Doing more with less: integrating small biopsies in cytology practice.

Cytopathologists are at the forefront of specimen acquisition during many different procedures while providing rapid on site evaluation (ROSE). This has added pressure to cytopathologists as more and more ancillary testing is being requested on smaller amounts of tissue. By focusing on the most common organ sites: lung, head and neck, and pancreas, there is a discussion of what the cytopathologist needs to know to triage tissue successfully. Finally, there is a discussion of the logistical aspects of integrating small biopsies into everyday practice.

Recurrent Wnt Pathway and ARID1A Alterations in Sinonasal Olfactory Carcinoma.

Sinonasal tumors with neuroepithelial differentiation, defined by neuroectodermal elements reminiscent of olfactory neuroblastoma (ONB) and epithelial features such as keratin expression or gland formation, are a diagnostically challenging group that has never been formally included in sinonasal tumor classifications. Recently, we documented that most of these neuroepithelial neoplasms have distinctive histologic and immunohistochemical findings and proposed the term "olfactory carcinoma" to describe these tumors. However, the molecular characteristics of olfactory carcinoma have not yet been evaluated. In this study, we performed targeted molecular profiling of 23 sinonasal olfactory carcinomas to further clarify their pathogenesis and classification. All tumors included in this study were composed of high-grade neuroectodermal cells that were positive for pankeratin and at least 1 specific neuroendocrine marker. A significant subset of cases also displayed rosettes and neurofibrillary matrix, intermixed glands with variable cilia, peripheral p63/p40 expression, and S100 protein-positive sustentacular cells. Recurrent oncogenic molecular alterations were identified in 20 tumors, including Wnt pathway alterations affecting CTNNB1 (n = 8) and PPP2R1A (n = 2), ARID1A inactivation (n = 5), RUNX1 mutations (n = 3), and IDH2 hotspot mutations (n = 2). Overall, these findings do demonstrate the presence of recurrent molecular alterations in olfactory carcinoma, although this group of tumors does not appear to be defined by any single mutation. Minimal overlap with alterations previously reported in ONB also adds to histologic and immunohistochemical separation between ONB and olfactory carcinoma. Conversely, these molecular findings enhance the overlap between olfactory carcinoma and sinonasal neuroendocrine carcinomas. A small subset of neuroepithelial tumors might better fit into the superseding molecular category of IDH2-mutant sinonasal carcinoma. At this point, sinonasal neuroendocrine and neuroepithelial tumors may best be regarded as a histologic and molecular spectrum that includes core groups of ONB, olfactory carcinoma, neuroendocrine carcinoma, and IDH2-mutant sinonasal carcinoma.

Binary subclassification scheme (AUS-Nuclear versus AUS-Other) adequately risk-stratifies thyroid fine needle aspiration specimens classified as Atypia of Undetermined Significance.

INTRODUCTION: The Bethesda System for Reporting Thyroid Cytopathology previously described 4 subclasses of atypia within the Atypia of Undetermined Significance (AUS) category: nuclear (AUS-Nuc), architectural (AUS-A), oncocytic (AUS-Onc), and atypia not otherwise specified (AUS-NOS). Accumulating evidence supports a binary AUS subclassification scheme based primarily on the presence of nuclear atypia only. The purpose of this study is to compare the risk stratification of binary versus 4-tier AUS subclassification systems among AUS nodules with molecular and/or histologic follow-up. MATERIALS AND METHODS: Thyroid aspirates classified as AUS and tested using Afirma (Veracyte, Inc.) between 6/2013 and 7/2021 were included. For resected nodules, histological classification was considered as the final outcome. For unresected nodules, benign Afirma results were considered low-risk outcomes, similar to histologically benign nodules. Suspicious or nondiagnostic Afirma results were considered indeterminate outcomes. The prevalence of outcomes warranting surgery (noninvasive follicular thyroid neoplasm with papillary-like nuclear features [NIFTP] or cancer) was calculated for each AUS subclass. RESULTS: A total of 559 AUS nodules with Afirma testing were identified. Excluding nodules with indeterminate molecular outcomes, NIFTP/cancer prevalence for AUS-Nuc was 21% (57/266), which was higher than that for AUS-A (6%, 11/188), AUS-Onc (8%, 4/53), and AUS-NOS (0%, 0/9). A binary AUS subclassification scheme based on nuclear atypia showed a significant difference in NIFTP/cancer prevalence (21% versus 6%, P < 0.0001). CONCLUSIONS: Binary reporting of AUS subclasses based on nuclear atypia distinguishes cases with a higher risk of NIFTP/cancer. There is a low but non-negligible prevalence of NIFTP/cancer in cases without nuclear atypia.

