DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner.

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.

Prognostic Assessment of BRAF Mutation in Preoperative Thyroid Fine-Needle Aspiration Specimens.

OBJECTIVES: We investigate the potential role of BRAF testing in guiding surgical intervention in papillary thyroid carcinoma (PTC). METHODS: Thyroid fine-needle aspiration (FNA) cases with available BRAF result and follow-up thyroidectomy for PTC were included in the study. Cytology and surgical diagnoses were correlated with BRAF status. RESULTS: There were 151 cases of thyroid FNA specimens with BRAF testing (70 mutant and 81 wild-type BRAF) and histologically confirmed unilateral, unifocal PTCs. There were no differences in age, sex, tumor size, or lymphovascular invasion on thyroidectomy specimens between mutant and wild-type BRAF cases. BRAF mutation was significantly associated with cytology diagnosis (P < .001), PTC subtype (P < .001), extrathyroidal extension (ETE) (P = .006), and higher tumor (T) stage (P = .04). However, an analysis within the histologic subtypes of PTC revealed no significant association between BRAF mutation and ETE or higher T stage. There was also no difference in central (P = .847) or lateral (p = 1) neck lymph node (LN) metastasis. CONCLUSIONS: BRAF mutation identified in thyroid FNA specimens correlates with histologic subtypes but is not an independent factor for predicting PTC biological behavior and should not be used to guide the extent of LN dissection.

5-Endo Trig Oxidative Radical Cyclizations of Ugi-3CR Products toward 1,4-Imidazolidinones.

A 5-endo trig oxidative radical cyclization of benzylamine-derived Ugi three-component reaction products rapidly affords imidazolidinones with three diversity elements. This adaptation of our previously described multicomponent reaction-oxidation methodology further showcases manipulation of the diversity elements in multicomponent reaction products via oxidative radical cyclizations, which generates highly decorated privileged heterocycles.

The Value of Negative Diagnosis in Thyroid Fine-Needle Aspiration: a Retrospective Study with Histologic Follow-Up.

The Bethesda System for reporting thyroid cytopathology (BSRTC) predicts an incidence of malignancy of less than 5% in thyroid nodules with a benign diagnosis on fine-needle aspiration (FNA). However, recent series have suggested that the true rate of malignancy might be significantly higher in this category of patients. We reviewed our experience by performing a retrospective analysis of patients with benign thyroid FNA results who underwent thyroidectomy between 2008 and 2013 at a large academic center. Information including demographics, ultrasound features, FNA diagnosis, and surgical follow-up information were recorded. Slides were reviewed on cytology-histology discrepant cases, and it was determined whether the discrepancy was due to sampling or interpretation error. A total of 802 FNA cases with a benign diagnosis and surgical follow-up were identified. FNA diagnoses included 738 cases of benign goiter and 64 cases of lymphocytic thyroiditis. On subsequent surgical resection, 144 cases were found to be neoplastic, including 117 malignant cases. False negative, defined as interpretation error and inadequate biopsy of the nodule harboring malignancy, was 6%. When cases of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) were excluded from the analysis, false-negative rate was 5%. When microPTC cases were excluded, false-negative rate was 3% and was slightly less than 3% when both microPTC and NIFTP cases were excluded from the analysis. Retrospective review of neoplastic cases showed that 57% were due to sampling error and 43% were due to interpretation error. Interpretation error was more likely to occur in follicular patterned neoplasms (75%), while sampling error was more common in non-follicular variants of papillary thyroid carcinoma (non-FVPTC) (61%). With the exclusion of microPTC, interpretation errors were still more likely to occur in follicular neoplasms (79%) but there was no significant difference in sampling error between non-FVPTC (37%) and follicular patterned neoplasms (42%). Tumor size was larger in cases with interpretation error (mean = 2.3 cm) compared to cases with sampling error (mean = 1.4 cm). This study shows that the false-negative rate of thyroid FNA at our institution is not significantly above the rate suggested by the BSRTC. Interpretation errors were more likely to occur in follicular patterned neoplasms, while non-FVPTC was more frequently found in false negative cases due to inadequate sampling.

A Comparison of the Roche Cobas HPV Test With the Hybrid Capture 2 Test for the Detection of High-Risk Human Papillomavirus Genotypes.

