Stewart M. Knoepp

The Application of Molecular Diagnostic Studies Interrogating EGFR and KRAS Mutations to Stained Cytologic Smears of Lung Carcinoma

EGFR and KRAS mutation analyses are of increasing importance for guiding the treatment of non-small cell lung carcinomas. Insufficient cellularity of cell blocks can represent an impediment to the performance of these tests. We investigated the usefulness of cytologic direct smears as an alternative specimen source for mutation testing. Tumor cell-enriched areas from freshly prepared and archived rapid Romanowsky-stained direct smears in 33 cases of lung carcinoma were microdissected for DNA isolation and evaluated for EGFR and KRAS mutations. EGFR mutations were detected in 3 adenocarcinomas; 2 tumors had the L858R substitution and 1 an exon 19 deletion. KRAS mutations affecting codon 12, 13, or 61 were detected in 11 cases (8 adenocarcinomas and 3 non-small cell carcinomas). EGFR and KRAS mutations were mutually exclusive. Hence, archived and freshly prepared direct smears represent a robust and valuable specimen source for molecular studies, especially when cell blocks exhibit insufficient cellularity.

Ancillary techniques on direct‐smear aspirate slides

Numerous cytologic techniques aimed at effectively acquiring patient material for molecular testing have been proposed. Such techniques are becoming ever more important in an age of personalized medicine. In this commentary, the authors explored some more commonly proposed techniques to aid in the molecular testing of cytologic specimens. These techniques include the use of cell blocks, direct cytologic smears, filter paper storage, frozen samples, and enriched cellular techniques such as ThinPrep and cytospin preparations. Direct‐smeared slides demonstrate excellent preservation of DNA, are easy to prepare, and are amenable to immediate adequacy at the time of the fine‐needle aspiration (FNA) procedure as well as effective subsequent tumor purity estimation. Cell block methods cannot be assessed at the time of FNA and often demonstrate insufficiency, whereas filter paper and frozen techniques do not allow for the direct assessment of the presence and purity of tumor cells in the sample. Direct‐smeared slides are emerging as the most effective preparation and storage medium of cytologic material to be used for molecular testing. Their cost‐effectiveness, ease of use, and reliability have cemented them as the optimal solution for cytopathologists to fulfill the role of providing advanced molecular testing on patient samples. Cancer (Cancer Cytopathol) 2013.

Reply to Ancillary techniques on direct-smear aspirate slides: a significant evolution for cytopathology techniques.

We thank da Cunha Santos et al for their interest in our commentary, and agree that the use of FTA cards is most likely a sound choice for long-term DNA storage. In our experience, the use of Diff-Quik–stained cytologic smears, stored for years, is also satisfactory for the isolation of high-quality DNA. Several points deserve clarification regarding these methods. As the authors point out, the breakage of slides during transport is a potential issue; nonetheless, careful packing of slides can prevent this and we do not believe that this is a serious drawback to the use of smears. With regard to the possible problem with the stability of unstained smears, we acknowledge heat and humidity as potential problematic issues. This can be circumvented by storing the slides in climate-controlled rooms. In our practice, any remaining air-dried, unstained slides are stained with Diff-Quik, coverslipped, and stored long term after ancillary tests have been completed. If additional DNA is needed in the future, one of the extra Diff-Quik–stained slides can be decoverslipped for tumor cell microdissection and DNA purification. The authors fail to provide convincing data that filter papers are any less susceptible than unstained smears to heat and humidity. Because it is possible that the filter papers can attract moisture, a side-by-side comparison would be interesting. More clarification is needed regarding the authors’ insistence that analysis of a corresponding stained cytospin slide obtained from the same needle rinse as the FTA card helps ensure the identity of the material in the FTA card. When assessing tumor purity in a specimen, or whether tumor cells are even present, there is no better substitute than the ability to examine the actual cells to be extracted with the microscope. This is possible with stained slides. Slides are not homogenous; they often display contaminating, benign cells as well as areas of relatively pure tumor cells. Microdissection of tumorenriched areas is possible with smears whereas this is not possible with FTA cards. The use of cytospin preparations to infer tumor cellularity and purity on FTA cards is still an extrapolative exercise; extrapolation is not necessary with smears because “what you see is what you get.” More data are needed to conclude whether FTA cards or stained slides provide higher quality nucleic acids on extraction. Molecular testing methodology is constantly evolving, and the requirements of nucleic acid quality will require constant evaluation. During this very exciting era, cytopathologists are starting to investigate various cytopreparatory platforms (smears, FTA cards, ThinPrep slides, etc.) and slide staining conditions for the isolation of nucleic acids. We believe that no method is perfect and each can be continually improved. A major advantage of cytologic sample processing lies in the versatility of the sample slide preparation methods available, and we believe these different platforms are complementary. Collaborative efforts to continuously optimize nucleic acid purification from various specimen preparations is essential for cytopathologists worldwide to better serve their patients in this constantly evolving era of precision medicine.