Risk stratification of cytologically indeterminate thyroid nodules with nondiagnostic or benign cytology on repeat FNA: Implications for molecular testing and surveillance.

BACKGROUND: Evidence guiding the management of cytologically indeterminate thyroid nodules with nondiagnostic (ND) or benign cytology on repeat fine-needle aspiration (FNA) is limited. This study evaluates the utility of molecular testing and estimates the risk of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and cancer among such nodules. METHODS: This was a retrospective single-institution review of thyroid nodules from adults that were classified as atypia of undetermined significance (AUS) or follicular neoplasm (FN) on initial FNA and underwent repeat FNA for cytology and Afirma testing (June 2013-July 2021). The association between repeat FNA cytology and RNA yield for Afirma was determined. Histologic outcomes were integrated with Afirma results to define end points for each nodule. RESULTS: A total of 691 AUS and FN nodules underwent repeat FNA and Afirma testing. Diagnostic Afirma results were obtained in 98% of cases overall and in 91% of nodules with ND cytology on repeat FNA. Using combined molecular and histologic end points, the NIFTP and/or cancer prevalence for nodules with ND cytology on repeat FNA was 9% (95% confidence interval [CI], 0.042-0.182), falling between those nodules classified as benign (5%; 95% CI, 0.029-0.094) and those classified as AUS or FN (18%; 95% CI, 0.140-0.218) on repeat FNA, although not reaching statistical significance from either subgroup (p = .38 and .10, respectively). CONCLUSIONS: AUS and FN nodules that are ND on repeat FNA have low but nonnegligible risk of NIFTP and/or cancer and may benefit from molecular testing, given the low test failure rate in this subgroup. Conversely, AUS and FN nodules reclassified as benign on repeat FNA have a very low risk of NIFTP and/or cancer and are unlikely to benefit from molecular testing.

Follicular Neoplasm of Thyroid Revisited: Current Differential Diagnosis and the Impact of Molecular Testing.

The diagnosis of "follicular neoplasm" (FN) in thyroid cytopathology has a long history that originated not long after the practice of fine-needle aspiration (FNA) of thyroid nodules. From the outset, this interpretive category was intended to convey a set of differential diagnoses rather than a precise diagnosis, as key diagnostic features, such as capsular and vascular invasion, were not detectable on cytology preparations. Cytologic-histologic correlation studies over the past several decades have shown that FN interpretation can be applied to the spectrum of nonneoplastic tumors to carcinomas. Most tumors classified as FN include follicular adenoma, follicular carcinoma, noninvasive follicular thyroid tumor with papillary-like nuclear features, and follicular variant of papillary thyroid carcinoma. Less common entities that may be classified as FN on FNA include hyalinizing trabecular tumor (HTT), poorly differentiated thyroid carcinoma, medullary carcinoma, and nonthyroidal lesions such as parathyroid tissue, paraganglioma, and metastatic tumors. Advances in our ability to detect characteristic molecular alterations (eg, GLIS gene rearrangements for hyalinizing trabecular tumor) in FNA samples may assist in the identification of some of these entities. In this review, we summarize the pathophysiology, history, and evolution of the terminology and the current differential diagnosis according to the recently published 2022 World Health Organization classification, molecular testing, and management of nodules classified as FN.

