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Objectives Tuberculosis (TB) is a significant global health problem. In low-prevalence areas and low clinical suspicion, nucleic acid amplification tests (NAAT) for direct detection of Mycobacterium tuberculosis complex (MTBC) can speed therapy initiation and infection control. An NAAT assay (TBPCR) targeting MTBC IS6110 is used for detecting MTBC in our low-prevalence population. Methods Fifteen-year review of patient records identified 146 patients with culture-positive pulmonary tuberculosis (PTB) or extrapulmonary tuberculosis (EPTB). Laboratory-developed TBPCR was retrospectively compared with standard stain and cultures for PTB and EPTB diagnoses. Results TBPCR assay was used in 57% of patients with PTB and 33% of patients with EPTB. TBPCR detected 88.4% of all TB (smear-positive, 97%; smear-negative, 79%) with 100% specificity. Low bacterial load was indicated in TBPCR-negative PTB (P = .002) and EPTB (P < .008). Conclusions TBPCR performance was optimum but significantly underused. Guidelines are proposed for mandated use of TBPCR that capture patients with clinically suspected PTB. Focused TBPCR use in low prevalence populations will benefit patient care, infection prevention, and public health.

In the two decades since Accreditation Council for Graduate Medical Education-accredited Molecular Genetic Pathology fellowships began, the field of clinical molecular pathology has evolved considerably. The American Board of Pathology gathered data from board-certified molecular genetic pathologists assessing the alignment of skills and knowledge gained during fellowship with current needs on the job. The Association of Molecular Pathology conducted a parallel survey of program directors, and included questions on how various topics were taught during fellowship, as well as ranking their importance. Both surveys showed that most training aligned well with the practice needs of former trainees. Genomic profiling of tumors by next-generation sequencing, bioinformatics, laboratory management, and regulatory issues were topics thought to require increased emphasis in training. Topics related to clinical genetics and microbiology were deemed less important by those in practice, perhaps reflecting the increasing subspecialization of molecular pathologists. Program directors still viewed these topics as important to provide foundational knowledge. Parentage, identity, and human leukocyte antigen testing were less important to both survey audiences. These data may be helpful in guiding future adjustments to the Molecular Genetic Pathology curriculum and Accreditation Council for Graduate Medical Education program requirements.

Problems within the Pathology fellowship application process in the US have been recognized and reported for years. Recently, members of the Graduate Medical Education Committee (GMEC) of the Association of Pathology Chairs (APC) and collaborators collected survey data from the residents themselves and the fellowship programs, as represented by both the fellowship program directors (members of the Fellowship Directors Ad Hoc Committee, FDAHC) and the program administrators (members of the Graduate Medical Education Administrators Section, GMEAS). These data are presented and discussed, and potential steps to resolve some of the problems around fellowship applications in pathology are presented.

In an effort to develop a new therapy for cancer and to improve antiprogrammed death inhibitor-1 (anti-PD-1) and anticytotoxic T lymphocyte-associated protein (anti-CTLA-4) responses, we have created a telomerase reverse transcriptase promoter-regulated oncolytic adenovirus rAd.sT containing a soluble transforming growth factor receptor II fused with human IgG Fc fragment (sTGFβRIIFc) gene. Infection of breast and renal tumor cells with rAd.sT produced sTGFβRIIFc protein with dose-dependent cytotoxicity. In immunocompetent mouse 4T1 breast tumor model, intratumoral delivery of rAd.sT inhibited both tumor growth and lung metastases. rAd.sT downregulated the expression of several transforming growth factor β (TGFβ) target genes involved in tumor growth and metastases, inhibited Th2 cytokine expression, and induced Th1 cytokines and chemokines, and granzyme B and perforin expression. rAd.sT treatment also increased the percentage of CD8(+) T lymphocytes, promoted the generation of CD4(+) T memory cells, reduced regulatory T lymphocytes (Tregs), and reduced bone marrow-derived suppressor cells. Importantly, rAd.sT treatment increased the percentage of CD4(+) T lymphocytes, and promoted differentiation and maturation of antigen-presenting dendritic cells in the spleen. In the immunocompetent mouse Renca renal tumor model, similar therapeutic effects and immune activation results were observed. In the 4T1 mammary tumor model, rAd.sT improved the inhibition of tumor growth and lung and liver metastases by anti-PD-1 and anti-CTLA-4 antibodies. Analysis of the human breast and kidney tumors showed that a significant number of tumor tissues expressed high levels of TGFβ and TGFβ-inducible genes. Therefore, rAd.sT could be a potential enhancer of anti-PD-1 and anti-CTLA-4 therapy for treating breast and kidney cancers.

The advent of next generation DNA sequencing (NGS) has revolutionized clinical medicine by enabling wide‐spread testing for genomic anomalies and polymorphisms. With that explosion in testing, however, come several informatics challenges including managing large amounts of data, interpreting the results and providing clinical decision support. We present Flype, a web‐based bioinformatics platform built by a small group of bioinformaticians working in a community hospital setting, to address these challenges by allowing us to: (a) securely accept data from a variety of sources, (b) send orders to a variety of destinations, (c) perform secondary analysis and annotation of NGS data, (d) provide a central repository for all genomic variants, (e) assist with tertiary analysis and clinical interpretation, (f) send signed out data to our EHR as both PDF and discrete data elements, (g) allow population frequency analysis and (h) update variant annotation when literature knowledge evolves. We discuss the multiple use cases Flype supports such as (a) in‐house NGS tests, (b) in‐house pharmacogenomics (PGX) tests, (c) dramatic scale‐up of genomic testing using an external lab, (d) consumer genomics using two external partners, and (e) a variety of reporting tools. The source code for Flype is available upon request to the authors.

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor β-1 (TGFβ-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFβRIIFc, a TGFβ decoy that can inhibit TGFβ pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFβRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

This guest editorial highlights 20 years of education and training by JMD.

Seasonal influenza virus causes significant morbidity and mortality each year. Point-of-care (POC) testing using rapid influenza diagnostic tests (RIDTs), immunoassays that detect viral antigens, are often used for diagnosis by physician offices and urgent care centers. These tests are rapid but lack sensitivity, which is estimated to be 50 to 70%. Testing by PCR is highly sensitive and specific, but historically these assays have been performed in centralized clinical laboratories necessitating specimen transport and increasing the time to result. Recently, Clinical Laboratory Improvement Amendments (CLIA)-waived, POC PCR influenza assays have been developed with >95% sensitivity and specificity compared to centralized PCR assays. To determine the clinical impact of a POC PCR test for influenza, we compared antimicrobial prescribing patterns of one urgent care location using the Cobas LIAT Influenza A/B assay (LIAT assay; Roche Diagnostics, Indianapolis, IN) to other urgent care centers in our health system using traditional RIDT, with negative specimens being reflexed to PCR. Antiviral prescribing was lower in patients with a negative LIAT PCR result (2.3%) than in patients with a negative RIDT result (25.3%; P < 0.005). Antivirals were prescribed more often in patients that tested positive by LIAT PCR (82.4%) than in those testing positive by either RIDT or reflex PCR (69.9%; P < 0.05). Antibacterial prescriptions for patients testing negative by LIAT PCR were higher (44.5%) than for those testing negative by RIDT (37.7%), although the difference was not statistically significant. In conclusion, having results from a PCR POC test during the clinic visit improved antiviral prescribing practices compared to having rapid results from an RIDT.

The rapid implementation of new molecular methods in support of precision medicine has been both exciting and challenging to pathology. Next-generation sequencing is coming to maturity as a clinical procedure, with concomitant development of guidelines for assay validation and performance, quality standards, and proficiency programs. Evolving issues include the type, target, and timing of genomic analysis of tumors and regulatory and reimbursement issues. Additionally, educational needs for pathology trainees, practicing pathologists, and clinical colleagues should be addressed.

Context.— Developing skills related to use of computer-based tools is critical for practicing genomic pathology. However, given the relative novelty of genomics education, residency programs may lack faculty members with adequate expertise and/or time to implement training. A virtual team-based learning (TBL) environment would make genomic pathology education available to more trainees. Objective.— To translate an extensively implemented in-person TBL genomic pathology workshop into a virtual environment and to evaluate both knowledge and skill acquisition. Design.— Using a novel interactive simulation approach, online modules were developed translating aspects of the TBL experience into the virtual environment with a goal of acquisition of necessary computer-related skills. The modules were evaluated at 10 postgraduate pathology training programs using a pre-post test design with participants deidentified. A postmodule anonymous survey obtained participant feedback on module quality and efficacy. Results.— There were 147 trainees who received an email request to voluntarily participate in the study. Of these, 43 trainees completed the pretest and 15 (35%) subsequently completed the posttest. Mean overall scores were 45% on the pretest compared with 70% on the posttest (P < .001; effect size = 1.4). Posttest improvement of results was similar for questions testing acquisition of knowledge versus skills. Regarding the 19 participants who took the survey, 18 (95%) would recommend the modules to others and believed they met the stated objectives. Conclusions.— A simulation-based approach allows motivated pathology trainees to acquire computer-related skills for practicing genomic pathology. Future work can explore efficacy in a nonvoluntary setting and adaptation to different specialties, learners, and computer tools.

In-system clinical laboratories have proven themselves to be a fundamentally important resource to their institutions during the COVID-19 pandemic of the past year. The ability to provide SARS-CoV-2 molecular testing to our hospital system allowed us to offer the best possible care to our patients, and to support neighboring hospitals and nursing homes. In-house testing led to significant revenue enhancement to the laboratory and institution, and attracted new patients to the system. Timely testing of inpatients allowed the majority who did not have COVID-19 infection to be removed from respiratory and contact isolation, conserving valuable personal protective equipment and staff resources at a time that both were in short supply. As 2020 evolved and our institution restarted delivery of routine care, the availability of in-system laboratory testing to deliver both accurate and timely results was absolutely critical. In this article, we attempt to demonstrate the value and impact of an in-system laboratory during the COVID-19 pandemic. A strong in-house laboratory service was absolutely critical to institutional operational and financial success during 2020, and will ensure resiliency in the future as well.

