Insulinoma-associated protein-1 (INSM-1) is a useful diagnostic marker for the evaluation of primary thymic neuroendocrine neoplasms: an immunohistochemical study of 27 cases.

Insulinoma-associated protein 1 (INSM1) immunohistochemistry has been established as a sensitive and reliable immunohistochemical marker for detecting neuroendocrine differentiation in tumors across various organ systems. However, this marker has not been adequately investigated in primary thymic neuroendocrine tumors. We have studied a series of 27 cases of primary neuroendocrine carcinomas of the thymus, including 3 typical carcinoids, 18 atypical carcinoids, 4 large cell neuroendocrine carcinomas, and 2 small cell carcinomas. Immunostaining on whole tissue sections for INSM-1 was evaluated. Results of immunostaining for chromogranin and synaptophysin were also evaluated. 26/27 tumors (96%) demonstrated nuclear positivity for INSM1. 18 tumors (67%) showed strong and diffuse nuclear staining (3 +), 3 tumors (11%) moderate (2 +) nuclear staining, and 5 tumors (19%) showed weak (1 +) nuclear staining. The average percentage of tumor cells positive for INSM1 was 76%. Only one tumor, a small cell carcinoma, was negative. All tumors were positive for synaptophysin, and 26/27 (96%) were positive for chromogranin A. This study confirms that INSM1 immunohistochemistry is a sensitive marker of neuroendocrine differentiation in primary thymic neuroendocrine neoplasms and demonstrates similar performance characteristics compared to other organ systems. The nuclear staining with this marker offers the advantage of eliminating some of the ambiguity in the interpretation sometimes encountered with other markers. An added advantage is the consistent staining across the entire spectrum of neuroendocrine tumors of this organ.

Comparative genomic and immunopathologic analysis of lung adenocarcinomas with and without cytology-proven malignant pleural effusions.

BACKGROUND: Lung cancer complicated by malignant pleural effusions (MPEs) is associated with significantly increased morbidity and mortality, yet the mechanisms of MPE development remain poorly understood. This study sought to elucidate whether there were specific genomic alterations and/or immunologic biomarkers associated with the presence of MPEs. METHODS: Analysis of comprehensive genomic and immunologic profiling for 275 locally advanced (stage III) or advanced (stage IV) lung adenocarcinomas was subcategorized into cytology-confirmed MPE-positive (MPE+; n = 139 stage IV) and MPE-negative (MPE-; n = 30 stage III + n = 106 stage IV) groups. RESULTS: Smoking frequency (p = .0001) and tumor mutational burden (p < .001) were demonstrated to be lower in the MPE+ group compared to the MPE- group. Median overall survival in the MPE+ group was shorter than in the MPE- group across all data (2.0 vs. 5.5 years; p < .0001) and for smokers (1.2 vs. 6.4 years; p < .0001). There were a number of differences at the genomic level across all cases and when stratifying by smoking status, including a higher frequency of EGFR mutations and a lower frequency of STK11 mutations in the MPE+ cohort. Finally, investigation of the comutational profiles of tumors by MPE status revealed differences in TP53- and STK11-mutant tumors between the two groups. CONCLUSIONS: Overall, these findings imply that there are both clinical and genetic factors associated with advanced lung adenocarcinoma MPEs. Future studies of these alterations may prove important both for understanding the pathophysiology of MPE development in advanced cancer and for the earlier detection of at-risk patients.

Imaging of Small Airways Disease.

Bronchiolitis refers to a small airways disease and may be classified by etiology and histologic features. In cellular bronchiolitis inflammatory cells involve the small airway wall and peribronchiolar alveoli and manifest on CT as centrilobular nodules of solid or ground glass attenuation. Constrictive bronchiolitis refers to luminal narrowing by concentric fibrosis. Direct CT signs of small airway disease include centrilobular nodules and branching tree-in-bud opacities. An indirect sign is mosaic attenuation that may be exaggerated on expiratory CT and represent air trapping. Imaging findings can be combined with clinical and pathologic data to facilitate a more accurate diagnosis.

Rapid examination of lung tissues by nonlinear microscopy.

OBJECTIVES: Traditional histopathology is a time-intensive and labor-intensive process involving tissue formalin fixation, paraffin embedding, and microtoming into thin sections for H&E staining. Frozen section analysis is a modality used during surgery to quickly evaluate tissue, but it has limitations, such as the size and number of the specimens that can be analyzed as well as difficulties with fatty and bony tissues. Our objective was to investigate the performance of nonlinear microscopy, a fluorescence microscopy technique, for the rapid examination of resected lung tumors. METHODS: In this proof-of-principle study, nonlinear microscopy imaging of resected lung tissue was performed on a total of 73 tissue specimens collected from 13 patients who underwent lobectomy, segmentectomy, or wedge resection for pulmonary nodules. RESULTS: Two pathologists reviewed the digital nonlinear microscopy images in comparison to the corresponding histopathologic H&E slides from a variety of pulmonary pathologies. CONCLUSIONS: This study demonstrated that nonlinear microscopy readily replicates traditional H&E staining for both lung tumors and nonneoplastic pulmonary structures. Nonlinear microscopy provides many advantages over frozen section analysis and is an optical imaging platform that has the potential to augment rapid pathologic evaluation of resected tissues in the age of digital pathology.

A Real-World Study of Patient Characteristics and Clinical Outcomes in EGFR Mutated Lung Cancer Treated with First-Line Osimertinib: Expanding the FLAURA Trial Results into Routine Clinical Practice.

Osimertinib is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) that is used for first-line therapy in EGFR mutated non-small cell lung cancer (NSCLC) based on the results of the randomized FLAURA trial (ClinicalTrials.gov number NCT02296125). We performed a retrospective analysis of baseline characteristics and clinical outcomes in 56 real-world patients treated with osimertinib. In total, 45% of patients were determined to be FLAURA-eligible and 55% were FLAURA-ineligible based on the published inclusion/exclusion criteria of the aforementioned trial. For clinical outcomes, the median osimertinib time to treatment discontinuation (TTD) for all patients was 16.9 months (95% CI: 12.6-35.1), whereas the median TTD was 31.1 months (95% CI: 14.9-not reached) in the FLAURA-eligible cohort and the median TTD was 12.2 months (95% CI: 8.1-34.6 months) in the FLAURA-ineligible cohort. Re-biopsy at acquired resistance disclosed both on- and off-target mechanisms. The most common therapies following osimertinib included local therapies followed by post-progression osimertinib, platinum-doublet chemotherapy with or without osimertinib, and osimertinib combinatory targeted therapies. The median overall survival for all patients was 32.0 months (95% CI: 15.7-not reached), the median survival was not reached for the FLAURA-eligible cohort, and it was 16.5 months for the FLAURA-ineligible cohort. Our data support the use of osimertinib in real-word settings and highlight the need for designing registration trials that are more inclusive of patient/disease characteristics seen in routine clinical practice. It is yet to be determined if the use of evolving first-line EGFR inhibitor combination strategies (either platinum-doublet chemotherapy plus osimertinib or amivantamab plus lazertinib) will similarly translate from clinical trials to real-word settings.

Gastrointestinal stromal tumors in fine-needle aspiration biopsies.

