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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.

Objectives We previously developed and reported on a prototype clinical decision support system (CDSS) for cervical cancer screening. However, the system is complex as it is based on multiple guidelines and free-text processing. Therefore, the system is susceptible to failures. This report describes a formative evaluation of the system, which is a necessary step to ensure deployment readiness of the system. Materials and methods Care providers who are potential end-users of the CDSS were invited to provide their recommendations for a random set of patients that represented diverse decision scenarios. The recommendations of the care providers and those generated by the CDSS were compared. Mismatched recommendations were reviewed by two independent experts. Results A total of 25 users participated in this study and provided recommendations for 175 cases. The CDSS had an accuracy of 87% and 12 types of CDSS errors were identified, which were mainly due to deficiencies in the system's guideline rules. When the deficiencies were rectified, the CDSS generated optimal recommendations for all failure cases, except one with incomplete documentation. Discussion and conclusions The crowd-sourcing approach for construction of the reference set, coupled with the expert review of mismatched recommendations, facilitated an effective evaluation and enhancement of the system, by identifying decision scenarios that were missed by the system's developers. The described methodology will be useful for other researchers who seek rapidly to evaluate and enhance the deployment readiness of complex decision support systems.

Cytologic evaluation of pancreatobiliary brushings is specific but poorly sensitive for malignancy. Detection of polysomic cells by fluorescence in situ hybridization (FISH) is significantly more sensitive than routine cytology with similar specificity. The purpose of this study was to identify cytologic criteria most associated with malignancy in specimens unaffected by sample failure. Endoscopic brushings were split equally for routine cytologic and FISH analyses per clinical practice. We retrospectively evaluated 16 cytologic criteria on Papanicolaou-stained slides. We assumed that the presence of polysomic cells by FISH indicated successful tumor sampling in specimens from patients with pathologic evidence of malignancy on follow-up. We compared cytologic criteria of malignant brushings with corresponding positive FISH results (positive control, n = 39) with those without evidence of malignancy and corresponding negative FISH results (negative control, n = 30). The presence of single abnormal cells, irregular nuclear membranes, and enlarged nuclei were independent predictors of malignancy by logistic regression (P < .05).

The goal of this study was to assess the performance characteristics of the Automated Cellular Imaging System (ACIS III) for HER2 immunohistochemical analysis. The study was performed on 187 biopsy slides from patients who underwent HER2 testing between January and February 2008. Three scoring methods by the ACIS III were compared with the manual score and fluorescence in situ hybridization (FISH) results for HER2 amplification. The equal distribution score (EQD) method, in which 2 areas each of high-, moderate-, and low-intensity staining were measured, most closely matched the FISH HER2 amplification result. The numbers of immunohistochemically negative (0 or 1+)/ FISH+ cases were equivalent for all methods. The EQD method had significantly fewer 2+ cases (n = 16) (P < .001) vs the manual method (n = 35) and yielded a higher positive predictive value (38%) for HER2 amplification compared with the manual method (20%). The EQD method may more accurately identify FISHamplified HER2 cases with fewer 2+ cases that would be “reflexed” to FISH compared with the manual method. The proto-oncogene HER2 is amplified in 20% to 30% of breast cancers, resulting in overexpression of the HER2 protein. HER2+ breast cancer is associated with rapid tumor growth, increased risk of recurrence after surgery, poor response to conventional chemotherapy, and significantly shorter disease-free survival and overall survival than HER2– breast cancer. 1-3 Trastuzumab (Herceptin, Genentech, San Francisco, CA), a humanized monoclonal antibody, has been shown to improve clinical outcomes in the setting of HER2+ metastatic disease or adjuvant treatment for HER2+ invasive breast cancer when used in combination with chemotherapy as a first line. 4 Currently, 2 laboratory methods used to assess HER2 status include immunohistochemical analysis and fluorescence in situ hybridization (FISH), which assess protein overexpression and gene amplification, respectively. Immunohistochemical analysis and FISH are commonly used assays for determining HER2 status because morphologic examination is used to ensure that HER2 analysis is localized within tumor cells. Unfortunately, recent studies have demonstrated less-than-optimal concordance rates between immunohistochemical analysis and FISH among laboratories performing HER2 testing, which has been attributed to lack of standardized testing protocols and test interpretation by inexperienced personnel. 4-6

The MonoPrep Pap Test (MPPT; MonoGen, Lincolnshire, IL) is a novel, liquid-based specimen collection and processing technology for cytologic and molecular testing. Its usefulness in the detection of cervical cancer and its precursors was evaluated in a multicenter, masked, adjudicated, split-sample study of 10,739 samples. After preparation of a conventional smear, the residuum on the collection device was rinsed into a collection vial from which an MPPT slide was prepared. Accuracy was assessed by masked reference interpretation by an independent pathologist. Slides prepared by MPPT, compared with smears, yielded statistically significant increases in relative sensitivity for atypical squamous cells of undetermined significance and worse, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion/atypical glandular cells and worse, and low-grade squamous intraepithelial lesion and worse. There was no significant difference in relative specificity. MPPT provided a 58% reduction in unsatisfactory slides. There was no significant difference in the presentation of endocervical/transformation zone component or the detection of benign conditions. The MPPT is a promising new liquid-based technology for cervical cancer screening.

Many DNA-probe assays utilize oligonucleotide-coated microparticles for capture of complementary nucleic acids from solution. During development of these assays, as well as in other particle-based nucleic acid applications, it is useful to know both the amount of duplex formation expected under various experimental conditions and the coating density of the capture oligonucleotide on the particle surface. We examined the simplest form of a DNA-probe microparticle assay: hybridization of a particle-bound capture oligonucleotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the amount of labeled oligonucleotide remaining in solution at equilibrium was measured. We present a simple two-state, all-or-none model for bimolecular hybridization of non-self-complementary sequences that can be used to calculate the equilibrium dissociation constant ( Kd ) from hybridization data. With experimental conditions where both the Kd value and the concentration of capture probe in the reaction are small relative to the concentration of labeled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for particle-based hybridization were different from those obtained from solution-phase thermodynamic parameters. At higher temperatures, hybridization on particles was more efficient than hybridization in solution.