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1.  Three-Gene Molecular Diagnostic Model for Thyroid Cancer 
Thyroid  2012;22(3):275-284.
Background
The preoperative diagnosis of thyroid nodules primarily depends upon fine needle aspiration (FNA) cytology. However, up to 25% of FNA samples have associated “suspicious or indeterminate”, but not diagnostic cytologic reports, resulting in difficulty deciding appropriate clinical management for these patients. We hypothesize that the use of molecular markers as an adjunct to FNA cytology can improve the distinction of benign from malignant nodules that have associated suspicious or indeterminate cytology.
Methods
Using microarray analysis, we previously identified and reported on 75 genes useful in the distinction of benign versus malignant thyroid nodules. In the present study, we have further validated the expression of 14 of these markers in a large number of thyroid samples by immunohistochemistry (IHC) analysis of 154 thyroid tumors and quantitative real-time RT-PCR (QRT-PCR) analysis of 95 FNA samples. Of the 154 tumors analyzed by IHC, 44 samples (29%) had associated suspicious or indeterminate FNA cytology.
Results
Receiver operating characteristic using three-gene model, (HMGA2, MRC2, and SFN) analysis for the detection of malignant nodules resulted in areas under the curve (AUCs) of≥0.95 (80% sensitivity; 100% specificity) and≥0.84 (71% sensitivity; 84% specificity) for the IHC data in tumors, and QRT-PCR data in FNA samples, respectively.
Conclusions
Our results suggest that a three-gene model for the cytological diagnosis of indeterminate thyroid nodules is both feasible and promising. Implementation of this as an adjunct to thyroid cytology may significantly impact the clinical management of patients with suspicious or indeterminate thyroid FNA nodules.
doi:10.1089/thy.2011.0169
PMCID: PMC3286810  PMID: 22280184
2.  Associations between oral HPV16 infection and cytopathology: evaluation of an oropharyngeal “Pap-test equivalent” in high-risk populations 
Human papillomavirus (HPV) is responsible for the rising incidence of oropharyngeal squamous cell cancers (OSCC) in the United States (U.S.), and yet, no screening strategies have been evaluated. Secondary prevention by means of HPV detection and cervical cytology has led to a decline in cervical cancer incidence in the U.S. Here, we explored an analogous strategy by evaluating associations between HPV16 infection, cytopathology and histopathology in two populations at elevated risk for OSCC. In the first, a cross-sectional study population (PAP1), cytology specimens were collected by means of brush biopsy from patients presenting with oropharyngeal abnormalities. In the second (PAP2), a nested case-control study, bilateral tonsillar cytology samples were collected at 12-month intervals from HIV-infected individuals. The presence of cytopathological abnormality in HPV16-positive tonsil brush biopsies (cases) was compared to HPV16-negative samples (controls) matched on age and gender. HPV16 was detected in samples by consensus primer PCR and/or type-specific PCR. Univariate logistic regression was used to evaluate associations. In PAP1, HPV16 alone (OR 6.1, 95%CI 1.6–22.7) or in combination with abnormal cytology (OR 20, 95%CI 4.2–95.4) was associated with OSCC. In PAP2, 4.7% (72 of 1524) of tonsillar cytology specimens from HIV-infected individuals without oropharyngeal abnormalities were HPV16-positive. Tonsillar HPV16 infection was not associated with atypical squamous cells of unknown significance (ASCUS), the only cytological abnormality identified. Therefore, HPV16 was associated with OSCC among individuals with accessible oropharyngeal lesions, but not with cytological evidence of dysplasia among high-risk individuals without such lesions. An oropharyngeal Pap-test equivalent may not be feasible, likely due to limitations in sampling the relevant tonsillar crypt epithelium.
doi:10.1158/1940-6207.CAPR-11-0284
PMCID: PMC3380432  PMID: 21836021
oral HPV; tonsillar HPV; head and neck squamous cell cancer; cervical HPV; head and neck cancer screening
3.  Biospecimen Reporting for Improved Study Quality (BRISQ) 
Journal of proteome research  2011;10(8):3429-3438.
Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.
doi:10.1021/pr200021n
PMCID: PMC3169291  PMID: 21574648
4.  Biospecimen Reporting for Improved Study Quality 
Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The Biospecimen Reporting for Improved Study Quality guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.
doi:10.1089/bio.2010.0036
PMCID: PMC3142856  PMID: 21826252
5.  Analysis of Nondiagnostic Results after Image-guided Needle Biopsies of Musculoskeletal Lesions 
Background/rationale
Image-guided needle biopsies are commonly used to diagnose musculoskeletal tumors, but nondiagnostic (ND) results can delay diagnosis and treatment. It is important to understand which factors or diagnoses predispose to a ND result so that appropriate patient education or a possible change in the clinical plan can be made. Currently it is unclear which factors or specific lesions are more likely to lead to a ND result after image-guided needle biopsy.
