Cell free DNA in patients with pancreatic adenocarcinoma: clinicopathologic correlations.

Detection of circulating tumor DNA (ctDNA) from plasma cell free DNA (cfDNA) has shown promise for diagnosis, therapeutic targeting, and prognosis. This study explores ctDNA detection by next generation sequencing (NGS) and associated clinicopathologic factors in patients with pancreatic adenocarcinoma (PDAC). Patients undergoing surgical exploration or resection of pancreatic lesions were enrolled with informed consent. Plasma samples (4-6 ml) were collected prior to surgery and cfDNA was recovered from 95 plasma samples. Adequate cfDNA for NGS (20 ng) was obtained from 81 patients. NGS was performed using the Oncomine Lung cfDNA assay on the Ion Torrent S5 sequencing platform. Twenty-five patients (30.9%) had detectable mutations in KRAS and/or TP53 with allele frequencies ranging from 0.05 to 8.5%, while mutations in other genes were detected less frequently and always along with KRAS or TP53. Detectable ctDNA mutations were more frequent in patients with poorly differentiated tumors, and patients without detectable ctDNA mutations showed longer survival (medians of 10.5 months vs. 18 months, p = 0.019). The detection of circulating tumor DNA in pancreatic adenocarcinomas is correlated with worse survival outcomes.

The safety and efficacy of systemic delivery of a new liver-de-targeted TGFß signaling inhibiting adenovirus in an immunocompetent triple negative mouse mammary tumor model.

Aberrant TGFß signaling is linked to metastasis and tumor immune escape of many cancers including metastatic triple negative breast cancer (mTNBC). Previously, we have found that oncolytic adenoviruses expressing a TGFß signaling inhibitory protein (sTGFßRIIFc) induced immune activation in a mouse TNBC (4T1) immunocompetent subcutaneous model with intratumoral injection. Systemic administration of adenoviruses can be a superior route to treat mTNBC but faces the challenges of increased toxicity and viral clearance. Thus, we created a liver-de-targeted sTGFßRIIFc- and LyP-1 peptide-expressing adenovirus (mHAdLyp.sT) with enhanced breast cancer cell tropism. Its safety and immune response features were profiled in the 4T1 model. Our data showed that the systemic administration of mHAdLyp.sT resulted in reduced hepatic and systemic toxicity. mHAdLyp.sT was also effective in increasing Th1 cytokines and anti-tumor cell populations by cytokine analysis, spleen/tumor qRT-PCR, and flow cytometry. We further tested the therapeutic effects of mHAdLyp.sT alone and in combination with immune checkpoint inhibitors (ICIs). mHAdLyp.sT alone and with all ICI combinations elicited significant inhibition of lung metastasis by histological analysis. When mHAdLyp.sT was combined with both anti-PD-1 and anti-CTLA-4 antibodies, primary 4T1 tumor growth was also significantly inhibited. We are confident in advancing this new treatment option for mTNBC.

Molecular detection of bacteria, placental inflammation, and neonatal sepsis risk.

OBJECTIVE: To identify bacteria in umbilical cord tissue and investigate the association with placental inflammation and neonatal sepsis risk score. STUDY DESIGN: Retrospective cohort study from 2017-2019. RNA was extracted from umbilical cord tissue and NanoString nCounter used to identify seven bacteria genera. Sepsis risk score was calculated using the Kaiser sepsis calculator. Placental histopathology was abstracted from medical records. RESULTS: Detection of bacterial RNA in the umbilical cord (n = 96/287) was associated with high-stage maternal and fetal acute placental inflammation (maternal 35.4% vs 22.5%, p = 0.03 and fetal 34.4% vs 19.4%, p < 0.01) and maternal vascular malperfusion (36.5% vs 23.0%, p = 0.02). Detection of Ureaplasma spp. was also associated with increased sepsis risk score (1.5/1000 [0.6, 8.6] vs 0.9/1000 [0.2, 2.9], p = 0.04). CONCLUSION: Umbilical cord bacterial pathogens are linked to fetal and maternal placental inflammation and maternal vascular malperfusion during gestation and associated with increased sepsis risk score in the neonate.

Personalized medicine in a community health system: the NorthShore experience.