Training in surgical pathology specimen dissection and microscopic diagnosis is an integral part of pathology residency training, as surgical pathology is one of the defining activities of most pathologists. The Accreditation Council for Graduate Medical Education and the American Board of Pathology policies delineate guidelines and requirements for residency training. Both the ACGME and ABP require that residents are ready for “independent practice” upon completion of training (ACGME) and for board eligibility (ABP). This position paper, developed through a consensus process involving the Association of Pathology Chairs, including the Program Directors and Graduate Medical Education committee, expands on these guidelines and the importance of gross dissection as a part of training.

Background: Combined use of MammaPrint and a molecular subtyping profile (BluePrint) identifies disease subgroups with marked differences in long-term outcome and response to neo-adjuvant therapy [Glück et al. BCRT 2013]. The aim of this study is to compare tumor subtyping between clinical immunohistochemistry (IHC)/fluorescent in-situ (FISH) hybridization and MammaPrint and BluePrint and to determine which method of subtyping most accurately predicts long term outcome. Methods: 325 frozen/FFPE tumor samples from patients with AJCC stage TI-III, N0-Ib breast cancers were obtained from two separate cancer centers (NorthShore and Fox Chase Cancer Center) in the United States from 1992-2010. Median follow-up was 10.2 years. All patients underwent surgical excision and adjuvant therapy according to NCCN guidelines. Clinical subtyping included IHC and FISH whereas MammaPrint and BluePrint included microarray analysis. Results: Median age at diagnosis was 55 years (range 28-89 years) and 138 (42%) were AJCC Stage I, 119 (37%) Stage IIA, and 68 (21%) Stage IIB. One hundred three (33%) patients received adjuvant endocrine therapy (ET) alone, 84 (27%) received adjuvant chemotherapy (CT) alone, 104 (33%) received both modalities and 25 (7%) did not receive adjuvant treatment. Clinical Hormone Receptor (HR) and HER2 status assessment revealed that 208 (64%) of all tumors examined were luminal-like (ER/PR positive, HER2 negative), 52 (16%) were classified as HER2 positive and 65 (20%) as triple negative. Molecular classification using BluePrint demonstrated discordance in the following clinical groups: 20 out of 208 (10%) originally classified as luminal-like were reclassified as HER2-type (35%, n = 7) and as Basal-type (65%, n = 13) by BluePrint; 26 out of 52 (50%) previously identified as HER2 positive were reclassified as Luminal A (19%, n = 5), Luminal B (50%, n = 13), and 8 (31%) as Basal-type by BluePrint; 14 out of 65 (22%) patients originally identified as triple negative were reclassified as Luminal A (14%, n = 2), 5 (36%) as Luminal B and 7 (50%) as Basal-type by BluePrint. Molecular classification with BluePrint and MammaPrint identifies 40% of patients as Luminal A-type with 99% distant metastases free survival (DMFS) at 5 years. DMFS at 5-years for Luminal B tumors and HER2-type tumors was similar between clinical subtyping and BluePrint/MammaPrint subtyping but for triple negative tumors was different. For tumors using clinical subtyping, the DMFS for triple negative tumors was 84% compared to 93% for BluePrint/MammaPrint subtyping. Discordant cases are being centrally re-assessed for ER, PR and HER2. Conclusions: Molecular subtyping with BluePrint and MammaPrint leads to a more accurate molecular classification with clinically significant prognostic stratification. The use of MammaPrint and BluePrint in the management of patients with primary breast cancer should be considered to select adjuvant therapy in this era of personalized medicine. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-23.

Antimicrobial resistance to clarithromycin is a growing concern in the treatment of Helicobacter pylori and is associated with three major point mutations of the 23S rRNA, A2142C, A2142G, and A2143G. The use of traditional culture-based methods for determination of clarithromycin resistance in H. pylori are time consuming and lack sensitivity. We implemented a real-time PCR with melt curve analysis to detect and characterize H. pylori in formalin-fixed, paraffin-embedded gastric biopsy specimens to assess the frequency of clarithromycin resistance mutations in our study population. One hundred and fifty-three formalin-fixed, paraffin-embedded gastric biopsies were chosen on the basis of positive immunohistochemical staining for H. pylori and an accompanying histopathological diagnosis of Helicobacter-associated gastritis. New adjacent sections were taken for immunohistochemical staining and DNA extraction with subsequent testing by PCR assay and melt curve analysis using a primer and probe combination first described by Oleastro et al.(12) One hundred and forty-six samples demonstrated adequate amplification of a human DNA control target. Of these, there were 122 H. pylori immunohistochemistry-positive samples. In all, 103 out of 122 (84%) immunohistochemistry-positive samples demonstrated amplifiable H. pylori 23S rRNA gene target and 19 (16%) demonstrated no amplification of H. pylori. Twenty-two samples were negative for H. pylori by immunohistochemistry and PCR. Two were negative for H. pylori by immunohistochemistry, but were positive for H. pylori by PCR. In all, 52 out of 105 (50%) PCR-positive samples demonstrated resistance mutations, and it was determined that a heterogeneous population of mutated and unmutated organisms was present in 11 out of 52 samples. The use of PCR assays allows for a timely assessment of clarithromycin resistance status without the disadvantages of culture-based methods, and may lead to a decrease in treatment failure rates.