Application of immunocytochemistry and BRAF mutational analysis to direct smears of metastatic melanoma

The cytodiagnosis of melanoma in fine‐needle aspiration (FNA) specimens can be challenging, often requiring the use of immunocytochemistry. As constitutively activating mutations in the BRAF oncogene are present in at least 40% of melanomas, the use of FNA material to interrogate the BRAF mutational status is likely to increase. Because cell blocks, traditionally used for these studies, can occasionally exhibit insufficient tumor cellularity, the authors investigated the utility of direct smears for immunocytochemistry and BRAF mutational analysis.

Group consensus review minimizes the diagnosis of "follicular lesion of undetermined significance" and improves cytohistologic concordance

We conducted a group consensus review of thyroid aspirates that were previously interpreted as “atypia of undetermined significance/follicular lesion of undetermined significance” (AUS/FLUS) and followed by surgical interventions. The study aimed to investigate if consensus review would minimize the diagnosis of AUS/FLUS with an optimal interobserver agreement and also promote a better cytohistologic concordance. A group of reviewers who were blinded to the corresponding histologic findings simultaneously evaluated a total of 50 aspirates at a multiheaded light microscope. Using the Bethesda System for Reporting Thyroid Cytopathology as a guideline, a consensus interpretation was reached upon review of each aspirate. Interobserver agreement was calculated and recorded. The cytohistologic correlation was then performed between the consensus interpretation and the corresponding histologic diagnosis. The consensus review reclassified 26 (52%) aspirates as non‐neoplasia/benign, 10 (20%) as follicular neoplasm/suspicious for a follicular neoplasm, 1 (2%) as papillary thyroid carcinoma, and 2 (4%) as nondiagnostic. Eleven (22%) aspirates remained AUS/FLUS. The interobserver agreement across the five diagnostic categories ranged from 71.6% to 100% with an average level of 88.8%. Cytohistologic concordance was achieved in 24 of 26 (92.3%) and 9 of 11 (81.8%) aspirates that were reclassified as non‐neoplasia/benign and neoplasia/malignancy, respectively. A diagnostic accuracy of 89.2% (33/37) was obtained in reclassified cases. In conclusion, the group consensus review minimized AUS/FLUS, offered an optimal level of interobserver agreement, and most importantly, promoted excellent cytohistologic concordance in reclassified cases and, therefore, could play a substantial role in the future in reducing reaspiration and/or unnecessary surgeries. Diagn. Cytopathol. 2012.

Utility of PAX8 and PAX2 immunohistochemistry in the identification of renal cell carcinoma in diagnostic cytology