Questions/purposes
We therefore identified specific factors and diagnoses most likely to yield ND results. We also asked whether an image-guided needle biopsy of bone and soft tissue lesions is an accurate and clinically useful tool.
Methods
We retrospectively reviewed data from a prospectively collected database for a case-control study of 508 image-guided needle biopsies of patients with suspected musculoskeletal tumors between 2003 and 2008.
Results
The interpretations of 453 of the 508 (89%) needle biopsies were accurate and clinically useful. Forty-five biopsies (9%) were ND and 10 (2%) were incorrect (IC). Bone lesions had a higher ND rate than soft tissue lesions (13% vs. 4%). The specific diagnosis with the highest ND rate was histiocytosis. Elbow and forearm locations had higher ND rates than average. Malignant tumors had a higher IC rate than benign tumors (5% vs. 0%); fibromyxoid sarcoma and rare subtypes of osteosarcoma had higher IC rates than other diagnoses. Repeat needle or open biopsies were performed in 71 (14%) patients. Bone lesions were more likely than soft tissue lesions to require repeat biopsies (18% vs. 9%).
Conclusions
A high rate of accuracy and clinical usefulness is possible with image-guided needle biopsies of musculoskeletal lesions. We believe these biopsies appropriate in selected circumstances but a key factor for appropriate use is an experienced musculoskeletal tumor team with frequent communication to correlate clinical, radiographic, and histologic information for each patient.
Level of Evidence
Level II, prognostic study. See the Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-010-1337-1
PMCID: PMC2947700  PMID: 20383617
6.  Identification of Genes Differentially Expressed in Benign versus Malignant Thyroid Tumors 
Purpose
Although fine-needle aspiration biopsy is the most useful diagnostic tool in evaluating a thyroid nodule, preoperative diagnosis of thyroid nodules is frequently imprecise, with up to 30% of fine-needle aspiration biopsy cytology samples reported as “suspicious” or “indeterminate.” Therefore, other adjuncts, such as molecular-based diagnostic approaches are needed in the preoperative distinction of these lesions.
Experimental Design
In an attempt to identify diagnostic markers for the preoperative distinction of these lesions, we chose to study by microarray analysis the eight different thyroid tumor subtypes that can present a diagnostic challenge to the clinician.
Results
Our microarray-based analysis of 94 thyroid tumors identified 75 genes that are differentially expressed between benign and malignant tumor subtypes. Of these, 33 were overexpressed and 42 were underexpressed in malignant compared with benign thyroid tumors. Statistical analysis of these genes, using nearest-neighbor classification, showed a 73% sensitivity and 82% specificity in predicting malignancy. Real-time reverse transcription – PCR validation for 12 of these genes was confirmatory. Western blot and immunohistochemical analyses of one of the genes, high mobility group AT-hook 2, further validated the microarray and real-time reverse transcription – PCR data.
Conclusions
Our results suggest that these 12 genes could be useful in the development of a panel of markers to differentiate benign from malignant tumors and thus serve as an important first step in solving the clinical problem associated with suspicious thyroid lesions.
doi:10.1158/1078-0432.CCR-07-4495
PMCID: PMC3086681  PMID: 18519760
7.  NKX3.1 as a Marker of Prostatic Origin in Metastatic Tumors 
NKX3.1 is a prostatic tumor suppressor gene located on chromosome 8p. Although most studies have shown that staining for NKX3.1 protein is positive in the majority of primary prostatic adenocarcinomas, it has been shown to be downregulated in many high-grade prostate cancers, and completely lost in the majority of metastatic prostate cancers (eg, in 65% to 78% of lesions). A recent study showed that NKX3.1 staining with a novel antibody was highly sensitive and specific for high-grade prostatic adenocarcinoma when compared with high-grade urothelial carcinoma. This raised the question that this antibody may perform better than earlier used antibodies in metastatic prostate tumors. However, the sensitivity and specificity for prostate carcinomas for this antibody in metastatic lesions was not determined. Although prostate-specific antigen (PSA) and prostatic-specific acid phosphatase (PSAP) are excellent tissue markers of prostate cancer, at times they may be expressed at low levels, focally, or not at all in poorly differentiated primary and metastatic prostatic adenocarcinomas. The purpose of this study was to determine the performance of NKX3.1 as a marker of metastatic adenocarcinoma of prostatic origin. Immunohistochemical staining against NKX3.1, PSA, and PSAP was carried out on a tissue microarray (TMA) (0.6-mm tissue cores) of hormone naïve metastatic prostate adenocarcinoma specimens from lymph nodes, bone, and soft tissue. To determine the specificity of NKX3.1 for prostatic adenocarcinoma, we used TMAs that contained cancers from various sites including the urinary bladder, breast, colon, salivary gland, stomach, pancreas, thyroid, and central nervous system, and standard paraffin sections of cancers from other sites including the adrenal cortex, kidney, liver, lung, and testis. Overall 349 nonprostatic tumors were evaluated. Any nuclear staining for NKX3.1 was considered positive and the percentage of cells with nuclear staining and their mean intensity level were assessed visually. Sensitivity was calculated by considering a case positive if any TMA core was positive. The sensitivity for identifying metastatic prostatic adenocarcinomas overall was 98.6% (68/69 cases positive) for NKX3.1, 94.2% (65/69 cores positive) for PSA, and 98.6% (68/69 cores positive) for PSAP. The specificity of NKX3.1 was 99.7% (1/349 nonprostatic tumors positive). The sole positive nonprostatic cancer case was an invasive lobular carcinoma of the breast. NKX3.1 seems to be a highly sensitive and specific tissue marker of metastatic prostatic adenocarcinoma. In the appropriate clinical setting, the addition of IHC staining for NKX3.1, along with other prostate-restricted markers, may prove to be a valuable adjunct to definitively determine prostatic origin in poorly differentiated metastatic carcinomas.