Context.—Recently, robotic-assisted laparoscopic prostatectomy has replaced open retropubic radical prostatectomy as the surgical procedure of choice. This less-invasive approach offers many advantages but exposes prostate tissue to longer periods of warm ischemia that may affect subsequent analysis of biomarkers. Objective.—To analyze the nucleic acid quality and quantity isolated from open versus laparoscopic prostatectomies. Design.—Nucleic acids were isolated from 10 open-obtained and 10 laparoscopic-obtained tissues stored in our prostate sample repository. Nucleic acid integrity was assessed via electrophoresis and polymerase chain reaction amplification of RNA and DNA targets ranging in size from 125 to 939 base pairs. Results.—The DNA yield, integrity, and polymerase chain reaction amplification were identical between samples obtained from both surgical approaches. The RNA integrity number and yield were similar, as was β-2 microglobulin mRNA amplification up to 652 base pairs. However, 2 of 10 samples (20%) collected robotically showed decreased real-time reverse transcriptase-polymerase chain reaction amplification of prostate-specific antigen messenger RNA, especially with targets larger than 300 base pairs. Conclusions.—Generally, the quality and quantity of nucleic acids isolated from prostate tissue obtained via open or laparoscopic approaches are equivalent, suggesting that procurement of tissues is appropriate from either procedure. However, some loss of reverse transcriptase-polymerase chain reaction amplification of larger RNA targets was noted in the laparoscopic samples; appropriate design of assays to keep amplicon sizes small and the use of internal controls to assess RNA integrity is recommended.

One of the key roles performed by pathologists is determination of the presence or absence of tumor in clinical samples. This is the basis for most approaches to staging, monitoring response to treatment, and detecting relapse of neoplasia and, as such, is a critical step in determining the course of patient management. Pathologists have utilized a variety of methods, continually seeking to improve assay performance and thus patient outcome. The literature reflects this quest, including reports assessing the increased sensitivity afforded by immunohistochemistry (IHC), flow cytometry, and, more recently, molecular approaches for the detection of tumor cells and nucleic acids in blood and bone marrow samples. The goal is, of course, the more accurate detection of disease spread and, ultimately, better patient care.

Background Herpetic dermatitis due to herpes simplex virus (HSV) and varicella zoster virus (VZV) can present with similar clinical and histopathologic features. Further confounding matters, viral cytopathic changes are not always observed in biopsy specimens. Therefore, use of polymerase chain reaction (PCR) analysis can play an integral role in the definitive diagnosis of herpetic dermatitis and in the distinction of HSV-1/HSV-2 from VZV. Methods Forty patients with skin biopsies (2004–2011) had PCR analysis performed to detect HSV-1/2 or VZV. Patient demographics, clinical impression and histopathologic characteristics were reviewed and correlated with PCR findings. Results Overall, there was complete correlation between clinical impression, histopathology and PCR results in 21 of 40 cases. In 19 cases, clinical impression and histopathology were discrepant and in 15 of these cases PCR confirmed HSV or VZV infection. We also describe 3 cases of herpetic dermatitis without viral change that histopathologically demonstrate the pattern of a dermal hypersensitivity reaction. Conclusions The results of this study suggest that routine use of PCR for definitive diagnosis of herpetic dermatitis should be considered when there is a clinical suspicion of herpes virus infection, even when there is a lack of specific histopathologic findings. Additionally, a dermal hypersensitivity reaction should be recognized as one histopathologic manifestation of herpes incognito.

Genomic technologies are dramatically changing the practice of medicine. Next-generation sequencing has allowed prognostic stratification of cancer patients, personalized drug therapy and the identification of genetic risk factors for a multitude of diseases. As the physicians who oversee tissue- and laboratory-based diagnostic testing, pathologists must understand and utilize this new technology for the benefit of patients; however, only a minority of pathology residency programs currently provide training in genomics. In response to this urgent need, the Training Residents in Genomics (TRIG) Working Group has made significant progress towards creating, implementing, evaluating and disseminating a national curriculum in genomic pathology. Although presented in the context of pathology training, the approach described in this review can serve as model for education in genomic medicine of students, trainees or professionals in other areas of healthcare.

The current best serum marker for pancreatic cancer, CA 19-9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19-9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19-9 antigen, we immunoprecipitated the CA 19-9 antigen from pooled sera and identified the associated proteins using mass spectrometry. Among the high-confidence identifications, we confirmed the presence of the CA 19-9 antigen on Apolipoprotein B-100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19-9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. 10–25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19-9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of mass spectrometry and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.