The diagnosis of metastatic renal cell carcinoma (RCC) in cytology specimens may be difficult to confirm on the basis of cytomorphology alone. Often, immunohistochemistry serves as an important adjunct in confirming this diagnosis. Recently, PAX2 was shown to be useful in this regard. In this study, we sought to compare the utility of PAX8 to that of PAX2 immunohistochemistry in the diagnosis of RCC in cytology specimens. First, we verified the performance of PAX8 immunohistochemistry on a tissue microarray (TMA) composed of 54 cases of RCC; PAX8 immunoreactivity was seen in at least 10% of the tumor cells in all cases. Next, we applied PAX8 immunohistochemistry to cell block sections prepared from 24 cases of RCC, obtained from fine‐needle aspirates and effusion specimens. PAX2 immunohistochemistry was performed for comparison. Immunopositivity was defined as the presence of nuclear staining in at least 10% of tumor cell nuclei. Immunoreactivity for PAX8 and PAX2 was seen in 21 (88%) and 20 (83%) of the 24 cases, respectively. The presence of either PAX8 or PAX2 immunostaining was present in 22 of 24 cases, thus showing a total sensitivity of 92%. Overall, the results indicate that PAX8 and PAX2 are diagnostically useful adjuncts in confirming the diagnosis of RCC in cytology specimens. Diagn. Cytopathol. 2012.

Molecular diagnostics of melanoma fine-needle aspirates: a cytology-histology correlation study.

Patients with advanced-stage melanoma harboring a BRAF mutation are candidates for BRAF inhibition as a therapeutic strategy. The use of fine-needle aspiration (FNA) to diagnose metastatic melanoma is increasing. Studies examining the predictive value of BRAF mutation analysis on melanoma FNAs via correlation with follow-up excision findings are lacking. We examined 37 consecutive FNA cases of metastatic melanoma in which the aspirated lesion was subsequently excised. DNA was purified from Diff-Quik-stained FNA smears and tissue blocks from corresponding excisions in parallel. BRAF mutation status was successfully obtained from both specimen types in 34 (92%) of 37 cases. BRAF mutations were detected in 12 (35%) of 34 cases-11 V600E and 1 V600K. Results of BRAF mutational analysis were concordant in all 34 FNA smear/tissue excision pairs. Thus, melanoma FNA for molecular diagnostics represents a rapid, minimally invasive, and effective management strategy in this era of precision medicine.

The application and diagnostic utility of immunocytochemistry on direct smears in the diagnosis of pulmonary adenocarcinoma and squamous cell carcinoma.

The importance of subclassifying pulmonary nonsmall cell carcinoma (NSCLC) in cytologic material is becoming increasingly paramount. Occasionally, cell blocks traditionally used for ancillary studies are sparsely cellular or acellular. Hence, we investigated the diagnostic utility of immunocytochemistry for Napsin‐A, TTF‐1, and p63 on direct smears of NSCLC. Immunohistochemistry for Napsin‐A was initially tested on a tissue microarray (TMA) composed of pulmonary adenocarcinoma. Subsequently, in 25 cases, immunocytochemistry for Napsin‐A, TTF‐1, and p63 was performed on cytologic direct smears. Smears were prepared from tumor cells scraped from lung resection specimens (n = 10), endobronchial ultrasound‐guided transbronchial fine‐needle aspirates (n = 13), and pelleted cell material from pleural effusions (n = 2). Immunohistochemistry utilizing the TMA revealed Napsin‐A positivity in 73% of pulmonary ADCs. Next, immunocytochemistry on direct cytologic smears demonstrated a Napsin‐A(+)/TTF‐1(+) immunophenotype in 15 of 18 adenocarcinomas; p63 was completely negative (n = 12) or only focally positive (n = 3) in these 15 adenocarcinomas. The remaining three adenocarcinomas were negative for all three markers. All six squamous cell carcinomas were Napsin‐A(−)/TTF‐1(−) and diffusely p63(+). In conclusion, direct smears represent a feasible and robust source of cellular material for immunocytochemical studies to diagnose pulmonary ADC and SQC. Our method allows the cytologist to confirm on site that material for diagnostic immunocytochemistry is present thereby serving as a safeguard in instances where the cell block is of insufficient cellularity. Diagn. Cytopathol. 2012.

