. 90%, p = 0.25), while the specificity of HC2 was significantly lower than that of FISH (28% vs. 48%, p=0.003). FISH diagnosed fewer specimens as positive as compared to HC2 (62% vs. 79%). CONCLUSION: These preliminary data suggest that FISH testing may be useful for determining which patients with LSIL are most likely to have CIN 2+ on clinical follow-up.

Prospective cytological assessment of gastrointestinal luminal fluid acquired during EUS: a potential source of false-positive FNA and needle tract seeding.

OBJECTIVES: Endoscopic ultrasound (EUS) fine needle aspiration (FNA) can result in false-positive cytology and can also cause needle tract seeding. Our goal was to evaluate a potential cause, namely, the presence of malignant cells within gastrointestinal (GI) luminal fluid, either as a result of tumor sloughing from luminal cancers or secondary to FNA of extraluminal sites. METHODS: During EUS, luminal fluid that is usually aspirated through the echoendoscope suction channel and discarded was instead submitted for cytological analysis among patients with cancer and benign disease. Pre- and post-FNA luminal fluid samples were collected to discern the role of FNA in inducing a positive cytology. When not performing FNA, one sample was collected for the entire examination. The final diagnosis was based on strict clinicopathological criteria and >or=2-year follow-up. This study was conducted in a tertiary referral center. RESULTS: We assessed the prevalence of luminal fluid-positive cytology among patients with luminal (e.g., esophageal), extraluminal (e.g., pancreatic), and benign disease. Among the 140 patients prospectively enrolled with sufficient sampling and follow-up, an examination of luminal fluid cytology showed positive results for malignancy in luminal and extraluminal cancer patients, 48 and 10%, respectively. This included 8 out of 23 esophageal, 4 of 5 gastric, and 9 of 15 rectal cancers. The positive luminal fluid cytology rate with luminal cancers was not affected by performing FNA. Post-FNA luminal fluid cytology was positive in 3 out of 26 with pancreatic cancers. Cytological examination of luminal fluid aspirates did not demonstrate malignant cells in any patient with nonmalignant disease. CONCLUSIONS: Malignant cells are commonly present in the GI luminal fluid of patients with luminal cancers and can also be found in patients with pancreatic cancer after EUS FNA. Further study is needed to determine the impact of these findings on cytological interpretation, staging, risk of needle tract seeding, and patient care and outcomes.

Improving cellularity and quality of liquid-based cytology slides processed from pancreatobiliary tract brushings.

Cytology has been reported to have suboptimal sensitivity for detecting pancreatobiliary tract cancer in biliary tract specimens partly as a result of low specimen cellularity and obscuring noncellular components. The goal of this study was to determine if the use of a glacial acetic acid wash prior to processing would increase the cellularity and improve the quality of ThinPrep slides when compared to standard non-gyn ThinPrep processing. Fifty consecutive pancreatobiliary tract specimens containing 20 ml of sample/PreservCyt were divided equally for standard non-gyn ThinPrep (STP) and glacial acetic acid ThinPrep processing (GATP). A manual drop preparation was also performed on residual STP specimen to determine the number of cells left in the vial during STP processing. Twenty-six (52%) specimens had more epithelial cell groupings with the GATP methodology while 19 (38%) had equivalent cellularity with both methods. The STP method produced more epithelial cell groupings in 5 (10%) of the specimens. Of the 26 specimens that had less cells with the STP method, 14 (54%) had > or = 50 cell groupings on the manual drop slide processed from the residual STP specimen suggesting that many cells remain in the vial after STP processing. The GATP method was preferred in 25 (50%) of the specimens, the STP method in 5 (10%), while both methodologies provided similar findings in the remaining 20 (40%) of specimens. The data from this study suggests that the GATP method results in more cells being placed on the slide and was preferred over the STP method in a majority of specimens.

A prospective pilot study of circulating endothelial cells as a potential new biomarker in portal hypertension.

BACKGROUND/AIMS: Peripheral circulating endothelial cells (CEC) have been proposed as a prognostic marker in cardiovascular diseases. Cirrhosis and portal hypertension are associated with vascular injury yet little is known about CEC count in these conditions. Therefore, we evaluated CEC count in patients with cirrhosis, and correlated it with markers of portal hypertension/disease severity. PATIENTS/METHODS: Fifteen patients with cirrhosis/portal hypertension and 15 matched controls were prospectively recruited for study participation. An automated rare cell analysis system was used to enumerate CEC from peripheral blood and correlated with clinical features. RESULTS: Median CEC levels were significantly higher in patients with cirrhosis as compared with controls (median [interquartile range (IQR)]; cirrhosis: 73.7 cells/4 ml [53.7-140.3]; controls: 28.7 cells/4 ml [21-58.7]; P=0.021). Ratio of CEC to platelet count (CEC/PC) also distinguished patients with cirrhosis from controls (IQR; cirrhosis: 0.723 [0.396-1.672]; controls: 0.126 [0.103-0.333]; P<0.001). Receiver operator characteristic analysis revealed that CEC cut-off of 42 cells/4 ml showed sensitivity of 87% and specificity of 74% for differentiating cirrhosis from controls (AUC: 0.74), while CEC/PC ratio at 0.21 showed sensitivity of 100% and specificity of 73% (AUC: 0.89). Furthermore, CEC/PC index was significantly elevated in patients with hepatic decompensation as defined by Child B/C (P<0.05). The intra- and interobserver variability correlation coefficients for CEC measurement were 0.9989 and 0.9986 respectively. CONCLUSION: Median CEC count and CEC/PC ratio are significantly elevated in patients with cirrhosis, with CEC/PC also increased in patients with decompensated cirrhosis. These data provide rationale for larger validation studies to assess if CEC may have prognostic utility in patients with cirrhosis and portal hypertension.

Comparison of fluorescence in situ hybridization, hybrid capture 2 and polymerase chain reaction for the detection of high-risk human papillomavirus in cervical cytology specimens.

OBJECTIVE: To compare a recently developed fluorescence in situ hybridization (FISH) high-risk human papillomavirus (HR-HPV) assay to Hybrid Capture 2 (HC2) (Digene Corporation, Gaithersburg, Maryland, U.S.A.) and polymerase chain reaction (PCR) for the detection of HR-HPV subtypes in cervical cytology specimens. STUDY DESIGN: One hundred forty-one liquid-based cytology specimens were used to produce a thin-layer slide for FISH analysis. The remaining material was sent for HC2 and PCR HR-HPV testing. Thin-layer slides were hybridized with a FISH probe set containing a biotin-labeled HR-HPV cocktail and were manually screened for HR-HPV-infected cells. Specimens with > or = 1 HPV-positive cell by FISH were considered positive for HR-HPV infection. RESULTS: There was complete concordance between HC2, FISH and PCR in 104 (75%) specimens. FISH was concordant with HC2 and PCR in 120 (85%) and 115 (82%) specimens, respectively. HC2 and PCR were concordant in 118 (84%) specimens. CONCLUSION: The concordance of HR-HPV detection between FISH and HC2/PCR appears similar to concordances between HC2 and PCR. This suggests that FISH may be another method of detecting HR-HPV while having the potential to add additional information such as integrated/episomal staining and the ability to detect chromosomal abnormalities in individual cells.