Influenza virus subtyping has emerged as a critical tool in the diagnosis of influenza. Antiviral resistance is present in the majority of seasonal H1N1 influenza A infections, with association of viral strain type and antiviral resistance. Influenza A virus subtypes can be reliably distinguished by examining conserved sequences in the matrix protein gene. We describe our experience with an assay for influenza A subtyping based on matrix gene sequences. Viral RNA was prepared from nasopharyngeal swab samples, and real-time RT-PCR detection of influenza A and B was performed using a laboratory developed analyte-specific reagent-based assay that targets a conserved region of the influenza A matrix protein gene. FluA-positive samples were analyzed using a second RT-PCR assay targeting the matrix protein gene to distinguish seasonal influenza subtypes based on differential melting of fluorescence resonance energy transfer probes. The novel H1N1 influenza strain responsible for the 2009 pandemic showed a melting profile distinct from that of seasonal H1N1 or H3N2 and compatible with the predicted melting temperature based on the published novel H1N1 matrix gene sequence. Validation by comparison with the Centers for Disease Control and Prevention real-time RT-PCR for swine influenza A (novel H1N1) test showed this assay to be both rapid and reliable (>99% sensitive and specific) in the identification of the novel H1N1 influenza A virus strain.

Neuroendocrine (NE) phenotype, seen in >30% of prostate adenocarcinomas (PCa), and NE prostate tumors are implicated in aggressive prostate cancer. Formation of NE prostate tumors in the TRAMP mouse model was suppressed in mice lacking the ubiquitin ligase Siah2, which regulates HIF-1alpha availability. Cooperation between HIF-1alpha and FoxA2, a transcription factor expressed in NE tissue, promotes recruitment of p300 to transactivate select HIF-regulated genes, Hes6, Sox9, and Jmjd1a. These HIF-regulated genes are highly expressed in metastatic PCa and required for hypoxia-mediated NE phenotype, metastasis in PCa, and the formation of NE tumors. Tissue-specific expression of FoxA2 combined with Siah2-dependent HIF-1alpha availability enables a transcriptional program required for NE prostate tumor development and NE phenotype in PCa.

Although patients with chronic pancreatitis (CP) have an increased risk of pancreatic cancer (PanCa), the timely detection of PanCa often is difficult, because the symptoms of CP and PanCa are very similar. Moreover, secondary inflammation may be identified in PanCa, further complicating diagnosis. To improve the survival of patients with PanCa, a reliable test to differentiate CP from PanCa is needed. In this article, the authors describe a methylation profile of cell-free plasma DNA that distinguished CP from PanCa with90% accuracy.: Methylation in cell-free, plasma DNA was compared among 30 samples from patients with CP, 30 samples from patients with PanCa, and 30 samples from healthy controls (N) using a microarray-mediated methylation analysis of 56 fragments in each sample (MethDet56). Statistical analysis was done by using the Fisher exact test, a naive Bayes algorithm, and 25 rounds of 5-fold cross-validation.: The MethDet56 methylation analysis technique identified 17 gene promoters as informative (8 for distinguishing N from CP and 14 for distinguishing CP from PanCa). It achieved 81.7% sensitivity and 78% specificity (P.01) in the detection of CP (N vs CP) and 91.2% sensitivity and 90.8% specificity (P.01) in the differential detection of PanCa (PanCa vs CP).: The current data suggested that, among patients with pancreatic disease, the methylation profiles of inflammatory disease and cancer are different and open a new venue for the development of biomarkers for differential diagnosis. Further investigation of diagnostic biomarkers for pancreatic cancer based on methylation in cell-free, circulating DNA appears to be warranted. Cancer 2010. (c) 2010 American Cancer Society.

In many families with histories suggestive of BRCA1- or BRCA2-related disease, the proband is deceased. Reliable assessment of archived tissue blocks not amenable to full gene sequencing would be helpful. In this study, a polymerase chain reaction (PCR) assay using primers that bracket the BRCA mutation site and microfluidics-based detection of heteroduplex/amplicon size differences was developed to circumvent artifacts associated with low quality DNA from formalin-fixed paraffin-embedded (FFPE) tissue. Genomic DNA was extracted from 100 FFPE specimens from patients that had previously undergone BRCA gene sequence analysis on blood specimens. Conventional PCR amplification products were differentiated using the Agilent 2100 Bioanalyzer. One FFPE specimen failed to amplify the wild-type alleles for all three sites and was therefore called indeterminate. All 62 FFPE specimens with known Ashkenazi Jewish founder mutations had both the wild-type and the correct mutated allele amplified, including one specimen that failed to amplify the mutant allele in other real-time PCR assays. Appropriately, 21 FFPE specimens known to have other BRCA1/2 mutations and 16 without any mutation had only the wild-type allele correctly amplified for each target. Therefore, by changing the primer location and detecting amplicons via heteroduplexes formed by size differences, we identified mutations from FFPE tissues missed using real-time methods.

We compared two real-time PCR assays (both by the use of melting curve analysis) for their ability to identify Staphylococcus species directly from 200 positive blood culture bottles. The PCR assays correctly identified 83% to 94% of the Staphylococcus isolates to species clusters. Molecular testing significantly outperformed commercially available latex tests (sensitivity for both latex tests

Nasal and perianal swab specimens were tested for detection of Staphylococcus aureus and vancomycin-resistant Enterococcus species (VRE) using a laboratory-developed real-time PCR test and microbiological cultures. The real-time PCR and culture results for S. aureus were similar. PCR had adequate sensitivity, but culture was more specific for the detection of VRE.

