Overtreatment of Nonneoplastic Gallbladder Polyps due to Inadequate Routine Ultrasound Assessment.

BACKGROUND: The primary aim was to assess the diagnostic accuracy of routine ultrasound assessment for gallbladder polyps. The secondary aim was to identify the characteristics that differentiate neoplastic polyps from nonneoplastic polyps. METHODS: A total of 156 patients with histopathologically proven gallbladder polyps in 4 Dutch hospitals between 2003 and 2013 were included. Sensitivity and specificity of ultrasound for polyp size, number of polyps, and polyp type were assessed using histopathological findings as a reference standard. In addition, diagnostic accuracy of sonographic size ≥1 cm for neoplasia was assessed. Subgroup analysis for patients with polyps as primary indication for cholecystectomy was performed. The sonographic polyp characteristics on preoperative routine ultrasound were described. RESULTS: Fifty-six percent of gallbladder polyps were preoperatively identified on ultrasound, of which 31% were neoplastic. Sensitivity and specificity of ultrasound to estimate polyp size were 93 and 43% (subgroup; 92 and 33%). Sensitivity and specificity of sonographic polyp size ≥1 cm for neoplasia were 86 and 32% (subgroup; 94 and 26%). No specific sonographic characteristics for neoplastic polyps could be established due to lack of reporting. CONCLUSION: Routine ultrasound assessment of polyps is associated with overestimation of polyp size and low specificity of sonographic size ≥1 cm for neoplasia, which contributes to surgical overtreatment of nonneoplastic polyps.

Performance analysis of high-throughput HPV testing on three automated workflows.

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene® HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas® HPV test (DNA amplification), and (3) Hologic Aptima® HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory's demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.

Allelic imbalance at the HER2/TOP2A locus in breast cancer.

BACKGROUND: Breast cancer is a heterogeneous disease with various histological features and molecular markers. These are utilized for the prediction of clinical outcome and therapeutic decision making. In addition to well established markers such as HER2 overexpression and estrogen and progesterone receptor (ER and PR) status, chromosomal instability is evolving as an important hallmark of cancers. The HER2/TOP2A locus is of great importance in breast cancer. The copy number variability at this locus has been proposed to be a marker for the degree of chromosomal instability. We therefore developed a Single Nucleotide Polymorphism (SNP) assay to evaluate allelic imbalance at the HER2/TOP2A locus in three different entities of primary breast tumors. METHODS: Eleven SNPs were carefully selected and detected by real time PCR using DNA extracted from paired (histologically normal and tumor) paraffin-embedded tissues. Primary breast tumors of 44 patients were included, 15 tumors with HER2 overexpression, 16 triple negative tumors, defined by the absence of HER2 overexpression and a negative ER and PR status and 13 ER and PR positive tumors without HER2 overexpression. As controls, histologically normal breast tissues from 10 patients with no breast tumor were included. RESULTS: Allelic imbalance was observed in 13/15 (87 %) HER2 positive tumors, the remaining 2 being inconclusive. Of the 16 triple negative tumors, 12 (75 %) displayed instability, 3 (19 %) displayed no instability, and 1 was inconclusive. Of the 13 hormone receptor positive tumors, 5 (38 %) displayed allelic imbalance, while 8 did not. CONCLUSIONS: We conclude that the SNP assay is suitable for rapid testing of allelic (im)balance at the HER2/TOP2A locus using paraffin-embedded tissues. Based on allelic imbalance at this locus, both triple negative and ER and PR positive breast tumors can be subcategorized. The clinical relevance of the allelic (im)balance status at the HER2/TOP2A locus in breast cancer is subject of future study. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2086062232155220.

Single nucleotide polymorphism (SNP)-based loss of heterozygosity (LOH) testing by real time PCR in patients suspect of myeloproliferative disease.

During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n=6 25-50% and n=6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25-50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci.

Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications.

BACKGROUND: A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE). These FFPE-archived tissues are an extremely valuable source for retrospective (genetic) studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues. FINDINGS: We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1) heat-treatment, 2) QIAamp DNA-blood-mini-kit, 3) EasyMAG NucliSens and 4) Gentra Capture-Column-kit.Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp). QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG. CONCLUSIONS: We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications.