Characterization of PD-L1 Immunohistochemical Expression in Cell Blocks With Different Specimen Fixation and Processing Methods.

Interpretative criteria for programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) have been largely based on data from formalin-fixed, paraffin-embedded tissues, despite the fact that cytologic specimens, especially cell blocks, are often the only or most readily available tissue for testing. Unlike biopsy specimens, however, cytology sample processing methods can vary markedly. The purpose of this study was to evaluate the effects of several common preanalytic variables on PD-L1 IHC. Two cell lines with strong expression of PD-L1 (H441) and no expression (MCF7) were cultured in vitro. Harvested cells were collected in PreservCyt, CytoLyt, cell culture media (RPMI), saline, and formalin. Cell blocks were prepared by the plasma-thromboplastin method or Cellient automated system and stained with the FDA-approved 28-8 PD-L1 antibody per protocol. PD-L1 expression was scored manually by 3 pathologists for stain intensity and localization and compared across preparation methods. Several IHC staining patterns were observed: complete membranous, partial membranous, globular, and cytoplasmic, with some overlap. Cellient blocks had the best interobserver agreement and cytomorphology, highest proportion of strong complete membranous staining (82%), and least amount of cytoplasmic (11%) and globular staining (8%). RPMI, saline, and formalin samples demonstrated increased amounts of cytoplasmic and globular staining relative to Cellient, while CytoLyt exhibited the poorest performance overall. Interpretation of PD-L1 IHC on cell blocks is feasible for most processing methods examined, but may require recognition of increased cytoplasmic and globular staining in some sample types. Cellient cell blocks demonstrated superior performance compared with other methods.

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.

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

OBJECTIVES: To determine the accuracy and reproducibility of differentiating between squamous cell carcinoma (SCC) and adenocarcinoma (ADC) on fine-needle aspiration (FNA) specimens. METHODS: Fifty cases of NSCLC diagnosed by FNA having either concurrent core biopsies or resection as a diagnostic reference standard were selected. FNA slides were reviewed independently by five blinded observers. Two rounds of review were performed. Cases were initially categorized as SCC, favor SCC, NSCLC (type indeterminate), favor ADC, or ADC; while the indeterminate category was eliminated in the second round of review. RESULTS: The interobserver agreement was 0.22 and 0.1 with and without the indeterminate category, respectively. The overall accuracy for differentiating between SCC and ADC of the lung was 65% with the indeterminate category and 66% without. CONCLUSION: Overall, the low interobserver agreement in our study indicates that accurate subclassification between the NSCLCs often cannot be made by cytomorphology alone.

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

BACKGROUND: 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. METHODS: Twenty sequential brushing specimens of bile or pancreatic ducts from each of the categories "atypical" and "suspicious for malignancy" were identified and the slides retrieved. All 40 cases were reviewed independently by four cytopathologists blinded to the original diagnoses. Resulting review diagnoses were statistically analyzed for agreement and the Kappa statistic calculated. Agreement of the observers' diagnoses with original diagnoses was also evaluated. RESULTS: Interobserver agreement was graded as slight to fair with observers agreeing in about 50% of cases. The corresponding kappa statistic for the category "atypical" was 0.21 and 0.18 for the category "suspicious for malignancy." Reviewer agreement with the original reference diagnosis occurred in approximately one half of review diagnoses. CONCLUSION: Analysis of agreement shows that interobserver agreement was only slight to fair. Despite the categories "atypical" and "suspicious for malignancy" having distinct risks of malignancy (62% versus 74%), the reproducibility of these categories is relatively poor. A single intermediate category may improve reproducibility over the scheme proposed by the Papanicolaou Society of Cytopathology while maintaining an ability to stratify risk of malignancy.

Significance of atypia in pancreatic and bile duct brushings: follow-up analysis of the categories atypical and suspicious for malignancy.

Brushing cytology is frequently utilized for the investigation of pancreatic and biliary strictures but is associated with low diagnostic sensitivity. The Papanicolaou Society of Cytopathology has presented a system for diagnostic classification which includes the categories benign, atypical, suspicious for malignancy and malignant. We studied a series of 216 pancreatic and biliary brushings with either histologic follow-up or a minimum of 6 months clinical follow-up to determine outcomes for the diagnostic categories ("benign," "atypical, favor reactive," "atypical, not otherwise specified," "atypical, suspicious" and "malignant"). Eighty-six of the 216 (39.8%) were designated "atypical" with 10 of these designated as "atypical favor reactive." Forty-five were called "atypical not otherwise specified" and 31 were interpreted as "atypical suspicious for malignancy." On follow-up, 2 of 10 (20%) "atypical favor reactive" were eventually associated with a malignant diagnosis and 23 of 31 (74.2%) "atypical, suspicious for malignancy" demonstrated a malignant outcome. The remaining 45 brushings in the "atypical" category were "atypical not otherwise specified," and 62% of these were associated with malignancy on follow-up. Stratification of the "atypical" category into "atypical favor reactive," "atypical, not otherwise specified" and "atypical, suspicious for malignancy" improves diagnostic accuracy. The "atypical suspicious for malignancy" category has a follow-up similar to the "malignant" category while the "atypical favor reactive" category is associated with a clinical outcome similar to that of the "benign" category.

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.

Beyond cytomorphology: expanding the diagnostic potential for biliary cytology.

Malignancy of the extrahepatic biliary tract is a difficult and crucial diagnosis, both clinically and pathologically. Cytologic evaluation of brushings obtained endoscopically from the biliary tree is currently the standard of care in most institutions. However, bile duct brushing cytology has been plagued by low sensitivity and interpretative difficulties in differentiating reactive from malignant cytology. This review outlines both the difficulties presented by cytomorphology and the potential of new diagnostic techniques that promise to increase sensitivity without sacrificing the high specificity of cytomorphology.

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%.

Comparison of ThinPrep UroCyte and cytospin slide preparations for gastrointestinal specimens: evaluation and retrospective performance review.

Gastrointestinal (GI) tract cytology has high specificity but poor sensitivity for detecting GI tract cancer. Newer methods of slide preparation may improve cytology performance and additionally permit molecular slide-based assays that could improve diagnostic accuracy. A split-sample validation study compared slides prepared using ThinPrep UroCyte filters or a cytocentrifuge method with respect to cellularity, stain quality, and interpretation. In this 15-slide split-sample study, UroCyte slide preparations were judged to be superior to cytocentrifuge preparations, and the method was implemented for GI cytology in December 2006. To assess diagnostic performance for GI cytology, we retrospectively reviewed outcomes for one year before and after implementation of UroCyte filter slide preparation. Sensitivity, specificity, positive predictive value, and negative predictive value for both slide preparations were largely equivalent to one another and compared favorably with values in the literature, but varied greatly depending on how atypical and suspicious-atypical cases were defined for calculations. For biopsied biliary samples, the highest sensitivities were observed when all atypical and suspicious-atypical cases were considered positive for malignancy, but were lower when suspicious-atypical cases were considered positive and atypical cases were considered negative for malignancy. This highlights the difficulty with comparing studies that define atypical classes differently, and points to the need for a well-defined approach to performance evaluation that relates directly to how diagnostic information is used clinically. We conclude that the UroCyte filter slide preparation is valid for evaluation of GI cytology specimens and may simplify adjunct molecular testing such as FISH. This is the first reported use of UroCyte filters for preparation of GI specimens.

Check Sample Abstracts.

The following abstracts are compiled from Check Sample exercises published in 2008. These peer-reviewed case studies assist laboratory professionals with continuing medical education and are developed in the areas of clinical chemistry, cytopathology, forensic pathology, hematology, microbiology, surgical pathology, and transfusion medicine. Abstracts for all exercises published in the program will appear annually in AJCP.

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.