Gastrointestinal stromal tumors (GISTs), although rare, are the most common mesenchymal neoplasms of the gastrointestinal tract. Their potential for malignancy underscores the significance of identifying them through cytomorphologic findings and pertinent immunohistochemical markers. GISTs can emerge anywhere along the gastrointestinal tract with a predilection for the stomach. The clinical manifestations vary from nonspecific abdominal symptoms to incidental discovery during diagnostic interventions for unrelated signs and symptoms. Cytologically, GIST aspirates contain spindle or epithelioid cells with immunoreactivity for CD117/c-KIT, DOG-1, and CD34. Molecularly, KIT or PDGFRA mutations are prevalent, guiding targeted therapy with tyrosine kinase inhibitors. Distinct subtypes like succinate dehydrogenase-deficient GISTs pose challenges, often affecting younger individuals and displaying unique features. Histologically, GISTs are graded by mitotic rates, aiding prognostication. Distinguishing GISTs from similar entities is pivotal, necessitating attention to their immunostaining patterns for making an accurate diagnosis and molecular alterations for effectively planning treatment. Common differential diagnoses include leiomyoma, schwannoma, and solitary fibrous tumor. This article presents a classic GIST case and showcases relatively simple diagnostic clues for identifying similar lesions that may occur in diverse locations.

The Impact of On-Target Resistance Mediated by EGFR-T790M or EGFR-C797S on EGFR Exon 20 Insertion Mutation Active Tyrosine Kinase Inhibitors.

Introduction: Mechanisms of resistance to EGFR exon 20 insertion mutation active inhibitors have not been extensively studied in either robust preclinical models or patient-derived rebiopsy specimens. We sought to characterize on-target resistance mutations identified in EGFR exon 20 insertion-mutated lung cancers treated with mobocertinib or poziotinib and evaluate whether these mutations would or would not have cross-resistance to next-generation inhibitors zipalertinib, furmonertinib, and sunvozertinib. Methods: We identified mechanisms of resistance to EGFR exon 20 insertion mutation active inhibitors and then used preclinical models of EGFR exon 20 insertion mutations (A767_V769dupASV, D770_N771insSVD, V773_C774insH) plus common EGFR mutants to probe inhibitors in the absence/presence of EGFR-T790M or EGFR-C797S. Results: Mobocertinib had a favorable therapeutic window in relation to EGFR wild type for EGFR exon 20 insertion mutants, but the addition of EGFR-T790M or EGFR-C797S negated the observed window. Zipalertinib had a favorable therapeutic window for cells driven by EGFR-A767_V769dupASV or EGFR-D770_N771insSVD in the presence or absence of EGFR-T790M. Furmonertinib and sunvozertinib had the most favorable therapeutic windows in the presence or absence of EGFR-T790M in all cells tested. EGFR-C797S in cis to all EGFR mutations evaluated generated dependent cells that were resistant to the covalent EGFR tyrosine kinase inhibitors mobocertinib, zipalertinib, furmonertinib, sunvozertinib, poziotinib, and osimertinib. Conclusions: This report highlights that poziotinib and mobocertinib are susceptible to on-target resistance mediated by EGFR-T790M or -C797S in the background of the most prevalent EGFR exon 20 insertion mutations. Furmonertinib, sunvozertinib, and to a less extent zipalertinib can overcome EGFR-T790M compound mutants, whereas EGFR-C797S leads to covalent inhibitor cross-resistance-robust data that support the limitations of mobocertinib and should further spawn the development of next-generation covalent and reversible EGFR exon 20 insertion mutation active inhibitors with favorable therapeutic windows that are less vulnerable to on-target resistance.

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.

EGFR exon 20 insertion mutations and ERBB2 mutations in lung cancer: a narrative review on approved targeted therapies from oral kinase inhibitors to antibody-drug conjugates.