Sinonasal Tumors With Neuroepithelial Differentiation (Olfactory Carcinoma): Delineation of Their Pathologic and Clinical Features With Insights into Their Relationship to Olfactory Neuroblastoma and Sinonasal Carcinoma.

Olfactory carcinoma is one of many names applied to sinonasal malignancies with histologic similarity to olfactory neuroblastoma (ONB) but cytokeratin expression or gland formation. It is unclear whether these neuroepithelial tumors represent a unified category and if they are separate from ONB and currently-recognized sinonasal carcinomas. This study aims to explore their clinicopathologic characteristics based on a large collective experience. A total of 53 sinonasal tumors with neuroepithelial differentiation were identified affecting 41 men and 12 women, median age 47 years (range: 12 to 82 y). The vast majority arose in the superior nasal cavity and presented at the high Kadish-Morita stage. Frequent histologic findings included (1) lobulated and solid growth, (2) rosettes and/or neurofibrillary stroma, (3) high-grade cytology, (4) complex, often ciliated glands, (5) nonfocal pancytokeratin expression, (6) neuroendocrine pos+itivity, and (7) variable S100-positive sustentacular cells. Twelve patients with available follow-up (48%) developed progressive disease at a median 8 months (range: 0 to 114 mo to progression), and 7 (28%) died of disease. Despite disparate historical terminology, neuroepithelial differentiation is a recurrent and recognizable histologic pattern that is associated with aggressive behavior in sinonasal tumors. While tumors with this phenotype may originate from olfactory mucosa, well-developed epithelial features warrant separation from conventional ONB and neural elements distinguish them from most sinonasal carcinomas. Although their full histogenesis remains uncertain and some heterogeneity may exist, we propose that this pattern is sufficiently distinctive to merit separate recognition as olfactory carcinoma. Use of consistent nomenclature may facilitate greater recognition of tumors with this phenotype and understanding of their pathogenesis and classification.

All in for patient safety: a team approach to quality improvement in our laboratories.

Patient safety and quality improvement initiatives are integral parts of every cytopathology laboratory. The need to revisit our approaches to patient safety are essential in light of the expanding test menu, ancillary studies, comprehensive diagnostic reports, and emergence of new technologies for augmenting cytologic diagnosis. Our interview with Drs. Yael Heher, Adam Seegmiller, and Paul VanderLaan explores recent developments that have shaped their perspectives in patient safety, test usage, and laboratory quality. The practical strategies presented provide tools for enhanced patient safety and improved outcomes in a new era of ancillary and molecular testing and standardized reporting in the cytopathology laboratory.

Molecular testing results as a quality metric for evaluating cytopathologists' utilization of the atypia of undetermined significance category for thyroid nodule fine-needle aspirations.

INTRODUCTION: The use of the indeterminate category of atypia of undetermined significance (AUS) for thyroid fine-needle aspirations (FNAs) should be kept to a minimum. Here, we investigate the utility of combining AUS utilization rates with Afirma (Veracyte, Inc., South San Francisco, CA) genomic sequencing classifier (GSC) molecular testing results as a quality improvement metric for describing cytopathologist practice patterns. MATERIALS AND METHODS: Thyroid FNAs evaluated in our laboratory by 9 cytopathologists from December 2017 to July 2021 were stratified by Bethesda diagnostic category, and Afirma GSC testing results for AUS cases were compiled and correlated with AUS call rates. RESULTS: Over this period, the laboratory AUS rate was 22.3% (672 of 3008), with an individual cytopathologist range of 11.6% to 39.3%. Afirma GSC testing had suspicious (GSC-S) results in 29% (48 of 167) of cases, with a cytopathologist range of 5% to 67%. Linear regression analysis of individual AUS rates versus Afirma GSC-S rates demonstrated no significant relationship between these 2 variables. However, based on the pattern of AUS use and GSC-S rates, a novel conceptual framework for understanding cytopathologist practice patterns is proposed. CONCLUSIONS: Combining molecular testing results with AUS call rates of thyroid nodules can provide a more nuanced explanation of cytopathologist practice patterns, and can be utilized to provide directed feedback to bring individual cytopathologist diagnostic category use in line with laboratory averages or published benchmarks.