Background. Clostridium difficile-associated diarrhea (CDAD) is the major cause of health care-associated infectious diarrhea. Current laboratory testing lacks a single assay that is sensitive, specific, and rapid. The purpose of this work was to design and validate a sensitive and specific real-time polymerase chain reaction (PCR) diagnostic test for CDAD.Methods. This observational validation study of a new real-time PCR assay occurred from July 2004 through April 2006 and involved the testing of 1368 stool samples. As the final validation portion of the investigation, 350 inpatients were prospectively interviewed for clinical findings for 365 episodes of diarrheal illness. Test results and clinical criteria were used to assess the performance of 4 assays.Results. Using clinical criteria requiring at least 3 loose stools in 1 day as part of the reference standard for a positive test result supporting CDAD, the sensitivity, specificity, and positive and negative predictive values were 73.3%, 97.6%, 73.3%, and 97.6%, respectively, for enzyme immunoassay; 93.3%, 97.4%, 75.7%, and 99.4%, respectively, for real-time PCR; 76.7%, 97.1%, 69.7%, and 97.9%, respectively, for cell culture cytotoxin assay; and 100.0%, 95.9%, 68.2%, and 100.0%, respectively, for anaerobic culture (for toxigenic C. difficile strains). The real-time PCR and anaerobic culture assays were significantly more sensitive than the enzyme immunoassay (P < .01 to P < .05).Conclusions. With an assay turnaround time of

We evaluated the use of the BD GeneOhm MRSA real-time PCR assay (BD Diagnostics, San Diego, CA) for the detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA). The initial evaluation consisted of 403 paired nasal swabs and was done using the specimen preparation provided with the kit and an in-house lysis method that was specifically developed to accommodate large-volume testing using a minimal amount of personnel time. One swab was placed in an achromopeptidase (ACP) lysis solution, and the other was first used for culture and then prepared according to the kit protocol. PCR was performed on both lysates, and results were compared to those for culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay were 98%, 96%, 77%, and 99.7% with the kit lysate and 98%, 95%, 75%, and 99.7% with the ACP lysate ( P , not significant), respectively. The second evaluation was done after implementation of all-admission surveillance using PCR with ACP lysis and a sampling of 1,107 PCR-negative samples and 215 PCR-positive samples that were confirmed by culture. The results of this sampling showed an NPV of 99.9% and a PPV of 73.5% (prevalence, 6%), consistent with our initial findings. The BD GeneOhm MRSA assay is an accurate and rapid way to detect MRSA nasal colonization. When one is dealing with large specimen numbers, the ACP lysis method offers easier processing without negatively affecting the sensitivity or specificity of the PCR assay.

Screening assays for Neisseria gonorrhoeae exhibit low positive predictive values, particularly in low-prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae , the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive for cppB . However, cppB was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or porA pseudogene (2%) assays. The porA pseudogene assay had the highest specificity for both genders but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppB , due to poor specificity. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non- N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay.

Nasal carriage of Staphylococcus aureus is considered a source of subsequent infection in health care settings. Utilization of real-time polymerase chain reaction (PCR) for detection of S. aureus has the potential to dramatically affect infection control practice by rapidly identifying S. aureus-colonized patients. We developed and validated the use of real-time PCR for detection of S. aureus colonization in two patient populations. Paired nasal swabs were collected from 299 neonates and from 151 adult patients at Evanston Hospital. One swab was used for culture and the other placed into a bacterial lysis solution containing achromopeptidase. The DNA liberated was used as the template for real-time PCR with primers for the femA gene. SYBR Green was used for amplicon detection. In the neonatal population the sensitivity, specificity, predictive value positive and predictive value negative for culture and PCR was 92% versus 96%, 100% versus 100%, 100% versus 100%, and 98% versus 99%, respectively. In the adults the results were 90% versus 100%, 100% versus 98%, 100% versus 96%, and 95% versus 100%, respectively. Real-time PCR was able to detect S. aureus in 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.

This invention provides compositions and methods for detecting HPV in a sample. This invention also provides related kits, systems, and computers.