Background and Objective: This review will provide an overview of EGFR and ERBB2 mutations in non-small-cell lung cancer (NSCLC) with a focus on recent clinical approvals. Methods: We obtained data from the literature in accordance with narrative review reporting guidelines. Key Content and Findings: EGFR mutations are present in up to 15-20% of all NSCLCs; amongst these, 10% correspond to kinase domain insertions in exon 20. Structurally similar, ERBB2 (HER2) mutations occurs in 1-4% of NSCLCs, mostly consisting of insertions or point mutations. The majority of EGFR exon 20 insertions occur within the loop following the regulatory C-helix and activate the kinase domain of EGFR without generating a therapeutic window to gefitinib, erlotinib, afatinib, dacomitinib or osimertinib. Mobocertinib represents a novel class of covalent EGFR inhibitors with a modest therapeutic window to these mutants and induces anti-tumor responses in a portion of patients [at 160 mg/day: response rate of <30% with duration of response (DoR) >17 months and progression-free survival (PFS) of >7 months] albeit with mucocutaneous and gastrointestinal toxicities. The bi-specific EGFR-MET antibody amivantamab-vmjw has modest but broad preclinical activity in EGFR-driven cancers and specifically for EGFR exon 20 insertion-mutated NSCLC has response rates <40% and PFS of <8.5 months at the cost of both infusion-related plus on-target toxicities. Both drugs were approved in 2021. The clinical development of kinase inhibitors for ERBB2-mutated NSCLC has been thwarted by mucocutaneous/gastrointestinal toxicities that preclude a pathway for drug approval, as the case of poziotinib. However, the activation of ERBB2 has allowed for repurposing of antibody-drug conjugates (ADCs) that target ERBB2 with cytotoxic payloads. The FDA approved fam-trastuzumab deruxtecan-nxki in 2022 for NSCLC based on response rate of >55%, DoR >9 months, PFS >8 months and manageable adverse events (including cytopenias, nausea and less commonly pneumonitis). Other therapies in clinical development include sunvozertinib and zipalertinib, among others. In addition, traditional cytotoxic chemotherapy has some activity in these tumors. Conclusions: The approvals of mobocertinib, amivantamab, and trastuzumab deruxtecan represent the first examples of precision oncology for EGFR exon 20 insertion-mutated and ERBB2-mutated NSCLCs.

The 2023 Bethesda System for reporting thyroid cytopathology.

Since the publication of the first edition in 2010, The Bethesda System for Reporting Thyroid Cytopathology has allowed cytopathologists to use a standardized, category-based reporting system for thyroid fine needle aspirations. The third edition builds on the success of the 2 earlier editions and offers several key updates. The most important is the assignment of a single name for each of the 6 diagnostic categories: (i) nondiagnostic; (ii) benign; (iii) atypia of undetermined significance; (iv) follicular neoplasm; (v) suspicious for malignancy; and (vi) malignant. Each of the categories has an implied risk of malignancy (ROM), which has been updated and refined based on data reported after the second edition. The third edition offers an average ROM for each category, in addition to the expected range of cancer risk. The atypia of undetermined significance subcategorization is simplified into 2 subgroups based on the implied ROM and molecular profiling. A discussion of pediatric thyroid disease has been added, and pediatric ROMs and management algorithms are discussed in the relevant sections. Nomenclature has been updated to align with the 2022 World Health Organization Classification of Thyroid Neoplasms. Two new chapters have been added: one that addresses the significant and expanded use of molecular and ancillary testing in thyroid cytopathology, and another that summarizes clinical perspectives and imaging findings in thyroid disease.

The 2023 Bethesda System for Reporting Thyroid Cytopathology.

Since the publication of the first edition in 2010, The Bethesda System for Reporting Thyroid Cytopathology has allowed cytopathologists to use a standardized, category-based reporting system for thyroid fine needle aspirations. The third edition builds on the success of the 2 earlier editions and offers several key updates. The most important is the assignment of a single name for each of the 6 diagnostic categories: (i) nondiagnostic; (ii) benign; (iii) atypia of undetermined significance; (iv) follicular neoplasm; (v) suspicious for malignancy; and (vi) malignant. Each of the categories has an implied risk of malignancy (ROM), which has been updated and refined based on data reported after the second edition. The third edition offers an average ROM for each category, in addition to the expected range of cancer risk. The atypia of undetermined significance subcategorization is simplified into 2 subgroups based on the implied ROM and molecular profiling. A discussion of pediatric thyroid disease has been added, and pediatric ROMs and management algorithms are discussed in the relevant sections. Nomenclature has been updated to align with the 2022 World Health Organization Classification of Thyroid Neoplasms. Two new chapters have been added: one that addresses the significant and expanded use of molecular and ancillary testing in thyroid cytopathology, and another that summarizes clinical perspectives and imaging findings in thyroid disease.