Cytomorphologic features of intraductal salivary gland carcinoma: A multi-institutional study of 13 FNA cases with histologic, molecular, and clinical correlations.

BACKGROUND: Intraductal carcinoma of the salivary gland (IDC) is a rare cancer with potential actionable targets, including RET fusions. Histologic and molecular features of IDC were recently reported, but cytomorphologic data are limited. In the largest multi-institutional fine-needle aspiration (FNA) series, the authors describe the cytomorphologic features of 13 IDC cases with available clinical, radiologic, histopathologic, and molecular data. METHODS: The cases included 13 FNAs for 9 low-grade (LG) IDCs and 4 high-grade (HG) IDCs with corresponding histopathology and available molecular, imaging, and clinical data. Smears and liquid-based preparations available for 12 FNAs were semiquantitatively scored for key cytomorphologic findings and correlated with the corresponding resection. RESULTS: LG IDC FNAs showed a cellular, biphasic population of large, atypical ductal cells with mildly pleomorphic nuclei in a clean background and a minor population of small, uniform myoepithelial cells. In contrast, all HG IDC FNAs showed predominantly ductal cells with marked nuclear pleomorphism, coarse chromatin, and necrosis. With the Milan system, most LG and HG IDC FNAs were classified as either salivary gland neoplasms of uncertain malignant potential (54%) or malignant (31%). Immunohistochemistry showed ductal epithelial reactivity with mammaglobin, androgen receptor, and S100, whereas myoepithelial cells were positive for p63 and/or calponin. Among cases with next-generation sequencing, 4 LG IDCs showed NCOA4-RET gene fusions, whereas an HG IDC showed HRAS and PIK3CA mutations. CONCLUSIONS: The cytomorphology of IDC overlaps with other benign and malignant salivary gland neoplasms. Immunohistochemistry limits the differential diagnosis, but definitive classification requires molecular analysis. A diagnosis of IDC has potential implications for patient management.

Combined molecular and histologic end points inform cancer risk estimates for thyroid nodules classified as atypia of undetermined significance.

BACKGROUND: Thyroid nodules classified as atypia of uncertain significance (AUS) on fine-needle aspiration cytology are heterogeneous. Prior studies reported a higher risk of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP)/cancer among AUS nodules that had cytologic (AUS-C) versus architectural (AUS-A) atypia; however, such studies were generally confined to resected cohorts, introducing bias into risk calculations. The authors hypothesized that combined histologic and molecular end points would permit clinically meaningful calculations of NIFTP/malignancy risk among AUS nodules. METHODS: The study consisted of 279 thyroid nodules classified as AUS on initial fine-needle aspiration and tested by the Afirma Gene Expression Classifier (GEC) between June 2013 and October 2017. Results of GEC testing and histopathologic diagnoses were stratified by AUS classifiers. The AUS-A category was further subclassified as 1) hypocellular microfollicular or 2) cellular with mixed but predominantly microfollicular architecture. NIFTP/cancer risk was calculated for each subgroup, with the inclusion of unresected nodules that had benign GEC results as low-risk end points comparable to histologically benign nodules. RESULTS: When only histologic end points were considered, there was no difference in NIFTP/cancer risk (25% vs 23%; P = .82). By using molecular and histologic end points, AUS cases with cytologic atypia trended toward higher NIFTP/cancer risk than AUS-A cases (14% vs 6%; P = .06). Furthermore, AUS-A cases showed a trend toward lower NIFTP/cancer risk for hypocellular microfollicular aspirates (3%) compared with cellular samples that had mixed/predominantly microfollicular architecture (13%; P = .18). CONCLUSIONS: The inclusion of unresected benign GEC nodules in risk-of-malignancy calculations provides more accurate results, which may be helpful for informing patient management as well as quality improvement in the cytopathology laboratory.

