Application of Immunohistochemistry in Cytology.

Immunohistochemistry (IHC), also referred to as immunocytochemistry in cytology literature, has revolutionized the practice of cytopathology. Because of the complexity of cytology preparation and limited diagnostic material, performing IHC remains a challenge. Formalin-fixed paraffin-embedded (FFPE) cell block (CB) is the optimal choice for IHC. In this review, the approaches for improving CB preparation will be discussed. When CB material is not available, various cytology specimens can also be used for IHC. With the utilization of Antigen Retrieval (AR) technique, these nonformalin-fixed cytology specimens can achieve successful IHC staining, comparable with the results from FFPE tissue sections. In the last part of this review, we will discuss the use of positive controls and the important role of AR in standardization of IHC in cytology.

Evaluation of ACR TI-RADS cytologically indeterminate thyroid nodules and molecular profiles: a single-institutional experience.

INTRODUCTION: The American College of Radiology (ACR) Thyroid Imaging Reporting and Data Systems (TI-RADS) was developed to standardize thyroid ultrasound reports and predict the likelihood of malignancy. In our study, we aimed to correlate indeterminate thyroid fine needle aspiration cytology cases with preceding ultrasound (US) ACR TI-RADS scores and concurrent molecular testing results to examine how well the use of the ACR TI-RADS in our institution predicted which patients with indeterminate cytology might harbor molecular alterations. MATERIALS AND METHODS: We performed a retrospective review of thyroid nodules. Patients with US reports that included TI-RADS scores, fine needle aspiration specimens with indeterminate cytology (Bethesda class III-V), and molecular testing results were included. RESULTS: A total of 46 indeterminate cytology cases had had preceding US reports with TI-RADS scores and molecular testing (Bethesda class III, n = 37; Bethesda class IV, n = 6; Bethesda class V, n = 3). Most of the indeterminate cases had had a TI-RADS score of TR4 (31 of 46; 67.39%) or TR5 (9 of 46; 19.57%). RAS mutations were the most common alteration (n = 12). Of the 46 cases, 22 (47.85%) showed no alterations. Ten cases proceeded to surgery, of which seven displayed malignancies. CONCLUSIONS: Molecular testing in cytologically indeterminate thyroid nodules provided valuable information for TR4 and TR5 lesions; however, the TR2 and TR3 lesions often had no molecular alterations. These findings highlight the potential value of including US imaging features when assessing the significance of indeterminate cytology findings.

Myoepithelial carcinoma of soft tissue is a diagnostic challenge on fine-needle aspiration: Case report and review of literature.

Myoepithelial carcinoma (MEC) of soft tissue, also known as malignant myoepithelial tumor, is an uncommon malignancy. Cytologic diagnosis of this entity is challenging due to its rarity and heterogeneous morphology. We report a case of MEC in a 22-year-old man, who presented with a 6.5 cm soft tissue mass on his right distal forearm that has been enlarging over the past 3 months. Ultrasound-guided fine-needle aspiration (FNA) revealed abundant isolated neoplastic cells ranging from spindled cells to epithelioid and plasmacytoid morphology in a myxoid background. These cells showed moderate cytologic atypia characterized by high-nuclear/cytoplasmic ratio, irregular nuclear contours, and prominent nucleoli. The cytoplasm varied from dense to vacuolated and occasionally rhabdoid with intracytoplasmic inclusions. Scattered bi- and multinucleated cells were identified. A diagnosis of high-grade malignancy was made with the differential diagnosis including rhabdomyosarcoma and melanoma. A subsequent core biopsy of the tumor showed immunoreactivity for pan-cytokeratins, calponin, p63, and smooth muscle actin. INI-1 was lost. SOX-10 and Melan-A were negative. Molecular studies showed loss of SMARCB1 (INI-1) and CDKN2A. Gene fusion studies did not detect any fusion. A diagnosis of soft tissue MEC was made which is a challenge on FNA due to several cytologic mimickers including rhabdomyosarcoma, epithelioid sarcoma, extrarenal rhabdoid tumor, extra-axial chordoma and melanoma. Recognition of the biphasic cell population in a myxoid background and a battery of immunohistochemical stains are crucial for accurate diagnosis.

