Barbara Chadwick

Detection of c-kit exons 11- and 17-activating mutations in testicular seminomas by high-resolution melting amplicon analysis

A subgroup of testicular seminomas has been reported to contain activating mutations in KIT, the transmembrane tyrosine kinase receptor encoded by the c-kit gene. Most mutations are in exon 17, although exon 11-activating mutations have recently been described. For patients refractory to standard therapeutic protocols for seminoma, the presence of c-kit-activating mutations in some of these neoplasms might suggest an alternative therapy with KIT targeting drugs. We used the novel mutation scanning technique, high-resolution melting amplicon analysis, to screen a series of 22 testicular seminomas for c-kit-activating mutations. Four cases (18%) had exon 17-activating mutations and these included D816Y, D816V, Y823N and one case that contained both D816E and D820H. A single case (5%) had an exon 11-activating mutation. Interestingly, the exon 11-activating mutation was L576P, the same mutation that characterizes the rare c-kit mutation-positive cases of malignant melanoma. Fluorescence in situ hybridization (FISH) for c-kit suggested that most seminomas are probably polysomic for c-kit and there was not a significant difference in c-kit FISH characteristics between the mutation-positive and mutation-negative cases. The use of high-resolution melting amplicon analysis as a screening technique will allow for the rapid identification of patients with testicular seminomas whose tumors contain c-kit-activating mutations. This could benefit patients whose tumors are refractory to standard therapeutic protocols.

Histologic, immunohistochemical, and molecular classification of 52 IPMNs of the pancreas.

BackgroundIntraductal papillary mucinous neoplasms (IPMNs) of the pancreas account for approximately 5% of pancreatic neoplasms. Prognosis is superior to that of pancreatic invasive ductal carcinoma. IPMNs reveal a variety of epithelial linings expressing different mucin staining patterns and may progress along different oncogenic pathways. Materials and MethodsFifty-two IPMNs were studied for expression of MUC1, MUC2, p16, p21, HER2, cyclin D1, and p53 protein and for mutations in K-ras, HER2, p53, EGFR, and BRAF genes. The cases were evaluated for dysplasia, presence of invasion, and morphology of lining epithelium. ResultsTwenty-six IPMNs appeared intestinal (IN). Five were low, 12 moderate, and 9 high grade. K-ras mutations were found in 15, EGFR mutations in 2, and BRAF mutation in 1. Seven cases were pancreaticobiliary (PB) and all showed moderate to high-grade dysplasia. Six K-ras mutations and 2 p53 mutations were found in PB tumors. p53 mutations were in cases with high-grade dysplasia. Nineteen IPMNs demonstrated a gastric foveolar (GF) pattern. The majority of GF cases had low or moderate dysplasia. Sixteen revealed K-ras mutations and 1 case each demonstrated a HER2 or p53 mutation. Five IPMNs revealed invasive adenocarcinoma, including a colloid carcinoma from an IN type epithelium. ConclusionsIN pattern IPMNs were the most common. Mixed histology was common. K-ras mutations were most common, but did not correlate with dysplasia. p53 mutations were seen in 6% of cases (only in GF and PB subtypes). A HER2 mutation was found in a GF IPMN. EGFR and BRAF mutations were restricted to IN IPMNs. These findings suggest the possibility of alternate pathways for carcinogenesis between epithelial subtypes of IPMNs.

Pathologic evaluation of a new endoscopic ultrasound needle designed to obtain core tissue samples: A pilot study

Background and Objectives: Standard endoscopic ultrasound-fine-needle aspiration (EUS-FNA) needles are in widespread use. Meaningful differences between the available needles have been difficult to identify. Recently, a new EUS needle (Shark Core®, Covidien, Dublin, Leinster, Ireland), has been introduced in an attempt to improve diagnostic accuracy, tissue yield, and to potentially obtain a core tissue sample. We performed a pilot study prospectively to evaluate this new needle when compared to a standard EUS-FNA needle. Materials and Methods: Analysis of the first 15 patients undergoing EUS-FNA with the Shark Core needle was performed and it was compared to EUS-FNA in 15 patients who underwent EUS-FNA with a standard needle. Results: The Shark Core needle required fewer needle passes to obtain diagnostic adequacy than the standard needle [(χ2(1) = 11.3, P < 0.001]. The Shark Core needle required 1.5 passes to reach adequacy, whereas the standard needle required three passes. For cases with cell blocks, the Shark Core needle produced diagnostic material in 85% of cases [95% confidence interval (CI): 54–98], whereas the standard needle produced diagnostic material in 38% of the cases (95% CI: 9-76). The Shark Core needle produced actual tissue cores 82% of the time (95% CI: 48–98) and the standard needle produced no tissue cores (95% CI: 0-71) (P = 0.03). Conclusion: This pilot study found that the Shark Core needle had a high rate of producing adequate cytologic material for the diagnosis of pancreatic and peri-pancreatic lesions sampled by EUS with fewer passes required to obtain a definitive diagnosis and with a high rate of tissue cores being obtained when compared to a standard FNA needle.

Identification of factors predictive of malignancy in patients with atypical biliary brushing results obtained via ERCP

Biliary brushings obtained during ERCP can have one of three cellular interpretations: benign, malignant, or “atypical.” Atypical interpretations usually result in further testing, and may cause controversy over management and increases in cost. We evaluated a large cohort of patients with atypical biliary brushings for analysis and risk stratification.

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.

A retrospective review of UroVysion fish interpretations over 8.6 years: a major shift in the patient test population.