CONTEXT: All Food and Drug Administration-approved methods in the United States for human papillomavirus testing including the Hybrid Capture 2 human papillomavirus assay and the Roche cobas human papillomavirus test are approved for cytology specimens collected into ThinPrep media but not for specimens collected into SurePath solution. OBJECTIVE: To compare the performance of the Roche cobas and Hybrid Capture 2 tests for the detection of high-risk human papillomavirus using both ThinPrep and SurePath preparations as part of a validation study. DESIGN: One thousand three hundred seventy-one liquid-based cytology samples, including 1122 SurePath and 249 ThinPrep specimens, were tested for high-risk human papillomavirus DNA using the Roche cobas human papillomavirus test and the Hybrid Capture 2 human papillomavirus assay. For cases with discrepant results, confirmatory testing was performed using Linear Array human papillomavirus testing. RESULTS: One hundred and fifty-six (11.38%) and 184 (13.42%) of the 1371 specimens tested positive for high-risk human papillomavirus DNA using the Hybrid Capture 2 human papillomavirus assay and Roche cobas human papillomavirus assay, respectively. In addition, 1289 (94.0%) of 1371 specimens demonstrated concordant high-risk human papillomavirus results with a κ value of 0.72 (95% confidence interval, 065-0.78). There was no statistically significant difference in the percentage of positive high-risk human papillomavirus results between the 2 liquid-based preparations with either assay. Discordant results between the 2 assays were noted in 82 of 1371 cases (6%). Twenty-seven of 82 cases (32.9%) were Hybrid Capture 2 positive/Roche cobas negative and 55 of 82 cases (67.1%) were Roche cobas positive/Hybrid Capture 2 negative. Two of 20 Hybrid Capture 2-positive/Roche cobas-negative cases (10%) and 26 of 37 Roche cobas-positive/Hybrid Capture 2-negative cases (70%) tested positive for high-risk human papillomavirus by Linear Array. CONCLUSIONS: Both assays showed good agreement and excellent specificity with either ThinPrep or SurePath preparations. The number of discordant results was relatively small. The performance of both assays was similar for ThinPrep specimens, but the Roche cobas test demonstrated higher sensitivity with SurePath specimens.

Radiologic and clinical predictors of malignancy in the follicular lesion of undetermined significance of the thyroid.

Various ultrasonographic characteristics of thyroid nodules have been associated with a higher likelihood of malignancy, and certain clinical features may also increase the likelihood of malignancy in patients. This study is designed to determine the ultrasonographic and clinical predictors of malignancy in the atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) category. A search through the cytology files at our institution was made for cases with diagnosis of AUS/FLUS. The clinical and radiologic findings were correlated with the final surgical pathology diagnosis. A total of 140 cases of AUS/FLUS with corresponding surgical intervention were identified (112 females and 28 males). There was a 79 % malignancy rate in nodules with irregular contours, compared to 51 % in nodules with regular outlines. Nodules demonstrating calcifications showed a 57 % malignancy rate, compared to 50 % in nodules without calcifications. Sixty-one percent of cases with an ultrasonographic diagnosis of indeterminate to suspicious were malignant following surgical resection. The rates of malignancy in patients with radiation exposure, symptomatic nodules, and positive family history of thyroid cancer were 22, 59, and 33 %, respectively. BRAF mutation was demonstrated in 57 % of malignant cases and in none of benign cases. No single clinical or ultrasonographic feature or combination of features is adequately sensitive or specific to identify all malignant nodules. However, a combination of solid nodules, nodules with irregular contours, symptomatic nodules, and positive BRAF mutation has high predictive value for malignancy in patients with a cytologic diagnosis of AUS/FLUS.

Increasing cytotechnologist workload above 100 slides per day using the BD FocalPoint GS imaging system negatively affects screening performance.

Studies examining the effects of increased workload on the performance of individual cytotechnologists are limited. Using FocalPoint GS, the performance of 3 cytotechnologists was evaluated. The study consisted of 3 phases. In phase I, cytotechnologists were asked to screen at their usual pace. In phase II, cytotechnologists were asked to screen as fast as possible without feeling that the quality of their work was diminished. In phase III, cytotechnologists were asked to screen at least 15% more than their daily workload from phase II. Productivity was increased by decreasing the percentage of cases that underwent full manual review (from 38% to 19%) and by decreasing the time spent on each slide (from 5.5 min to 3.7 min). Overall, the total abnormal rate decreased by 31.9% from phase I to phase III of the study. In addition, the false-negative fraction increased significantly, from 1% to 6.9%. Our results indicated a negative association between increased cytotechnologist daily workload with FocalPoint GS and CT screening performance. Workloads were increased by decreasing the time spent reviewing 10 fields of view and the percentage of cases that underwent full manual review.