Does a higher American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS) score forecast an increased risk of malignancy? A correlation study of ACR TI-RADS with FNA cytology in the evaluation of thyroid nodules.

BACKGROUND: Ultrasound has become the initial approach to evaluating thyroid nodules, facilitating the distinction between benign and malignant nodules based on composition, echogenicity, nodule border or margin, shape, the presence of calcifications, and nodule dimensions. The American College of Radiology (ACR) recommended the Thyroid Imaging Reporting and Data System (TI-RADS) as a classification system to standardize thyroid ultrasound reports and to predict the probability of malignancy in thyroid nodules using a scoring system (TR1-TR5) based on multiple ultrasound characteristics and nodule size. Fine-needle aspiration (FNA) is recommended as the next step for nodules that warrant further workup. The authors assessed the accuracy of the ACR TI-RADS based on the corresponding FNA cytology results (Bethesda system diagnoses I-VI). METHODS: ACR TI-RADS ultrasound reports and corresponding FNA cytology diagnoses from January 1, 2018 to August 30, 2018 were evaluated. RESULTS: From January 1, 2018 to August 30, 2018, 2306 thyroid ultrasound-guided FNAs were performed at our institution. Of 2306 cases, 361 had ACR TI-RADS reports available. The majority of FNAs were TR4 (180; 49.9%) or TR3 (108; 29.9%). No TR2 or TR3 nodules were associated with Bethesda category V or VI diagnoses. The majority of TR4 nodules (142 of 180; 78.9%) and TR5 nodules (42 of 65; 64.6%) exhibited benign (Bethesda category II) cytology. Fourteen TR5 cases (21.5%) had malignant (Bethesda category VI) cytology. CONCLUSIONS: Although there were no TR2 or TR3 malignant (Bethesda category VI) diagnoses, and there were only a few malignancies in the TR4 and TR5 categories, the current results reassert the notion that the ACR TI-RADS scoring system shows at least some correlation between benign or malignant cytology diagnoses, as illustrated by the greater number of malignant cases in the higher ACR TI-RADS categories.

Application of GATA 3 and TTF-1 in differentiating parathyroid and thyroid nodules on cytology specimens.

BACKGROUND: Differentiating parathyroid from thyroid lesions can be difficult on fine-needle aspiration (FNA) due to overlapping cytomorphologic features. While the traditional parathyroid hormone (PTH) assays can help in the distinction, these tests may be cumbersome, particularly when the lesion is unexpected clinically and a needle wash is not collected at the time of FNA. Therefore, we chose to investigate the application of immunohistochemical staining (IHC) with GATA 3 and thyroid transcription factor-1 (TTF-1) on air-dried cytology smears to distinguish parathyroid and thyroid lesions. METHODS: Air-dried touch preparation (TP) slides were prepared from consecutively selected parathyroid and thyroid specimens. Thirteen FNA cases with the clinical concern for parathyroid lesions were also included in the study. IHC was performed on unstained and ultrafast Papanicolaou (UFP) stained air-dried slides. RESULTS: On TP slides, GATA 3 expression was observed in all cases of parathyroid origin but no immunoreactivity was present in thyroid lesions. TTF-1 expression was observed in all cases of thyroid origin but not in parathyroid lesions. GATA 3 and TTF-1 expression of 13 FNA cases were consistent with the clinical impression or concurrent PTH tests. CONCLUSIONS: IHC with GATA 3 and TTF-1 on air-dried cytology smears is a simple and effective way to differentiate parathyroid vs thyroid lesions on FNA. Air-dried unstained and UFP-stained slides perform equally well with IHC, but UFP-stained slides provide the added benefit of morphologic evaluation and assessment of smear cellularity prior to IHC.