UroVysion FISH detects chromosomal aberrations associated with urothelial carcinoma. In our laboratory, UroVysion FISH was initially evaluated manually with a change to image‐aided interpretation using the BioView Duet imaging system. This retrospective study examined diagnostic findings over an 8.6 year period, with 1,869 manual interpretations over 4.8 years and 3,936 image‐aided interpretations over 3.8 years. Although the initial goal was to evaluate possible impacts of the imaging system on diagnostic interpretations, the most important finding was that the demographics of the test population changed significantly. Female specimens increased incrementally from an average of 29% compared to 43% of the samples during periods of manual interpretation versus image‐aided interpretation, respectively. The shift may reflect a gradual increase in the percentage of low‐risk hematuria patients being evaluated for initial diagnosis of bladder cancer, rather than bladder cancer recurrence. Interpretation rates, evaluated separately for males and females, changed significantly over the test period. Male interpretation results were negative (75.1 vs. 67%), positive (18.6 vs. 14.6%), unsatisfactory (5.0 vs. 16.9%), and equivocal (1.4 vs. 1.5%) during periods of manual versus image‐aided interpretation, respectively (Fisher Exact Test P‐value = <0.0001). For females, results were negative (86.1 vs. 79.3%), positive (9.2 vs. 11.1%), unsatisfactory (2.8 vs. 8.9%), and equivocal (1.8 vs. 0.7%) over the same periods (Fisher Exact Test P‐value = <0.0001). Logistic regression analysis identified the change in test population as the variable with the greatest impact on observed interpretation rate changes. Diagn. Cytopathol. 2013.

Cytomorphology is often insufficient to categorize non‐small‐cell lung carcinoma on FNA specimens

To determine the accuracy and reproducibility of differentiating between squamous cell carcinoma (SCC) and adenocarcinoma (ADC) on fine‐needle aspiration (FNA) specimens.

Interobserver reproducibility and agreement with original diagnosis in the categories “atypical” and “suspicious for malignancy” for bile and pancreatic duct brushings

The Papanicolaou Society of Cytopathology has developed a set of guidelines which include a diagnostic scheme with the categories “atypical” and “suspicious for malignancy.” These intermediate categories may help stratify risk of malignancy for samples obtained from the bile and pancreatic ducts. However, the reproducibility of these intermediate categories is currently unknown.

Evaluation of the SharkCore needle for EUS-guided core biopsy of pancreatic neuroendocrine tumors.

Background and Objectives: EUS guided core biopsy was once rarely performed but is now entering mainstream practice. Neuroendocrine tumors often warrant core biopsy as sufficient tissue must be obtained to allow for special staining to ensure a correct diagnosis. Traditionally these lesions were sampled with FNA needles. We performed a retrospective pilot study to evaluate the clinical value and efficacy of the a new EUS core needle biopsy needle as compared to a standard EUS FNA needle in the evaluation of patients with known or suspected neuroendocrine tumors. Methods: A retrospective analysis of the first 10 patients (between January 2015 and April 2016) to undergo EUS-FNA with the SharkCore® needle at the University of Utah School of Medicine/Huntsman Cancer Center with neuroendocrine tumors. Each case was retrospectively reviewed by a board certified cytopathologist (BLW) for the following cytologic parameters on the aspirate smears or touch/squash preparations: overall cellularity [1 (low) to 3 (high)], percentage of obtained cells that were lesional/representative (<25%, 26%-50%, and >50%), relative ease of interpretation [1 (difficult) to 3 (easy)]. Pathologic material and reporting records were also reviewed for each case to confirm the number of needle passes to achieve diagnostic adequacy, the presence or absence diagnostic material on H&E slide (from cell block, if prepared), whether a definitive diagnosis was able to be rendered, and the presence or absence of a true core/core fragments (within the cell block, if prepared). Results: A total of 20 patients underwent EUS-FNA for suspected neuroendocrine lesions. Ten patients underwent either transgastric or transduodenal EUS-FNA with the 22 gauge SharkCore® needle. The comparison cohort of 10 patients underwent either transgastric or transduodenal EUS-FNA with the standard 22 gauge Echotip® needle. The SharkCore® needle required a fewer mean number of needle passes to obtain diagnostic adequacy than the Echotip® (P=0.0074). For cases with cell blocks, the SharkCore® needle produced diagnostic material in 100% of cases, whereas Echotip® produced diagnostic material in 60% of cases. There was no significant difference between specimen cellularity, percentage of lesional material, or ease of interpretation between the two needle types. Conclusion: Our pilot investigation targeting patients with known or suspected pancreatic NETs indicates that the SharkCore® needle shows promise in obtaining suitable tissue for ancillary testing that can allow for more definitive pathologic interpretations on EUS FNA specimens. Fewer passes were needed with the core needle when compared to a standard needle.

Lymphoepithelioma-like carcinoma of the uterine cervix: Cytomorphologic features and diagnostic pitfalls by liquid-based cytology

Lymphoepithelioma‐like carcinoma (LELC) is a rare variant of cervical squamous cell carcinoma with unclear HPV and EBV association. Due to the accompanying and often obscuring lymphocytic inflammation, malignant cells may be difficult to recognize on cytologic samples and can mimic several more common benign and atypical cervical lesions. In this case report, we describe the cytomorphologic findings of LELC by liquid‐based cytology in a 59‐year‐old Caucasian woman who was positive for high risk HPV but had no visible cervical lesions. We also discuss the differential diagnosis, diagnostic pitfalls, and practical considerations for diagnosis.