doi:10.1097/PAS.0b013e3181e6cbf3
PMCID: PMC3072223  PMID: 20588175
NKX3.1 protein; metastatic prostatic carcinoma; immunohistochemistry
9.  Host Cell Fate on Cryptosporidium parvum Egress from MDCK Cells  
Infection and Immunity  2003;71(9):5422-5426.
Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness of unclear etiology. Also unclear is the fate of the host cell upon parasite egress. We show in an MDCK cell model that the host cell is killed upon parasite egress; this death is necrotic, rather than apoptotic, in nature.
doi:10.1128/IAI.71.9.5422-5426.2003
PMCID: PMC187295  PMID: 12933897
10.  Cryptosporidium parvum Infection Requires Host Cell Actin Polymerization 
Infection and Immunity  2001;69(9):5940-5942.
The intracellular protozoan parasite Cryptosporidium parvum accumulates host cell actin at the interface between the parasite and the host cell cytoplasm. Here we show that the actin polymerizing proteins Arp2/3, vasodilator-stimulated phosphoprotein (VASP), and neural Wiskott Aldrich syndrome protein (N-WASP) are present at this interface and that host cell actin polymerization is necessary for parasite infection.
doi:10.1128/IAI.69.9.5940-5942.2001
PMCID: PMC98718  PMID: 11500478
11.  Cryptosporidium parvum Induces Host Cell Actin Accumulation at the Host-Parasite Interface 
Infection and Immunity  2000;68(4):2315-2322.
Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that Cryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein α-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including α-catenin, β-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus, Cryptosporidium induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and α-actinin into a host-parasite junctional complex.
PMCID: PMC97419  PMID: 10722635
12.  New Insights into Human Cryptosporidiosis 
Clinical Microbiology Reviews  1999;12(4):554-563.
Cryptosporidium parvum is an important cause of diarrhea worldwide. Cryptosporidium causes a potentially life-threatening disease in people with AIDS and contributes significantly to morbidity among children in developing countries. In immunocompetent adults, Cryptosporidium is often associated with waterborne outbreaks of acute diarrheal illness. Recent studies with human volunteers have indicated that Cryptosporidium is highly infectious. Diagnosis of infection with this parasite has relied on identification of acid-fast oocysts in stool; however, new immunoassays or PCR-based assays may increase the sensitivity of detection. Although the mechanism by which Cryptosporidium causes diarrhea is still poorly understood, the parasite and the immune response to it probably combine to impair absorption and enhance secretion within the intestinal tract. Important genetic studies suggest that humans can be infected by at least two genetically distinct types of Cryptosporidium, which may vary in virulence. This may, in part, explain the clinical variability seen in patients with cryptosporidiosis.
PMCID: PMC88924  PMID: 10515902
14.  Enteric β-Defensin: Molecular Cloning and Characterization of a Gene with Inducible Intestinal Epithelial Cell Expression Associated with Cryptosporidium parvum Infection 
Infection and Immunity  1998;66(3):1045-1056.