Does Noninvasive Follicular Thyroid Neoplasm With Papillary-Like Nuclear Features (NIFTP) Have a Unique Molecular Profile?

OBJECTIVES: Recognizing preoperative characteristics of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is important for clinical management. Therefore, we assessed presurgical NIFTP molecular profiles using fine-needle aspiration (FNA) material. METHODS: Presurgical FNA reports of 39 surgically confirmed NIFTP cases from January 2013 through May 2017 were assessed for Afirma and ThyroSeq results. RESULTS: Twenty-one of 39 NIFTP nodules were preoperatively tested with Afirma with two benign and 19 suspicious results. Twenty-seven of 39 nodules were tested with ThyroSeq (nine of 39 had both Afirma and Thyroseq): 18 (67%) had RAS mutations (13 NRAS, four HRAS, one KRAS), and three of 18 had multiple alterations (NRAS + TP53, n = 1; NRAS + PTEN, n = 2). BRAF T599_R603 + EIF1AX mutation (n = 1), PTEN mutation (n = 1), MET overexpression (n = 1), PAX8/PPARG fusion (n = 3), and THADA/IGF2BP3 fusion (n = 3) comprised the remainder. CONCLUSIONS: NIFTP cases most commonly displayed suspicious Afirma results and RAS mutations on ThyroSeq, lacking aggressive/BRAF-V600E-like mutations. While NIFTP remains a surgical entity, the lack of aggressive/BRAF-V600E-like mutations can aid in determining the extent of surgery.

Educational Case: Head and Neck Neoplasia: Salivary Gland Tumors.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.

Cytomegalovirus (CMV) in cervical cancer screening tests: A series of 8 cases and review of the literature.

OBJECTIVE: Cytomegalovirus (CMV) is a ubiquitous infection typically affecting over 50% of the US population by age 40. We report 8 cases of CMV infections detected in cervical cancer screening tests, the largest series of cases thus far reported in gynecologic cytology specimens. METHODS: A retrospective review of our pathology archival computer database was performed from January 1, 1994 through December 31, 2016 for CMV infections reported in cervical cytology specimens. The slides were retrieved for review if available. RESULTS: The eight patients ranged in age from 21-46 years, with a median age of 27 years and average age of 29.5 years. Two patients were significantly immunocompromised with one patient having AIDS and one patient diagnosed with autoimmune disease. The remaining six patients are considered immunocompetent. Cases were identified most often in the fall and winter months (6 of 8 cases). Seven cases were found using Surepath Pap (SP) liquid-based processing and one case was reported in a classic Papanicolaou smear (CPS). CONCLUSION: There was no correlation of cytologic presentation of CMV with a distinct cytohormonal pattern or inflammatory constituents. Rare diagnostic cells with changes of CMV infection were seen in 75% of the cases. The presence of CMV did not usually result in adverse patient outcome, except where CMV was one of the multiple opportunistic infections detected in the patient with AIDS.

Self-clotting method improves cell block preparation.

BACKGROUND: The success of cell block preparation is crucial for ancillary diagnostic tests in cytology. However, achieving an optimal cell block can be challenging. The current study describes a self-clotting-based technique for fine-needle aspiration (FNA) cell block preparations and evaluates its usefulness in comparison with the conventional needle wash technique. METHODS: The clinical data, FNA procedure, and cellularity of cell blocks of the self-clotting group (37 cases) and the conventional needle wash group (33 cases) were compared. The cellularity was evaluated using a scoring system (0 indicated acellular, 1 indicated 1-50 cells, and 2 indicated >50 cells). RESULTS: Approximately 76% of cases in the self-clotting group received a score of 2 versus 36% in the conventional needle wash group. Approximately 14% received a score of 1 in the self-clotting group compared with 9% in the conventional needle wash group, whereas 11% in the self-clotting group received a score of 0 versus 55% in the conventional needle wash group. The differences between the 2 methods were statistically significant. CONCLUSIONS: The results of the current study demonstrate that the self-clotting method is superior to the conventional needle wash method for FNA samples. Cancer Cytopathol 2018;126:190-9. © 2017 American Cancer Society.