Comparing two methods of detection for Chlamydia trachomatis in liquid-based Papanicolaou tests.

This study compared the performance of Chlamydia trachomatis testing using 2 methods: the BD ProbeTec Chlamydia trachomatis Q(x) Amplified DNA Assay (CTQ) on the BD Viper System with XTR technology (CTQ assay) and the Hybrid Capture (HC) 2 assay. A total of 1,054 Surepath and ThinPrep specimens were tested for C trachomatis nucleic acids using the CTQ assay and the HC2 assay. For positive and discrepant C trachomatis test results, confirmatory test for C trachomatis was performed using a reverse transcriptase-polymerase chain reaction. Of 1,054 liquid-based gynecologic cytology samples tested for C trachomatis using both assays, 1,041 tested negative on both. In 6 (0.57%) samples, findings were discordant. The CTQ assay and the HC2 assay had sensitivity rates of 100% and 66.7%, respectively, with comparable specificity (99.9%). The positive predictive values were 92.3% and 88.9% with the CTQ and HC2 assays, respectively. In this study, the CTQ assay was found to be more sensitive than the HC2 assay in detecting chlamydial infection; the CTQ assay also demonstrated a higher positive predictive value.

Implementation of FocalPoint GS location-guided imaging system: experience in a clinical setting.

BACKGROUND: Recently, the Food and Drug Administration approved the use of the location-guided imaging system FocalPoint GS (FPGS), on SurePath Papanicolaou (Pap) tests for primary screening. The objective of the current study was to evaluate the impact of FPGS on the following: distribution of diagnostic categories; rate of high-risk human papillomavirus (HR-HPV)-positive ASC-US cases; and quality control (QC) data before and after FPGS implementation. METHODS: A search of the laboratory information system was performed to identify all SurePath Pap tests processed in our laboratory for the first 19 months after FPGS implementation. We also retrieved all SurePath specimens from a 16-month period prior to FPGS implementation to serve as the control. During the period from Janaury 2008 to April 2009, the FocalPoint Slide Profiler was used. RESULTS: Implementation of FPGS resulted in a significantly higher percentage of LSIL and ASC-US interpretations, as well as a significant increase in the detection of candidiasis and bacterial vaginosis. The ASC-to-SIL ratio was 1.4 and 1.9 before and after FPGS implementation, respectively. There was a decrease in the HR-HPV positive rate in ASC-US cases, and a decrease in the estimated false-negative fraction after FPGS implementation. CONCLUSIONS: In conclusion, our study seems to demonstrate a favorable performance of FPGS in the routine clinical setting. FPGS may have the potential to be a promising screening tool for gynecologic cytology in a low-risk patient population.

Thyroid follicular lesion of undetermined significance: Evaluation of the risk of malignancy using the two-tier sub-classification.

The Bethesda 2007 Thyroid Cytology Classification defines follicular lesion of undetermined significance as a heterogeneous category of cases that are not convincingly benign nor sufficiently atypical for a diagnosis of follicular neoplasm or suspicious for malignancy. In our institution, we refer to these cases as indeterminate, and they are further sub-classified into two: (1) low cellularity with predominant microfollicular architecture and absence of colloid (IN(a)) and (2) nuclear features not characteristic of benign lesions (nuclear atypia) (IN(b)). We reviewed these indeterminate cases to document the follow-up trend using this two-tier classification. A search of the cytology records was performed for the period between January 2008 and June 2009. All thyroid fine-needle aspiration (FNA) cases were reviewed and the ones diagnosed as indeterminate were identified. Correlating follow-up FNA and/or surgical pathology reports were reviewed. The percentage of cases showing a malignancy was calculated. One hundred and seventy-one indeterminate cases were identified, representing 2.8% of the 6,205 thyroid FNA cases examined during the time under review (107 IN(a), 64 IN(b)). Records of follow-up procedures were available in 106 (61%) cases. Malignancy was identified in 27% of all indeterminate cases. This was disproportionately more in the IN(b) (56%) compared to the IN(a) (7%) cases. A diagnosis of "IN(a)" does not carry the same implication as that of "IN(b)". The IN(b) category needs a more aggressive follow-up than the IN(a) category and may justify an immediate referral for lobectomy. Despite the vague morphologic criteria for this diagnostic category, the indeterminate rate remains relatively low and falls within the NCI recommendation (<7%).