A growing body of evidence suggests that endogenous antibiotics contribute to the innate defense of mammalian mucosal surfaces. In the cow, β-defensins constitute a large family of antibiotic peptides whose members have been previously isolated from the respiratory and oral mucosa, as well as circulating phagocytic cells. A novel bovine genomic clone with sequence related to those of these α-defensins was isolated and characterized. The corresponding cDNA was isolated from a small intestinal library; its open reading frame predicts a deduced sequence of a novel β-defensin, which we designate enteric β-defensin (EBD). Northern blot analysis of a variety of bovine tissues revealed that EBD mRNA is highly expressed in the distal small intestine and colon, anatomic locations distinct from those for previously characterized β-defensins. EBD mRNA was further localized by in situ hybridization to epithelial cells of the colon and small intestinal crypts. Infection of two calves with the intestinal parasite Cryptosporidium parvum induced 5- and 10-fold increases above control levels of EBD mRNA in intestinal tissues. An anchored-PCR strategy was used to identify other β-defensin mRNAs expressed in the intestine. In addition to that of EBD, several low-abundance cDNAs which corresponded to other β-defensin mRNAs were cloned. Most of these clones encoded previously characterized β-defensins or closely related isoforms, but two encoded a previously uncharacterized prepro-β-defensin. Northern blot evidence supported that all of these other β-defensin genes are expressed at levels lower than that of the EBD gene in enteric tissue. Furthermore, some of these β-defensin mRNAs were abundant in bone marrow, suggesting that in enteric tissue their expression may be in cells of hematopoietic origin. Extracts of small intestinal mucosa obtained from healthy cows have numerous active chromatographic fractions as determined by an antibacterial assay, and one peptide was partially purified. The peptide corresponded to one of the low-abundance cDNAs. This study provides evidence of β-defensin expression in enteric tissue and that the mRNA encoding a major β-defensin of enteric tissue, EBD, is inducibly expressed in enteric epithelial cells. These findings support the proposal that β-defensins may contribute to host defense of enteric mucosa.
PMCID: PMC108014  PMID: 9488394
15.  Association Between BRAF V600E Mutation and Mortality in Patients With Papillary Thyroid Cancer 
Importance
BRAF V600E is a prominent oncogene in papillary thyroid cancer (PTC), but its role in PTC-related patient mortality has not been established.
Objective
To investigate the relationship between BRAF V600E mutation and PTC-related mortality.
Design, Setting, and Participants
Retrospective study of 1849 patients (1411 women and 438 men) with a median age of 46 years (interquartile range, 34–58 years) and an overall median follow-up time of 33 months (interquartile range, 13–67 months) after initial treatment at 13 centers in 7 countries between 1978 and 2011.
Main Outcomes and Measures
Patient deaths specifically caused by PTC.
Results
Overall, mortality was 5.3% (45/845; 95% CI, 3.9%–7.1%) vs 1.1% (11/1004; 95% CI, 0.5%–2.0%) (P<.001) in BRAF V600E–positive vs mutation-negative patients. Deaths per 1000 person-years in the analysis of all PTC were 12.87 (95% CI, 9.61–17.24) vs 2.52 (95% CI, 1.40–4.55) in BRAF V600E–positive vs mutation-negative patients; the hazard ratio (HR) was 2.66 (95% CI, 1.30–5.43) after adjustment for age at diagnosis, sex, and medical center. Deaths per 1000 person-years in the analysis of the conventional variant of PTC were 11.80 (95% CI, 8.39–16.60) vs 2.25 (95% CI, 1.01–5.00) in BRAF V600E–positive vs mutation-negative patients; the adjusted HR was 3.53 (95% CI, 1.25–9.98). When lymph node metastasis, extrathyroidal invasion, and distant metastasis were also included in the model, the association of BRAF V600E with mortality for all PTC was no longer significant (HR, 1.21; 95% CI, 0.53–2.76). A higher BRAF V600E–associated patient mortality was also observed in several clinicopathological subcategories, but statistical significance was lost with adjustment for patient age, sex, and medical center. For example, in patients with lymph node metastasis, the deaths per 1000 person-years were 26.26 (95% CI, 19.18–35.94) vs 5.93 (95% CI, 2.96–11.86) in BRAF V600E–positive vs mutation-negative patients (unadjusted HR, 4.43 [95% CI, 2.06–9.51]; adjusted HR, 1.46 [95% CI, 0.62–3.47]). In patients with distant tumor metastasis, deaths per 1000 person-years were 87.72 (95% CI, 62.68–122.77) vs 32.28 (95% CI, 16.14–64.55) in BRAF V600E–positive vs mutation-negative patients (unadjusted HR, 2.63 [95% CI, 1.21–5.72]; adjusted HR, 0.84 [95% CI, 0.27–2.62]).
Conclusions and Relevance
In this retrospective multicenter study, the presence of the BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC. Because overall mortality in PTC is low and the association was not independent of tumor features, how to use BRAF V600E to manage mortality risk in patients with PTC is unclear. These findings support further investigation of the prognostic and therapeutic implications of BRAF V600E status in PTC.
doi:10.1001/jama.2013.3190
PMCID: PMC3791140  PMID: 23571588
16.  Functional Profiling of Live Melanoma Samples Using a Novel Automated Platform 
PLoS ONE  2012;7(12):e52760.
Aims
This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform.
Methods
A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma.
Results
Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy.
Conclusion
Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients.
doi:10.1371/journal.pone.0052760
PMCID: PMC3532357  PMID: 23285177

Results 1-16 (16)