Does noninvasive follicular thyroid neoplasm with papillary-like nuclear features have distinctive features on sonography?

BACKGROUND: The noninvasive encapsulated follicular variant of papillary carcinoma (nEFVPTC) has recently been reclassified to "noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP)," removing this entity from the malignant category. This re-categorization has had major implications for clinical management. NIFTP has overlapping cytohistologic features with papillary thyroid carcinoma (PTC) and with follicular adenomas (FA), but sonographic data comparing NIFTP to PTC and FA is lacking. Our study examines the sonographic features of NIFTP as compared with PTC and FA. METHODS: Ultrasound scans and Doppler blood flow from subjects who had pre-surgical sonograms and fine needle aspiration biopsies with final surgical pathology diagnoses of NIFTP/nEFVPTC, classical PTC, and FA between 01/2013-08/2016 were assessed. Sonographic and Doppler features as well as Bethesda System (TBS) diagnoses were recorded and analyzed. RESULTS: 40 NIFTP, 58 classical PTC, and 23 FA cases were included. The most common NIFTP pre-surgical TBS cytology diagnosis was Atypia of Undetermined Significance (AUS/FLUS) (40%). NIFTP cases predominantly displayed wider-than-tall shape (100%), smooth borders (75%), occurrence in multinodular glands (82.5%), heterogeneous echogenicity (50%), both perinodular and intranodular Doppler flow patterns (70%), minimal Doppler flow grade (62.5%), and no calcifications (90%). CONCLUSIONS: Our study demonstrates that NIFTP, PTC, and FA display several distinguishing and overlapping sonographic and Doppler features. Sonographic features appear to complement cytology findings and may help raise pre-operative concern for NIFTP in the proper clinical setting, potentially leading to a more conservative management approach.

"Low-grade squamous intraepithelial lesion, cannot exclude high-grade:" TBS says "Don't Use It!" should I really stop it?

BACKGROUND: The Bethesda System uses a two-tiered approach in the diagnosis of cervical squamous intraepithelial lesions (SILs). Occasionally, Papanicolaou (Pap) tests with evident low-grade SIL (LSIL) also have some features suggestive but not diagnostic of high-grade SIL (HSIL). This study reviews our experience with "Low-grade Squamous Intraepithelial Lesion, Cannot Exclude High-grade" (LSIL-H) and discusses the best approach to report such Paps if the LSIL-H interpretation is abandoned. METHODS: Abnormal Paps were identified between January and December 2014 that had surgical follow-up within 6 months. Their biopsy outcomes were compared. Statistical analysis was performed using Pearson's Chi-square and McNemar tests in SPSS software version 23. Statistical significance was defined as P ≤ 0.05. RESULTS: There were a total of 1049 abnormal Paps with follow-up. High-grade dysplasia/carcinoma (HGD+) was found in 8% of LSIL, 30% of LSIL-H, 52% of atypical squamous cells (ASCs), cannot rule out HSIL (ASC-H), and 77% of HSIL Paps. The detection rate of HGD+ for LSIL-H was between that of LSIL (Pearson's Chi-square test, P = 0.000) and ASC-H (P = 0.04). If LSIL-H cases are reported as ASC-H, the rate of HGD+ for the ASC-H category would decrease from 51.5% to 37.4% (McNemar test, P = 0.000). Alternatively, if LSIL-H cases are downgraded to LSIL, the rate of HGD+ for the LSIL category would rise from 7.7% to 10.4% (McNemar test, P = 0.000). Nearly 86.7% of LSIL-H cases were positive for high-risk HPV (HR-HPV) in comparison to 77.5% of LSILs, 100% of ASC-Hs, and 75% of HSILs. The sample size for HR-HPV and LSIL-H was too small for meaningful statistical analysis. CONCLUSIONS: "LSIL-H" category detects more HGD+ than LSIL, and fewer than ASC-H and HSIL. If LSIL-H is eliminated, Paps with this finding are best reported as ASC-H to ensure that women with potential HGD+ undergo colposcopy in a timely manner. Reporting LSIL-H as LSIL may delay colposcopy since management of LSIL Paps depends on multiple factors (age, HPV status, etc.).