The positive impact of simultaneous implementation of the BD FocalPoint GS Imaging System and lean principles on the operation of gynecologic cytology.

CONTEXT: Our cytology laboratory, like many others, is under pressure to improve quality and provide test results faster while decreasing costs. We sought to address these issues by introducing new technology and lean principles. OBJECTIVE: To determine the combined impact of the FocalPoint Guided Screener (GS) Imaging System (BD Diagnostics-TriPath, Burlington, North Carolina) and lean manufacturing principles on the turnaround time (TAT) and productivity of the gynecologic cytology operation. DESIGN: We established a baseline measure of the TAT for Papanicolaou tests. We then compared that to the performance after implementing the FocalPoint GS Imaging System and lean principles. The latter included value-stream mapping, workflow modification, and a first in-first out policy. RESULTS: The mean (SD) TAT for Papanicolaou tests before and after the implementation of FocalPoint GS Imaging System and lean principles was 4.38 (1.28) days and 3.20 (1.32) days, respectively. This represented a 27% improvement in the average TAT, which was statistically significant (P < .001). In addition, the productivity of staff improved 17%, as evidenced by the increase in slides screened from 8.85/h to 10.38/h. The false-negative fraction decreased from 1.4% to 0.9%, representing a 36% improvement. CONCLUSIONS: In our laboratory, the implementation of FocalPoint GS Imaging System in conjunction with lean principles resulted in a significant decrease in the average TAT for Papanicolaou tests and a substantial increase in the productivity of cytotechnologists while maintaining the diagnostic quality of gynecologic cytology.

BRAF mutation testing of thyroid fine-needle aspiration specimens enhances the predictability of malignancy in thyroid follicular lesions of undetermined significance.

BACKGROUND/OBJECTIVE: The Bethesda 2007 Thyroid Cytology Classification defines atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) as a heterogeneous category of cases that are neither convincingly benign nor sufficiently atypical for a diagnosis of follicular neoplasm or suspicious for malignancy. At our institution, we refer to these cases as 'indeterminate' and they are further subclassified into two categories. BRAF mutation occurs in 40-60% of papillary thyroid carcinoma (PTC). In this study, we examined cases in the AUS/FLUS category in correlation with BRAF mutation analysis and surgical pathology outcome. STUDY DESIGN: Thyroid fine-needle aspiration (FNA) cytology specimens interpreted as 'indeterminate' were selected from our files, and available remnants of thin-layer processed specimens were used for BRAF mutation analysis. Surgical pathology reports were reviewed for the final outcomes in these patients. RESULTS: Of the 84 indeterminate cases with BRAF mutation analysis, only 49 had follow-up with surgical intervention. Sixteen cases had BRAF mutation. All of the BRAF-positive cases had a final diagnosis of PTC. CONCLUSIONS: The sensitivity and specificity of BRAF mutation in detecting PTC in FNA specimens with indeterminate diagnosis was 59.3 and 100%, respectively, while the positive and negative predictive values were 100 and 65.6%, respectively. The limited data supports the use of BRAF mutation analysis to predict the risk of malignancy in patients with indeterminate thyroid FNAs.

Use of high-risk human papillomavirus testing in patients with low-grade squamous intraepithelial lesions.

BACKGROUND: The role of testing for high-risk human papillomavirus (HR HPV) when triaging women with a cytologic diagnosis of low-grade squamous intraepithelial lesion (LSIL) has not been well established. The objective of the current study was to correlate the status of HR HPV with the incidence of cervical intraepithelial neoplasia 2 and more severe lesions (CIN 2+) on tissue follow-up in women with LSIL. METHODS: A total of 1046 women with LSIL and HR HPV testing were identified in the database of a large teaching hospital within a 12-month period. HR HPV testing was performed using the Hybrid Capture 2 assay with 1 relative light unit/cutoff as the cutoff. RESULTS: Of the 1046 women with LSIL and concurrent HR HPV testing, 82.3% tested positive for HR HPV, 91.1% of whom were women aged < 30 years and 73% of whom were women aged ≥ 30 years (P < .001). Cytologic and/or histologic follow-up was available in 979 (93.6%) women; 25.5% had negative follow-up, 62.5% were found to have CIN 1 lesions, and 12.0% had CIN 2+ lesions. The sensitivity and negative predictive value of HR HPV status as a marker of CIN 2+ lesions were 98.3% and 98.9%, respectively. The colposcopy rate was 73.3% and 96.9% for women aged ≥ 30 years and women aged < 30 years, respectively (P = .01). CONCLUSIONS: Using 1 RLU/CO as the cutoff value, HR HPV testing was found to be highly sensitive for detecting CIN 2+ lesions in women with LSIL. The colposcopy rate was significantly lower in women aged ≥ 30 years compared with women aged < 30 years. Triaging with HR HPV testing may be indicated in women aged ≥ 30 years with LSIL cytology, but not in women aged < 30 years. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.