Genomic and personalized medicine implementation efforts have largely centered on specialty care in tertiary health systems. There are few examples of fully integrated care systems that span the healthcare continuum. In 2014, NorthShore University HealthSystem launched the Center for Personalized Medicine to catalyze the delivery of personalized medicine. Successful implementation required the development of a scalable family history collection tool, the Genetic and Wellness Assessment (GWA) and Breast Health Assessment (BHA) tools; integrated pharmacogenomics programming; educational programming; electronic medical record integration; and robust clinical decision support tools. To date, more than 225,000 patients have been screened for increased hereditary conditions, such as cancer risk, through these tools in primary care. More than 35,000 patients completed clinical genetic testing following GWA or BHA completion. An innovative program trained more than 100 primary care providers in genomic medicine, activated with clinical decision support and access to patient genetic counseling services and digital healthcare tools. The development of a novel bioinformatics platform (FLYPE) enabled the incorporation of genomics data into electronic medical records. To date, over 4,000 patients have been identified to have a pathogenic or likely pathogenic variant in a gene with medical management implications. Over 33,000 patients have clinical pharmacogenomics data incorporated into the electronic health record supported by clinical decision support tools. This manuscript describes the evolution, strategy, and successful multispecialty partnerships aligned with health system leadership that enabled the implementation of a comprehensive personalized medicine program with measurable patient outcomes through a genomics-enabled learning health system model that utilizes implementation science frameworks.

The Safety and Efficacy of Systemic Delivery of a New Liver-de-targeted TGFß Signaling Inhibiting Adenovirus in an Immunocompetent Triple Negative Mouse Mammary Tumor Model.

Aberrant TGFß signaling is linked to metastasis and tumor immune escape of many cancers including metastatic triple negative breast cancer (mTNBC). Previously, we have found that oncolytic adenoviruses expressing a TGFß signaling inhibitory protein (sTGFßRIIFc) induced immune activation in a mouse TNBC (4T1) immunocompetent subcutaneous model with intratumoral injection. Systemic administration of adenoviruses can be a superior route to treat mTNBC but faces the challenges of increased toxicity and viral clearance. Thus, we created a liver-de-targeted sTGFßRIIFc- and LyP-1 peptide-expressing adenovirus (mHAdLyp.sT) with enhanced breast cancer cell tropism. Its safety and immune response features were profiled in the 4T1 model. Our data showed that the systemic administration of mHAdLyp.sT resulted in reduced hepatic and systemic toxicity. mHAdLyp.sT was also effective in increasing Th1 cytokines and anti-tumor cell populations by cytokine analysis, spleen/tumor qRT-PCR, and flow cytometry. We further tested the therapeutic effects of mHAdLyp.sT alone and in combination with immune checkpoint inhibitors (ICIs). mHAdLyp.sT alone and with all ICI combinations elicited significant inhibition of lung metastasis by histological analysis. When mHAdLyp.sT was combined with both anti-PD-1 and anti-CTLA-4 antibodies, primary 4T1 tumor growth was also significantly inhibited. We are confident in advancing this new treatment option for mTNBC.

The Prevalence of SARS-CoV-2 in Autopsies Surrounding the Time of Pandemic Onset: A Retrospective Review of Cases.

CONTEXT.­: The first case of COVID-19 in the United States was confirmed in January 2020. Initially, little was known about the epidemiology and clinical course of the disease, and diagnostic testing was limited in the United States until March/April 2020. Since then, many studies have speculated that SARS-CoV-2 may have preexisted undiagnosed outside China before the known outbreak. OBJECTIVE.­: To evaluate the prevalence of SARS-CoV-2 in adult autopsy cases performed just before and during the beginning of the pandemic at our institution, where autopsy was not performed on known COVID-19 cases. DESIGN.­: We included adult autopsies performed in our institution from June 1, 2019, to June 30, 2020. Cases were divided into groups based on the likelihood of cause of death being related to COVID-19, presence of a clinical respiratory illness, and histologic findings of pneumonia. Archived formalin-fixed, paraffin-embedded lung tissue of all COVID-possible cases and COVID-unlikely cases with pneumonia was tested for SARS-CoV-2 RNA, using Centers for Disease Control and Prevention 2019-nCoV quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS.­: Eighty-eight cases were identified, and among those, 42 (48%) were considered COVID-possible cause of death, with 24 of those 42 cases (57%) showing respiratory illness and/or pneumonia. COVID-19 as cause of death was considered unlikely in 46 of 88 cases (52%), with 34 of those 46 cases (74%) showing no respiratory illness or pneumonia. SARS-CoV-2 real-time reverse transcription-polymerase chain reaction was performed on a total of 49 cases, 42 COVID-possible and 7 COVID-unlikely with pneumonia, and all cases were negative (0 of 49). CONCLUSIONS.­: Our data suggest that autopsied patients in our community who died between June 1, 2019, and June 30, 2020, without known COVID-19 were unlikely to have had subclinical and/or undiagnosed COVID-19 infection.