Can p40 (Polyclonal) Replace p63 (Clone 4A4) in the Cytologic Diagnosis of Pulmonary Non-Small Cell Carcinoma?

OBJECTIVES: Differentiating squamous cell carcinoma from adenocarcinoma (ACA) in cytology specimens can be challenging. Recent literature showed p40 had higher specificity than p63 for this purpose. METHODS: We identified 190 cytology cases with p40 (polyclonal) and p63 (monoclonal clone 4A4) immunohistochemistry, including specimens from fine-needle aspirations (FNAs) and effusions. RESULTS: ACAs of lung origin stained for p40 and p63 in 21% and 20% of cases, respectively, regardless of specimen site. Among lung FNAs of primary pulmonary ACAs (n = 42), 14% were positive for p40 and 24% were positive for p63. Of the 20 pulmonary ACAs in effusions, more cases showed p40 positivity (40%) compared with FNAs, whereas p63 were positive in 15%. Among metastatic ACAs from other sites (n = 14), more cases were positive for p40 than p63. CONCLUSIONS: Polyclonal p40 yields a level of false positivity in ACAs similar to p63, which is highest in effusions and is not limited to lung origin.

Primary anorectal mucosal melanoma detected by anorectal cytology.

The detection of primary anorectal melanoma on anal cytology is a rare and challenging diagnosis. We report a case where anorectal cytology showed isolated malignant cells with oval nuclei, prominent nucleoli, and elongated wispy cytoplasmic projections. There was no evidence of squamous dysplasia or melanin pigment identified. To the best of our knowledge, this is the first reported case of a primary anorectal melanoma detected in anorectal cytology. Detection of malignancies other than squamous cell carcinoma can be seen on anorectal cytology and should be considered when there is no evidence of anal intraepithelial neoplasia. Diagn. Cytopathol. 2017;45:452-455. © 2017 Wiley Periodicals, Inc.

Microcystic/Reticular Schwannoma Arising in the Submandibular Gland: A Rare Benign Entity that Mimics More Common Salivary Gland Carcinomas.

Microcystic/reticular schwannoma is a recently described variant of schwannoma with a predilection for the gastrointestinal tract, rarely involving the head/neck region. This is the first reported case involving the submandibular gland. We present a case in a 34 year old man with 4.5 cm submandibular mass. Fine needle aspiration suggested a spindle cell lesion. Frozen section evaluation raised the possibility of mucoepidermoid carcinoma. Resection showed a well circumscribed mass with a mucoid appearance. Histologic findings include a lobular architecture with fibrous septa, a lympho-plasmacytic infiltrate, and scattered lymphoid aggregates at the periphery. There are two distinct histologic patterns with solid areas of spindle cells and areas of spindle/ovoid cells with a microcystic pattern in a myxoid background. The tumor has a pushing border, with extension into adipose and adjacent parenchyma, without cytologic atypia or necrosis. Immunohistochemical stains are positive for S-100 and CD34, and negative for calponin, mammoglobin, ALK1, p63, ER, GFAP, SMA, desmin, cytokeratin 7, cytokeratin AE1/AE3, and C-Kit. Mucicarmine stain is negative. Recognition of this benign unusual variant of schwannoma is paramount for appropriate conservative treatment due to the morphologic and immunohistochemical overlap with primary salivary gland carcinomas.