Comparison of Affirm VPIII and Papanicolaou tests in the detection of infectious vaginitis.

To compare the Affirm VPIII molecular test (Becton Dickinson, Burlington, NC) with morphologic identification used in routine Papanicolaou (Pap) test screening in the detection and identification of Candida species, Trichomonas vaginalis, and Gardnerella vaginalis, we identified 431 cases with a concomitant Pap test and Affirm VPIII assay performed from the archives of a large academic institution. The study population consisted of women ranging in age from 17 to 79 years (mean and median ages, 33 and 31 years, respectively). With a routine Pap test, 60 patients (13.9%) were found to have bacterial vaginosis, 60 (13.9%) candidiasis, and 3 (0.7%) Trichomonas infection. With the Affirm VPIII assay, 183 (42.5%) patients tested positive for G vaginalis, 70 (16.2%) positive for Candida species, and 10 (2.3%) positive for T vaginalis. The differences were statistically significant. The results demonstrate that our patient population had a high incidence of bacterial vaginosis/Candida vaginitis; however, the Affirm VPIII was a more sensitive diagnostic test for the detection and identification of all 3 organisms compared with the Pap test.

The Bethesda thyroid fine-needle aspiration classification system: year 1 at an academic institution.

BACKGROUND: Fine-needle aspiration (FNA) may be the procedure of choice in the preoperative evaluation of thyroid nodules, yet it suffers as a modality both because of its inherent limitations as well as variability in its diagnostic terminology. The National Cancer Institute recently proposed a classification system. The objective of this study was to report our experience in using this new reporting system to review the distribution of diagnosis categories and to evaluate the specificity of the system based on the cytologic-histologic correlation. PATIENTS AND METHODS: A total of 3207 thyroid nodules underwent FNA, that is, 3207 FNAs from 2468 patients were examined at our institution between January 1, 2008 and December 31, 2008. All FNAs were classified prospectively into unsatisfactory, benign, indeterminate (cells of undetermined significance), follicular neoplasm (FN), suspicious for malignancy, and positive for malignancy. RESULTS: The distribution of these categories from 3207 evaluated nodules was as follows: 11.1% unsatisfactory, 73.8% benign, 3.0% indeterminate, 5.5% FN, 1.3% suspicious, and 5.2% malignant. Of the 2468 sampled patients, 378 (15%) underwent thyroidectomy. The distribution of diagnoses of patients who underwent surgery was as follows: 10% unsatisfactory, 4.6% benign, 30.3% indeterminate, 61.4% FN, 76.9% suspicious, and 77.2% malignant. There was an excellent association between the categories and in predicting benign versus malignant thyroid nodules (p < 0.0001). However, the false-negative rate cannot be calculated because only a small number of patients with benign diagnosis underwent surgery. The false-positive rate was 2.2%; all were diagnosed as suspicious cytologically. Given that only 15% of the patients underwent surgery, at this time the sensitivity of thyroid FNA for diagnosing malignant thyroid nodules cannot be calculated, nor can the sensitivity of thyroid FNA as a screening test for all neoplasms be accurately estimated. The specificity for diagnosing malignant thyroid nodules was 93%, whereas the specificity as a screening test for all neoplasms was 68%. The positive predictive values for an FN, suspicious, and positive cytologic diagnosis were 34%, 87%, and 100%, respectively. CONCLUSIONS: These data demonstrate that the recently proposed classification system is excellent for reporting thyroid FNAs. Each diagnostic category conveys specific risks of malignancy, which offers guidance for patient management.

Distinct human papillomavirus type 16 methylomes in cervical cells at different stages of premalignancy.