Background: Microarray-based gene expression profiling demonstrated that breast cancer is a heterogeneous group of different diseases characterized by distinct molecular aberrations, rather than one disease. Improved understanding of the molecular phenotypes of the disease has already shown prognostic and predictive value and, when prospectively applied, could have dramatic implications in establishing a more personalized approach to the management of early-stage breast cancer. Combined use of MammaPrint and a molecular subtyping profile (BluePrint) identifies disease subgroups with marked differences in long-term outcome and response to neo-adjuvant therapy [Glück et al. ASCO 2012]. The aim of this study was to evaluate the implication of accurate molecular subtyping using MammaPrint and BluePrint in women with early-stage breast cancer treated at US Institutions following National Comprehensive Cancer Network (NCCN) standard guidelines. Methods: 208 frozen tumor samples from consecutive BC patients (TI-III, N0-Ib) were obtained from 2 US centers. Median age at diagnosis was 56 years (range 28–89 years). Between 1992 and 2010 patients were treated either with breast-conserving therapy or mastectomy with axillary lymph node dissection followed by systemic adjuvant therapy when indicated. Sixty-three percent of patients received adjuvant endocrine therapy (ET), 58% received adjuvant chemotherapy (CT) and 32% received both. Hormone Receptor (HR) and HER2 status were assessed by immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH), following standard guidelines. Median follow-up was 11.3 years. Survival was assessed for patient groups according to local pathological assessment and compared with molecular classification of patients (centrally assessed full genome expression at Agendia laboratory). Results: Standard HR and HER2 status assessment revealed that 57% of all tumors examined were luminal-like (ER/PR positive, HER2 negative), 20% HER2 positive and 24% triple negative. Molecular classification demonstrated discordance in the following clinical groups: 16 out of 41 patients previously identified as HER2 positive were reclassified as Luminal-type, with 97% 5-year distant metastases-free survival (DMFS) for Luminal A (MammaPrint Low Risk/Luminal-type) and 98% for Luminal B (MammaPrint High Risk/Luminal-type). Ten patients identified with clinical triple-negative tumors were reclassified with molecular subtyping as HER2 positive (n = 6) and Luminal-type (n = 4). Of the patients classified with BluePrint Basal-type tumors, 58 (28%) had a 5-year DMFS of 82% (81% received adjuvant CT). Of those with HER2-type tumors, 25 (12%) had a 5-year DMFS of 76% (88% received adjuvant CT without trastuzumab). Discordant cases are being centrally re-assessed for ER, PR and HER2. Conclusions: This retrospective study showed that Molecular Subtyping with BluePrint and MammaPrint leads to clinically significant prognostic and molecular stratification. The use of MammaPrint and BluePrint in the management of patients with primary breast cancer should be considered for a more accurate selection of adjuvant therapies in this era of personalized medicine. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-01.

An explosion of knowledge and technology is revolutionizing medicine and patient care. Novel testing must be brought to the clinic with safety and accuracy, but also in a timely and cost-effective manner, so that patients can benefit and laboratories can offer testing consistent with current guidelines. Under the oversight provided by the Clinical Laboratory Improvement Amendments, laboratories have been able to develop and optimize laboratory procedures for use in-house. Quality improvement programs, interlaboratory comparisons, and the ability of laboratories to adjust assays as needed to improve results, utilize new sample types, or incorporate new mutations, information, or technologies are positive aspects of Clinical Laboratory Improvement Amendments oversight of laboratory-developed procedures. Laboratories have a long history of successful service to patients operating under Clinical Laboratory Improvement Amendments. A series of detailed clinical examples illustrating the quality and positive impact of laboratory-developed procedures on patient care is provided. These examples also demonstrate how Clinical Laboratory Improvement Amendments oversight ensures accurate, reliable, and reproducible testing in clinical laboratories.

Context.—Genomic medicine is revolutionizing patient care. Physicians in areas as diverse as oncology, obstetrics, and infectious disease have begun using next-generation sequencing assays as standard diagnostic tools. Objective.—To review the role of pathologists in genomic testing as well as current educational programs and future training needs in genomic pathology. Data Sources.—Published literature as well as personal experience based on committee membership and genomic pathology curricular design. Conclusions.—Pathologists, as the directors of the clinical laboratories, must be prepared to integrate genomic testing into their practice. The pathology community has made significant progress in genomics-related education. A continued coordinated and proactive effort will ensure a future vital role for pathologists in the evolving health care system and also the best possible patient care.

Background This study compares the predictability of 5 tumor markers for distant metastasis and mortality in pancreatic neuroendocrine tumors (PNETs). Methods A total of 128 patients who underwent pancreatectomy for nonfunctioning PNETs between 1998 and 2011 were evaluated. Tumor specimens were stained via immunochemistry for cytoplasmic and nuclear survivin, cytokeratin 19 (CK19), c-KIT, and Ki67. Univariate and multivariate regression analyses and receiver operating characteristics curve were used to evaluate the predictive value of these markers. Results A total of 116 tumors (91%) were positive for cytoplasmic survivin, 95 (74%) for nuclear survivin, 85 (66.4%) for CK19, 3 for c-KIT, and 41 (32%) for Ki67 >3%. Twelve (9%) tumors expressed none of the markers. Survivin, CK19, and c-KIT had no substantial effect on distant metastasis or mortality. Age >55 years, grade 3 histology, distant metastasis, and Ki67 >3% were associated with mortality (P 3% was the best predictor (83%) of mortality with an area under the curve of 0.85. Ki67 >3% also predicted occurrence of distant metastases with odds ratio of 9.22 and 95% confidence interval of 1.55–54.55 (P Conclusion Of the 5 markers studied, only Ki67 >3% was greatly associated with distant metastasis and death. Survivin, CK19, and c-KIT had no prognostic value in nonfunctioning PNETs.