Minimizing the diagnosis of “follicular lesion of undetermined significance” and identifying predictive features for neoplasia

We used proposed standard morphologic criteria as a guideline to conduct a 10‐year retrospective review of thyroid fine‐needle aspiration specimens that were originally interpreted as “follicular lesion of undetermined significance” and followed by surgical intervention. We sought to investigate whether the indeterminate diagnosis could be minimized by assessing various cytomorphologic features and identifying the features predictive of neoplasia. Using the standard morphologic criteria, we semi‐quantitatively assessed a total of 24 cytomorphologic features in 123 aspirates and recorded an overall interpretation on completion of the review. Cyto‐histologic correlation was evaluated and logistic regression model was performed to identify cytomorphologic features predictive of neoplasia. Although 32 of 123 aspirates remained in the indeterminate category, the retrospective review reclassified 64 aspirates as non‐neoplasia and 27 aspirates as neoplasia. Histologic confirmation was achieved in 47 (73.4%) non‐neoplastic and 15 (55.6%) neoplastic aspirates with a diagnostic accuracy of 68.1%. Furthermore, our analysis demonstrated that neoplasia is positively associated with the presence of syncytial tissue fragments, isolated microfollicles, follicles with scalloped borders, scant cytoplasm, irregular nuclear membranes, nuclear overlapping, coarse chromatin, and increased cellularity. On the contrary, the presence of honeycombing tissue fragments, background colloid, and histiocytes inversely correlated with neoplasia.

Resolution of equivocal results with the Hybrid Capture II high-risk HPV DNA test: a cytologic/histologic review of 191 cases.

IntroductionThe Hybrid Capture II (HC II, Digene) high-risk human papilloma virus (HPV) (hrHPV) DNA test is an in vitro nucleic acid hybridization assay that uses enhanced chemiluminescence for the qualitative and semiquantitative detection of hrHPV in cervical samples. Patient samples are concomitantly tested with positive and negative DNA controls and results reported as positive or negative on the basis of a ratio of relative light units to a cutoff value derived from the positive control (RLU/CO). Samples with a ratio <1.0 RLU/CO are expressed as negative for hrHPV, samples with a ratio >2.5 RLU/CO are expressed as positive for hrHPV, and samples with a ratio between these numbers are submitted for retesting. These “equivocal” values are resulted as positive for hrHPV if either of 2 subsequent test values equals or exceeds 1.0 RLU/CO. Samples that show <1.0 RLU/CO after 2 repeat tests are resulted as negative for hrHPV. MethodsIn this study, we evaluated all hrHPV test results over a 17-month period in our institution. Initial tests showing an equivocal result were analyzed for final retesting result, and for all corresponding and subsequent cytology and histology results. All hrHPV tests were conducted on SurePath (TriPath) or ThinPrep (Cytyc) cervical cytology specimens using the HC II hrHPV DNA test. Subsequent hrHPV tests also were correlated with incident and follow-up findings. ResultsA total of 4792 hrHPV test results were evaluated. Of these, 191 (4%) showed equivocal initial results. When retested, 178 of the 191 samples (93%) resulted positive for hrHPV on first retest and an additional 8 resulted positive for hrHPV on the second retest, bringing the total positive tests to 186 out of 191 (97.4%). Five samples (2.6%) out of 191 were finally expressed as negative for hrHPV. Corresponding cytologic interpretations for the 191 specimens were as follows: NILM-30, atypical squamous cell of undetermined significance (ASC-US)-138, atypical squamous cells—cannot exclude HSIL-13 (ASC-H-13), LSIL-9, and high-grade squamous intraepithelial lesion (HSIL)-1. Follow-up histology was available for 60 of the 191 equivocal cases and showed cervical intraepithelial neoplasia (CIN) II or CIN III in 7 cases, CIN I in 13 cases, and negative or reactive changes in 40 cases. ConclusionsOn the basis of the results, repeat testing of equivocal specimens might not be necessary as these specimens are overwhelmingly found to be positive for hrHPV. Additionally, hrHPV tests falling in the equivocal range should be considered as definite positive tests, as follow-up results in this cohort demonstrate that significant histologic abnormalities are associated with 10.5% of these cases (20/191), and with 33% of those biopsied (20/60) cases.