A brief review of the WHO reporting system for lung cytopathology.

The International Academy of Cytology has joined with the International Agency for Research on Cancer to bring together a group of experts in lung cytopathology to develop a WHO Reporting System for Lung Cytopathology (WHO System). This System aims to improve and standardize the reporting of cytopathology, facilitate communication between cytopathologists and clinicians, and improve patient care. The WHO System describes 5 categories for reporting lung cytopathology: 'Insufficient/Inadequate/Nondiagnostic', 'Benign', 'Atypical', 'Suspicious for malignancy', and 'Malignant', each one with a clear descriptive term, a definition, a risk of malignancy, and a suggested management algorithm. The key diagnostic cytopathologic features of each of the lesions within each category have been established by consensus through an Expert Editorial Board, who are also the authors of this review and selected for each reporting system and chosen based on their expertise in the field and/or diversity of geographical representation. Many other co-authors from around the world also contributed. The assignment of writing and editing responsibilities used the same model as that used for the WHO Classification of Tumours (https://whobluebooks.iarc.fr/about/faq/). The WHO System provides the best practice application of ancillary testing, including immunocytochemistry and molecular pathology, and guides in sampling and processing techniques to optimize the handling and preparation of specimens. The WHO System was created by the authors to be applicable globally and is based on cytomorphology with possibilities for additional diagnostic management of the patient. The authors are aware that local medical and pathology resources would differ, especially in low- and middle-income countries. The WHO Tumour Classification for Thoracic Tumors, Fifth Edition, is directly accessible through the online WHO System.

EGFR exon 19 insertion EGFR-K745_E746insIPVAIK and others with rare XPVAIK amino-acid insertions: Preclinical and clinical characterization of the favorable therapeutic window to all classes of approved EGFR kinase inhibitors.

BACKGROUND: The epidermal growth factor receptor (EGFR)-K745_E746insIPVAIK and others with XPVAIK amino-acid insertions are exon 19 insertion mutations, which, at the structural modeling level, resemble EGFR tyrosine kinase inhibitor (TKI)-sensitizing mutants. An important unmet need is the characterization of therapeutic windows plus clinical outcomes of exon 19 XPVAIK amino-acid insertion mutations to available EGFR TKIs. METHODS: We used preclinical models of EGFR-K745_E746insIPVAIK and more typical EGFR mutations (exon 19 deletion, L858R, L861Q, G719S, A763_Y764insFQEA, other exon 20 insertion mutations) to probe representative 1st (erlotinib), 2nd (afatinib), 3rd generation (osimertinib), and EGFR exon 20 insertion active (mobocertinib) TKIs. We also compiled outcomes of EGFR exon 19 insertion mutated lung cancers-from our institution plus the literature-treated with EGFR TKIs. RESULTS: Exon 19 insertions represented 0.3-0.8% of all EGFR kinase domain mutation in two cohorts (n = 1772). Cells driven by EGFR-K745_E746insIPVAIK had sensitivity to all classes of approved EGFR TKIs when compared to cells driven by EGFR-WT in proliferation assays and at the protein level. However, the therapeutic window of EGFR-K745_E746insIPVAIK driven cells was most akin to those of cells driven by EGFR-L861Q and EGFR-A763_Y764insFQEA than the more sensitive patterns seen with cells driven by an EGFR exon 19 deletion or EGFR-L858R. The majority (69.2%, n = 26) of patients with lung cancers harboring EGFR-K745_E746insIPVAIK and other mutations with rare XPVAIK amino-acid insertions responded to clinically available EGFR TKIs (including icotinib, gefitinib, erlotinib, afatinib and osimertinib), with heterogeneous periods of progression-free survival. Mechanisms of acquired EGFR TKI resistance of this mutant remained underreported. CONCLUSIONS: This is the largest preclinical/clinical report to highlight that EGFR-K745_E746insIPVAIK and other mutations with exon 19 XPVAIK amino-acid insertions are rare but sensitive to clinically available 1st, 2nd, and 3rd generation as well as EGFR exon 20 active TKIs; in a pattern that mostly resembles the outcomes of models with EGFR-L861Q and EGFR-A763_Y764insFQEA mutations. These data may help with the off-label selection of EGFR TKIs and clinical expectations of outcomes when targeted therapy is deployed for these EGFR mutated lung cancers.