Background: Nucleic acid amplification technologies such as PCR are revolutionizing the detection of infectious pathogens such as tuberculosis (TB). Amplification technology offers the potential for the diagnosis of TB in a few hours with a high degree of sensitivity and specificity. However, molecular assays neither replace nor reduce the need for conventional smear and culture, speciation, and antibiotic sensitivity assays. Methods: We undertook prospective studies of sputum samples to assess the performance of two PCR-based assays for the detection of TB as well as the impact of more rapid availability of test results on patient care. Results: The sensitivity of both the in-house and Amplicor PCR assays was 100% for smear-positive sputa. For smear-negative sputa (two sputum samples collected during the first 24 h of hospitalization), the sensitivity was 85% for our in-house PCR assay and 74% for the Roche PCR assay. Approximately 10% of the smear- and culture-negative sputa yielded positive PCR results; however, more than one-half of these were positive with both the in-house and Amplicor assays, suggesting the presence of TB DNA or organisms. Several of these came from patients whose other samples grew Mycobacterium tuberculosis during the same admission, and others came from patients who had previously treated TB. Overall, the specificities of the in-house and Amplicor PCR assays in smear-negative patients were 86% and 93%, respectively. Conclusions: Molecular detection of slow-growing pathogens such as M. tuberculosis have the potential to improve clinical care through a dramatic reduction in the time required for detection and may provide substantial savings in the overall cost of care of a patient compared with conventional smear, culture, and speciation alone, despite the fact that conventional assays must still be performed for speciation of nontuberculous mycobacteria and for full assessment of antibiotic sensitivity.

From the Editor: Our world is changing, and our field is changing with it. Each new advance in technology, each new application, each step forward in automation and reduction in time and work required to perform molecular testing expands the potential of molecular diagnostics to impact the clinical management of patients and their families. The completion of the Human Genome Project will propel our field forward rapidly, and has already had a major impact in raising the visibility of our field in the public eye. Concomitantly, we have become more visible to government and regulatory agencies as well. Many of you have carefully followed the discussions of the Food and Drug Administration (FDA) over the years, and more recently the deliberations of the Secretaries Advisory Committee on Genetic Testing (SACGT) and the Clinical Laboratory Improvement Advisory Committee (CLIAC). Both groups have been at work these past two years, discussing the means by which genetic testing should be ordered, performed, reported, and overseen in the United States. Obviously, the results of their discussions would have enormous implications for those involved in molecular diagnostics. The primary concern leading to the establishment of SACGT approximately two years ago was protection of the public from harm due to inappropriately used or performed genetic testing. SACGT is a federally mandated group acting under the auspices of the Secretary of Health and Human Services. They hold that increased oversight of genetic testing is necessary, particularly since much of genetic testing is currently done via methods developed by individual laboratories and not by FDA-approved kits, and have recommended that the FDA be the agency to provide this oversight. In their deliberations, the definition of genetic test is very broad and would include both somatic and germline alterations. They have worked to develop a mechanism by which tests could be stratified based on their intended use and potential impact on patient care. In theory, this would allow triaging of “high-risk” tests such as those for Huntington’s disease or BRCA1 into a high scrutiny category necessitating high level oversight, whereas less critical tests such as Factor V Leiden, for example, would require a lower intensity scrutiny. Such algorithms have thus far proven unwieldy and ineffective for risk stratification, however, in part due to the multiple intended uses for many of these assays (disease confirmation, prenatal assessment, carrier detection, population screening, etc) and thus are still under discussion. SACGT holds firmly that some sort of FDA approval mechanism is needed before these tests can be applied to clinical decision-making. Numerous professional groups have responded to SACGT and have worked with the FDA through the FDA-Professional Organization Roundtable discussions in an effort to help develop a practical oversight plan. Many of these groups, including the Association for Molecular Pathology (AMP), hold that existing mechanisms for laboratory accreditation and oversight are best suited to be the nidus for any new oversight programs. Laboratories performing clinical testing must currently be certified by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Act (CLIA). Inspection and accreditation of laboratories by CAP provides a very detailed assessment of laboratory and quality control practices in molecular diagnostics laboratories. For example, laboratories are required to keep on file details and data of validation studies for each assay performed. CLIA requires additional quality-oriented practices. At the FDA Roundtable discussions, a genetic “template” was developed that could be used to help gather data on the use, performance, interpretation, and reporting of a genetic test. The template was very well received by SACGT, and discussion is underway regarding utilization of this template. The template would certainly help to gather and organize information on genetic testing and detailed laboratory practices, but numerous questions remain unanswered regarding further benefits of implementing use of the template. Would this move truly improve the quality of genetic testing in the United States, and in doing so, protect the public? Or would it duplicate existing regulatory mechanisms such as CAP accreditation, add to the administrative burden and cost of the labs, and thus limit public access to genetic testing? The views of AMP were presented at the most recent SACGT hearing in May 2001 by Dr. Debra Leonard, Past-President of AMP; her comments follow this introduction. Another federal group active in this arena is CLIAC. CLIAC has focused on defining genetic testing and addressing pre- and postanalytic issues in genetic testing, such as informed consent and reporting of results. Of note is that the definition of a genetic test put forth by CLIAC is extremely broad, to include not only nucleic acids but also proteins and metabolites. The recommendations of CLIAC are discussed in more detail by Dr. Andrea Gonzalez, Chair of the AMP Policy Committee, in the update to follow. During the coming months, much will become clear with these various regulatory issues. I anticipate that we will be satisfied with some aspects of the increased oversight, and very dissatisfied with others. Regardless of the outcomes, we will have to find ways to live with the recommendations of these committees. In the meantime, we must remain actively involved, commenting and working with federal agencies when possible to effect workable solutions. In the end, better patient care is the goal for all of us.