Germline mutations in high penetrance genes are associated with worse clinical outcomes in patients with non-small cell lung cancer.

Objective: To determine the frequency of pathogenic mutations in high-penetrance genes (HPGs) in patients with non-small cell lung cancer (NSCLC) and identify whether such mutations are associated with clinicopathologic outcomes. Methods: Patients with NSCLC who had consented to participate in a linked clinical database and biorepository underwent germline DNA sequencing using a next-generation sequencing panel that included cancer-associated HPGs and cancer risk-associated single nucleotide polymorphisms (SNPs). These data were linked to the clinical database to assess for associations between germline variants and clinical phenotype using Fisher's exact test and multivariable logistic and Cox regression. Results: We analyzed 151 patients, among whom 33% carried any pathogenic HPG mutation and 23% had a genetic risk score (GRS) >1.5. Among the patients without any pathogenic mutation, 31% were at cancer stage II or higher, compared with 55% of those with 2 types of HPG mutations (P = .0293); 40% of patients with both types of HPG mutations had cancer recurrence, compared with 21% of patients without both types (P = .0644). In multivariable analysis, the presence of 2 types of HPG mutations was associated with higher cancer stage (odds ratio [OR], 3.32; P = .0228), increased recurrence of primary tumor (OR, 2.93; P = .0527), shorter time to recurrence (hazard ratio [HR], 3.03; P = .0119), and decreased cancer-specific (HR, 3.53; P = .0039) and overall survival (HR, 2.44; P = .0114). Conclusions: The presence of mutations in HPGs is associated with higher cancer stage, increased risk of recurrence, and worse cancer-specific and overall survival in patients with NSCLC. Further large studies are needed to better delineate the role of HPGs in cancer recurrence and the potential benefit of adjuvant treatment in patients harboring such mutations.

Umbilical cord miRNAs to predict neonatal early onset sepsis.

OBJECTIVE: To determine if miRNA (miR) expression in umbilical cord blood and umbilical cord tissue differs between neonates with early onset sepsis (EOS) versus neonates without true infection. METHODS: Retrospective case-control study design of human patients with EOS (n = 8), presumed sepsis (N = 12) and non-infected control patients (N = 21). Differential expression of >300 miRs was examined using the MIHS-3001ZE-miScript miRNA PCR Array Human miFinder 384HC. Expression levels of miRs were normalized using the global Ct mean of expressed miR and compared between groups. Data analysis was performed using GeneGlobe data analysis software. Ratios of over and under-expressed miRs were calculated and compared between groups using receiver operating characteristic (ROC) curves. RESULTS: Both umbilical cord plasma and umbilical cord tissue revealed several miRs with differential expression with little overlap between the two specimen types. The most overexpressed miR in plasma of EOS patients was miR-211-5p and the most overexpressed in EOS cord tissue was miR-223-5p. ROC curves comparing the ratios of over and under-expressed miRs for EOS patients and controls resulted in an area under the curve of 0.787 for cord plasma (miR-211-5p/miR-142-3p) and 0.988 for umbilical cord tissue (miR-223-5p/miR-22-3p), indicating good discrimination. CONCLUSIONS: miRs show differential expression in EOS versus non-infected controls and presumed sepsis. A ratio of over and under-expressed miRs can provide a potentially sensitive and specific diagnostic test for EOS.

LyP-1-Modified Oncolytic Adenoviruses Targeting Transforming Growth Factor ß Inhibit Tumor Growth and Metastases and Augment Immune Checkpoint Inhibitor Therapy in Breast Cancer Mouse Models.

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 ß-1 (TGFß-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 sTGFßRIIFc, a TGFß decoy that can inhibit TGFß 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 sTGFßRIIFc 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.

Performance and Utilization of a Laboratory-Developed Nucleic Acid Amplification Test (NAAT) for the Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in a Low-Prevalence Area.

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.

An Oncolytic Adenovirus Targeting Transforming Growth Factor ß Inhibits Protumorigenic Signals and Produces Immune Activation: A Novel Approach to Enhance Anti-PD-1 and Anti-CTLA-4 Therapy.

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 (sTGFßRIIFc) gene. Infection of breast and renal tumor cells with rAd.sT produced sTGFßRIIFc 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 ß (TGFß) 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 TGFß and TGFß-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.

Author Correction: Integrated molecular subtyping defines a curable oligometastatic state in colorectal liver metastasis.

In the originally published version of this Article, the affiliation details for Kevin P. White inadvertently omitted 'Tempus Labs, Chicago, IL, 60654, USA'. This has now been corrected in both the PDF and HTML versions of the Article.