Can Cytologic and Sonographic Features Help Prevent Overtreatment of Bethesda V Thyroid Nodules?

BACKGROUND: Although nearly half of thyroid nodules with Bethesda V cytology (suspicious for malignancy) may be benign or harbor low-grade neoplasms that can be sufficiently treated with lobectomy, many patients with Bethesda V cytology continue to be treated with total thyroidectomy. The objectives of this study were to establish whether cytomorphologic and ultrasonographic features can determine appropriate surgery for thyroid nodules with Bethesda V cytology and how often patients are overtreated with total instead of partial thyroidectomy. METHODS: Utilizing a 10-y prospective database starting January 1, 2004, cytomorphologic and ultrasonographic features of thyroid nodules with Bethesda V cytology were reviewed. Overtreatment was defined as total thyroidectomy when histopathology revealed benign nodule, noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) or a unilateral < 4 cm low risk cancer. RESULTS: Sixty-three patients were included in the study. Seventeen (27%) had benign, 14 (22%) NIFTP, and 32 (51%) malignant nodules. On cytology, nuclear pseudoinclusions, and on ultrasound, taller-than-wide configuration, were more common in malignant than benign or NIFTP nodules. Among 56 patients who underwent total thyroidectomy, 14 and 11 had a benign or NIFTP nodule, respectively, and 13 had a unilateral < 4 cm low risk cancer, suggesting that 68% (38/56) were overtreated. CONCLUSIONS: Total thyroidectomy for Bethesda V thyroid nodules may result in overtreatment in more than half of the patients. Although certain cytomorphologic and ultarsonographic features may be helpful in determining appropriate surgery for Bethesda V thyroid nodules, additional characteristics are needed to reduce overtreatment of these nodules.

Repeat Fine Needle Aspiration Cytology Refines the Selection of Thyroid Nodules for Afirma Gene Expression Classifier Testing.

Background: Molecular testing (MT) refines risk stratification for thyroid nodules that are indeterminate for cancer by fine needle aspiration (FNA) cytology. Criteria for selecting nodules for MT vary and remain largely untested, raising questions about the best strategy for maximizing the usefulness of MT while minimizing the harms of overtesting. We used a unique data set to examine the effects of repeat FNA cytology-based criteria for MT on management decisions and nodule outcomes. Methods: This was a study of adults (age 25-90 years; 281 women and 72 men) with cytologically indeterminate (Bethesda III/IV) thyroid nodules who underwent repeat FNA biopsy and Afirma Gene Expression Classifier (GEC) testing (N = 363 nodules from 353 patients) between June 2013 and October 2017 at a single institution, with follow-up data collected until December 2019. Subgroup analysis was performed based on classification of repeat FNA cytology. Outcomes of GEC testing, clinical/sonographic surveillance of unresected nodules, and histopathologic diagnoses of thyroidectomies were compared between three testing approaches: (i) Reflex (MT sent on the basis of the initial Bethesda III/IV FNA), (ii) SemiRestrictive (MT sent if repeat FNA is Bethesda I-IV), and (iii) Restrictive (MT sent only if repeat FNA is Bethesda III/IV) testing approaches. Results: Restricting MT to nodules that remain Bethesda III/IV on repeat FNA would have missed 4 low-risk cancers and 3 noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) (collectively 2% of the test population) but would have avoided diagnostic surgery for 42 benign nodules (12% of the test population). The Restrictive testing strategy was more specific (delta 0.126 confidence interval [CI 0.093 to 0.159] and 0.129 [CI 0.097 to 0.161], respectively) but less sensitive (delta -0.339 [CI -0.424 to -0.253] and -0.340 [CI -0.425 to -0.255], respectively) than the Reflex and SemiRestrictive approaches for detecting NIFTP or cancer. Conclusions: Repeat FNA cytology can guide the selection of cytologically indeterminate thyroid nodules that warrant MT. The Restrictive model of performing Afirma GEC only on nodules with two separate biopsies showing Bethesda III/IV cytology would reduce the rate of diagnostic surgery for histologically benign nodules while missing only rare low-risk tumors. Given the low but nontrivial risks of thyroidectomy, the higher specificity of the Restrictive testing approach disproportionately outweighs the potential harms.