Human papillomavirus (HPV) gene expression is dramatically altered during cervical carcinogenesis. Because dysregulated genes frequently show abnormal patterns of DNA methylation, we hypothesized that comprehensive mapping of the HPV methylomes in cervical samples at different stages of progression would reveal patterns of clinical significance. To test this hypothesis, thirteen HPV16-positive samples were obtained from women undergoing routine cervical cancer screening. Complete methylation data were obtained for 98.7% of the HPV16 CpGs in all samples by bisulfite-sequencing. Most HPV16 CpGs were unmethylated or methylated in only one sample. The other CpGs were methylated at levels ranging from 11% to 100% of the HPV16 copies per sample. The results showed three major patterns and two variants of one pattern. The patterns showed minimal or no methylation (A), low level methylation in the E1 and E6 genes (B), and high level methylation at many CpGs in the E5/L2/L1 region (C). Generally, pattern A was associated with negative cytology, pattern B with low-grade lesions, and pattern C with high-grade lesions. The severity of the cervical lesions was then ranked by the HPV16 DNA methylation patterns and, independently, by the pathologic diagnoses. Statistical analysis of the two rating methods showed highly significant agreement. In conclusion, analysis of the HPV16 DNA methylomes in clinical samples of cervical cells led to the identification of distinct methylation patterns which, after validation in larger studies, could have potential utility as biomarkers of neoplastic cervical progression.

High grade cervical intraepithelial neoplasia and viral load of high-risk human papillomavirus: significant correlations in patients of 22 years old or younger.

High-risk human papillomavirus (HR-HPV) is recognized as the primary cause for the development of cervical cancers and their precursor lesions. We investigated whether high-grade cervical dysplasia correlates with high viral load of HR-HPV in an age-dependent manner. Cases were retrospectively selected to include patients with a prior cytological diagnosis of ASCUS or higher grade squamous intraepithelial lesions, and a positive Digene Hybrid Capture II (HC-II) HR-HPV testing within 2 months before or after cervical biopsy. The quantitative viral load data was classified as negative, low, moderate and high according to the manufacturer's instruction. Cases were then stratified into 4 age groups: 40 years. Chi-Square analysis and logistic regression were performed where appropriate. A total of 995 patients were identified. Age categories were significantly associated with HPV loads (p=0.046). Moderate to high viral loads of HPV were significantly related to the histological grade of dysplasia (p=0.029). Logistic regression analysis further confirmed the association of HPV with histological grades, even after adjusting for age factor. In particular, high-grade dysplasia (p=0.011) but not low grade dysplasia (p=0.140) was significantly associated with moderate to high HPV loads. Patients of 22 years old or younger were the only group found significantly correlated with high viral loads of HPV (p=0.015). In conclusion, high-grade squamous intraepithelial lesions and patients' age of 22 years old or younger are significantly associated with a moderate to high viral load of HR-HPV.

Use of magnetic enrichment for detection of carcinoma cells in fluid specimens.

BACKGROUND: Ascites fluid or a pleural effusion are common events in metastatic carcinoma, but they also can be associated with several other medical conditions. The standard for determination of malignancy in these situations is cytologic evaluation of these fluids. Although this method is frequently successful, there are times when it fails, even when the patient has a malignancy, either because of insufficient cells in the fluid or for other reasons. This study addresses this problem taking advantage of the recent advances in technology for detection of rare epithelial cells in liquid specimens. METHODS: The authors examined fluid specimens from 59 patients to determine the frequency of recovery of epithelial cells compared with that achieved by conventional cytopathology. The Dynal CELLection Epithelial Enrich (Dynal AS, Oslo, Norway) method was used. This method is based on immunomagnetic selection of cells binding to EpCAM antibodies. Carcinoma cells were confirmed by morphology and, when there was sufficient material, by E-cadherin staining. RESULTS: Grouping the cases by cytologic diagnosis, the authors found malignant cells using the cell enrichment assay in 11 of 12 malignant cases, 2 of 5 atypical cases, and 3 of 42 negative cases. Further investigations were conducted on the five cases that were cytologically negative or atypical but yielded epithelial cells after immunomagnetic enrichment. Four cases ultimately were proven malignant by other methods and one had incomplete follow-up. CONCLUSIONS: The new methods available for epithelial cell enrichment in liquids may be used successfully on cytologic fluid specimens and may lead to increased sensitivity for detection of malignancy, and consequently more accurate staging.