Integrated molecular subtyping defines a curable oligometastatic state in colorectal liver metastasis.

The oligometastasis hypothesis suggests a spectrum of metastatic virulence where some metastases are limited in extent and curable with focal therapies. A subset of patients with metastatic colorectal cancer achieves prolonged survival after resection of liver metastases consistent with oligometastasis. Here we define three robust subtypes of de novo colorectal liver metastasis through integrative molecular analysis. Patients with metastases exhibiting MSI-independent immune activation experience the most favorable survival. Subtypes with adverse outcomes demonstrate VEGFA amplification in concert with (i) stromal, mesenchymal, and angiogenic signatures, or (ii) exclusive NOTCH1 and PIK3C2B mutations with E2F/MYC activation. Molecular subtypes complement clinical risk stratification to distinguish low-risk, intermediate-risk, and high-risk patients with 10-year overall survivals of 94%, 45%, and 19%, respectively. Our findings provide a framework for integrated classification and treatment of metastasis and support the biological basis of curable oligometastatic colorectal cancer. These concepts may be applicable to many patients with metastatic cancer.

Performance of 3 real-time PCR assays for direct detection of Staphylococcus aureus and MRSA from clinical samples.

We compared 3 real-time PCR assays: off-label use of 2 commercial assays (BD-GeneOhm™ MRSA assay for methicillin-resistant Staphylococcus aureus [MRSA] detection and BD-GeneOhm StaphSR™ for MRSA and methicillin-susceptible S. aureus detection) and an in-house real-time PCR assay for detection of total S. aureus from clinical specimens. Testing was performed on 200 distinct specimens. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using culture as the gold standard. The prevalence of S. aureus in the samples was 44.5%, and MRSA was 20%. For total S. aureus, the StaphSR-PCR and the in-house PCR assays had a sensitivity and specificity of 94.4% and 96.4% and 93.3% and 99.1%, respectively. For MRSA detection, the StaphSR and the BD GeneOhm assay had a sensitivity and specificity of 92.5% and 98.8% and 92.5% and 96.3%, respectively. This study demonstrates the potential use of tests like the StaphSR-PCR assay for rapid detection of S. aureus and MRSA directly from clinical specimens; however, culture follow-up would be needed to identify other potential pathogens in the specimen.

Comparison of tumor markers for predicting outcomes after resection of nonfunctioning pancreatic neuroendocrine tumors.

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 < .05). A cut-off of Ki67 >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 < .015). 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.

Trends in thyroid surgery in Illinois.

BACKGROUND: Endocrine surgery is an evolving subspecialty in general surgery. To determine whether this subspecialty is having an effect on practice patterns of thyroid surgery, we reviewed all thyroidectomies performed in Illinois over an 11-year period. METHODS: The Illinois COMPdata database from the Illinois Hospital Association was used to retrieve all the thyroid operations performed in the state of Illinois from 1999 to 2009. Univariate and multivariate logistic regression analyses were performed to assess the effects of surgeon and hospital type on practice patterns of thyroidectomies. RESULTS: In the early period (1999-2004), 5,824 operations were identified compared with 8,454 in the late period (2005-2009; P < .001). Total thyroidectomy represented 2,679 (46%) of the thyroid operations done in the early period compared with 4,976 (59%) in the late period (P < .001). Sixty-two percent of all the thyroid operations were done at community hospitals in the early period compared with 56% in the late period. Endocrine surgeons (ES) performed the greatest rate of thyroidectomies, 0.7 and 0.6/10(5) population, in both early and late periods, respectively. CONCLUSION: In Illinois, the volume of thyroid operations has increased significantly over the past 10 years with a shift toward total thyroidectomy. Although most thyroidectomies are still performed in community hospitals, this percentage has decreased. ES perform a minority of thyroid operations, but they have the greatest volume of thyroidectomies per surgeon. These findings may represent broader trends in thyroid surgery throughout the United States.

Multiple broad-spectrum Beta-lactamase targets for comprehensive surveillance.

Real-time PCR testing for blaKPC, blaNDM, blaVIM, blaIMP, and blaCTX-M was performed on rectal swabs obtained from residents of two long-term acute-care facilities. While blaKPC was detected in 69/102 swabs (67.6%), testing for four other targets increased the positivity rate for a broad-spectrum ß-lactamase to 73.5% (McNemar's P = 0.03).

PCR detection of clarithromycin-susceptible and -resistant Helicobacter pylori from formalin-fixed, paraffin-embedded gastric biopsies.

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.

Integrity and amplification of nucleic acids from snap-frozen prostate tissues from robotic-assisted laparoscopic and open prostatectomies.

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 ß-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.