Multi-institutional 10-year retrospective review of amoeba diagnosed on cytologic evaluation of anal pap tests: what is the significance?

INTRODUCTION: Intestinal amoebae are usually transmitted via ingestion of amoebic cysts in fecally contaminated water or food. However, other modes of transmission include sexual contact through anal-oral sex. While the primary role of anal cytology is the detection of anal cancer and precursor lesions, organisms can also be identified. Despite this, assessment of the clinical significance of cytologic identification of amoebae is lacking in the literature. MATERIALS AND METHODS: A 10-year retrospective review of the pathology archives of 2 institutions in Boston, Massachusetts was conducted. Anal Pap tests that identified amoeba were reviewed and correlated with the medical records for investigation into clinical parameters and patient management. RESULTS: A total of 46 cases were identified between the 2 sites. The majority of patients were male (95.7%) and endorsed having sex with men (84.8%). Only a minority endorsed recent travel (6.5%). Most of the patients were also HIV (human immunodeficiency virus)-positive (71.1%) with all of these patients being well-controlled on antiretroviral therapy. Most patients were asymptomatic (87.0%). On review of the anal Pap tests, the average organism number per case was 35.4. In the majority of cases, follow-up microbiology testing for confirmation and/or speciation was not performed (89.1%) and were not treated (93.5%). CONCLUSIONS: While identification of amoeba is possible on anal cytology, the clinical significance remains unclear as most patients were asymptomatic and not treated in this series. Ultimately, the clinical setting likely plays an important role in determination of management.

ALK Deletion Exons 2 to 19: Case Report of a Rare ALK Inhibitor-Responsive Lung Cancer Driver Oncogene.

ALK internal deletions of nonkinase domain exons occur in 0.01% of lung cancers with ALK genomic aberrations. We report a lung adenocarcinoma with a previously undescribed somatic ALK deletion of exons 2 to 19 with dramatic and sustained (>23 mo) response to alectinib. Our and other reported cases with ALK nonkinase domain deletions (between introns and exons 1-19) can display positive results in nonsequencing-based lung cancer diagnostic tests (such as immunohistochemistry) used to screen for more common ALK rearrangements. This case report emphasizes that "ALK-driven" lung cancers should be expanded to encompass those harboring not only ALK rearrangements with other genes but also ALK nonkinase domain deletions.

Massive Thymic Hyperplasia Masquerading as Cancer: A Case Report and Review of the Literature.

A 34-year-old woman presenting with abdominal pain, chest pressure, weight loss, and tachycardia was found to have an 11.4-cm anterior mediastinal mass associated with intrathoracic lymphadenopathy on chest computed tomography (Fig. 1A). Core needle biopsy was concerning for a type B1 thymoma. During this patient's initial workup, she was found to have both clinical and laboratory evidence of Graves' thyroiditis, raising diagnostic suspicion for thymic hyperplasia rather than thymoma. The case discussed here highlights the unique challenges that arise in the evaluation and management of thymic masses and serves as a prudent reminder that both benign and malignant disorders may present with mass-like changes.

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.