Fine needle aspiration of salivary gland carcinomas with high-grade transformation: A multi-institutional study of 22 cases and review of the literature.

BACKGROUND: High-grade transformation (HGT) is a rare process whereby conventional low- to intermediate-grade salivary gland carcinomas (SGC) transform into high-grade, poorly or undifferentiated malignancies with focal or complete loss of their conventional histomorphologic features. Because tumors with HGT are associated with a worse prognosis than their conventional counterparts, preoperative recognition of HGT may be of benefit for optimal patient management. Using a multi-institutional approach, we describe the largest fine needle aspiration (FNA) cohort of salivary gland carcinomas with HGT. METHODS: The archives of 9 large academic medical centers were searched, and 22 cases of SGC with HGT were identified by surgical excision accompanied by preoperative FNA. Clinical and cytomorphologic features were retrospectively reviewed. RESULTS: The male-to-female ratio was 14:8, and the mean patient age was 60.2 years. The average tumor size was 3.6 cm, and 19 cases were from the parotid gland. Acinic cell carcinoma with HGT was the most common tumor subtype, comprising 12 cases with HGT, followed by adenoid cystic carcinoma, secretory carcinoma, and other subtypes. Eighteen cases were classified as malignant; however, a specific diagnosis of HGT was not made. Sixteen cases contained a high-grade cytologic component, and 7 cases had a mixture of both conventional and high-grade components retrospectively. CONCLUSIONS: SGC with HGT should be considered in the differential diagnosis of a salivary gland aspirate exhibiting high-grade cytomorphologic features. The presence of distinct tumor populations, conventional and high-grade, should prompt consideration of HGT, especially when the conventional component is acinic cell carcinoma or adenoid cystic carcinoma.

Papillary thyroid carcinoma with prominent myofibroblastic stromal component: clinicopathologic, immunohistochemical and next-generation sequencing study of seven cases.

Papillary thyroid carcinoma with desmoid-type fibromatosis or nodular fasciitis-like stroma is an extremely unusual and poorly understood subtype of papillary thyroid cancer. Although prior studies have demonstrated alterations in the Wnt/ß-catenin signaling pathway in some of these tumors, controversy still exists regarding the nature of the stromal spindle component. We have studied seven cases of papillary thyroid carcinoma with prominent myofibroblastic stroma, including six men and one woman aged 20-65 years (mean age = 44). All cases displayed areas consistent with conventional papillary thyroid carcinoma embedded in abundant myofibroblastic-like stroma. The myofibroblastic stroma in six cases resembled desmoid-type fibromatosis and in one case it more closely resembled nodular fasciitis. By immunohistochemical staining, the stromal spindle component showed positivity for SMA and low MIB1 proliferation index in all cases, and there was at least patchy strong nuclear positivity for beta-catenin in six/seven cases. Stains for cytokeratin AE1/AE3 and PAX8 were positive in the epithelial elements but negative in the stromal component. Next-generation sequencing was performed on six of seven cases. CTNNB1 gene mutations were identified in six/seven cases. The epithelial component showed BRAF mutations in two cases and an NRAS mutation in one case. The case with fasciitis-like stroma was negative for beta-catenin by sequencing and immunostaining as well as negative for USP6 gene rearrangement. Our findings indicate that papillary thyroid carcinoma with prominent myofibroblastic stroma may represent more than one category of lesions.