A retrospective analysis of molecular testing in cytologically indeterminate thyroid nodules with histologic correlation: Experience at a heterogenous multihospital system.

INTRODUCTION: Thyroid malignancy is one of the most common types of cancer in developed nations. Currently, fine-needle aspiration cytology (FNAC) is the most practical screening test for thyroid nodules. However, cytologically indeterminate samples comprise approximately 15%-30% of cases. These include cases classified as atypia of undetermined significance (AUS), follicular neoplasm (FN), and suspicious for malignancy (SFM). Indeterminate cases can be sent for molecular testing for more definitive classification to help guide management and prevent overtreatment of benign thyroid nodules. We conducted a retrospective review on molecular testing of indeterminate thyroid FNAC and reviewed subsequent histologic diagnoses in resection specimens to assess how molecular testing supported a diagnosis and its effect on clinical management of patients at our institution. METHODS: A retrospective chart review was performed on all thyroid FNAC specimens, corresponding molecular testing, and subsequent surgical resection specimens over a 6-year period. RESULTS: A total of 10,253 thyroid FNAC were performed in our hospital system during our study period, of which 10% (n = 1102/10,253) had indeterminate FNAC results. Molecular testing was performed in 16% (n = 178/1102) of indeterminate cytology cases. Genetic alterations were identified in 39% (n = 69/178) of the cases sent for molecular testing. The majority of cytologically indeterminate cases sent for molecular testing were follicular-patterned lesions and their corresponding resection specimens revealed mostly low grade follicular derived neoplasms (i.e., follicular adenoma, non-invasive follicular thyroid neoplasm with papillary-like nuclear features, and follicular variant of papillary thyroid carcinoma). Of the cases with identified genetic alterations, 75% (n = 52/69) were treated surgically. In cases with no genetic alterations identified, only 18% (n = 20/109) were treated surgically. DISCUSSION/CONCLUSION: Molecular testing on cytologically indeterminate thyroid nodules can help provide a more accurate risk of malignancy assessment in patients with lesions that are difficult to diagnosis based solely on FNAC morphology. The types of genetic alterations identified in the resected thyroid lesions were consistent with what has been previously described in the literature. Additionally, we found that in the patients with indeterminate thyroid FNAC with adjunct molecular testing, more than half did not undergo surgical resection. This finding emphasizes the value of adding molecular testing in patients, particularly when attempting to reduce unnecessary surgical intervention.

Pre-analytical effects on whole transcriptome and targeted RNA sequencing analysis in cytology: The effects of prolonged time in storage of effusion specimens prior to preservation.

OBJECTIVES: To investigate the pre-analytics of the molecular testing of cytology specimens, we studied the effects of time in refrigerator storage (4°C) of malignant effusions on RNA sequencing (RNAseq) results. METHODS: Ten effusion specimens were stored in a refrigerator (4°C) for different durations (day 0, 1, 4, and 7). All specimens were prepared as cytospins fixed in either Carnoy's solution or 95% ethanol (EtOH) and in an RNA preservative for a fresh frozen (FF) high-quality reference. Whole transcriptome (wt) and targeted (t)RNAseq of two multigene expression signatures were performed. We then compared transcript expression levels (including mutant allele fraction) according to pre-analytical variables using a concordance correlation coefficient (CCC) and a mixed effect model. RESULTS: Sequencing results were mostly stable over increasing time in storage. Cytospins fixed in Carnoy's solution were more concordant with FF samples than cytospins fixed in 95% EtOH at all timepoints. This finding was consistent for both wtRNAseq (averages: day 0 CCC = 0.98 vs 0.91; day 7 CCC = 0.88 vs 0.78) and tRNAseq methods (averages: day 0 CCC = 0.98 vs 0.81; day 7 CCC = 0.98 vs 0.90). Cytospins fixed in Carnoy's solution did not show significant changes in expression over timepoints or between expression signatures, whereas 95% EtOH did. CONCLUSION: RNAseq can be accurately performed on effusion specimens after prolonged refrigerator storage. RNA extracted from scraped cytospin slides fixed in Carnoy's solution was marginally superior to 95% EtOH fixation, but either method had comparable analytic performance to high-quality FF RNA samples.

Molecular testing opportunities on cytology effusion specimens: the pre-analytic effects of various body fluid cytology preparation methods on RNA extraction quality and targeted sequencing.

INTRODUCTION: RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections. MATERIALS AND METHODS: We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC). RESULTS: The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods. CONCLUSIONS: Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.

Assessing the quality of cytopathology whole slide imaging for education from archived cases.

INTRODUCTION: Many institutions have cytopathology case archives for education. Unfortunately, these slides deteriorate over time and have limited accessibility. Whole slide imaging (WSI) can overcome these limitations. However, suboptimal image quality and scanning effort are barriers. MATERIALS AND METHODS: We selected 123 slides from cytopathology study sets for WSI scanning at 400x magnification without z-stacking. The Ventana DP 200 scanner and Virtuoso software were used. Slides were scanned in 2 rounds: the first round of slides was prepared for scanning with light cleaning, and the second round was performed only on slides that had unacceptable WSI quality after thorough cleaning. Slides were assessed with a 4-tier grading system created by the authors. Time to scan each slide was recorded. RESULTS: Within the first round, 96 of 123 (78%) slides scanned were determined to be of acceptable quality. After the second round of scanning, in total, 118 of 123 (95.9%) slides were determined to be of acceptable quality. The average time needed to scan each slide was 213 seconds. CONCLUSIONS: The majority of slides scanned were of acceptable quality in the first round of scanning. After cleaning and rescanning, nearly every slide investigated was of acceptable quality. The primary objective is to provide other institutions that may be considering a similar project a benchmark so that they know what to expect in terms of slide scan success rate and the amount of time needed to digitize slides for educational archiving. This pilot study demonstrates the feasibility of using WSI for cytology education cases.

The gray zone of thyroid nodules: Using a nomogram to provide malignancy risk assessment and guide patient management.

BACKGROUND: Thyroid nodules have a low prevalence of malignancy and most proven cancers do not behave aggressively. Thus, risk-stratification of nodules is a critical step to avoid surgical overtreatment. We hypothesized that a risk management system superior to those currently in use could be created to reduce the number of clinically indeterminate nodules (i.e., the "gray zone") by concurrently considering the malignancy risks conferred by clinical, ultrasonographic, and cytologic variables. METHODS: Thyroidectomy cases were reviewed from three institutions. Their benign versus malignant outcome was used to evaluate the variables for correlation. A binary logistic regression model was trained and, using indeterminate nodules with Bethesda III and IV results, validated. A scoring nomogram was designed to demonstrate the application of the model in clinical practice. RESULTS: One hundred thirty thyroidectomies (28% malignant) met inclusion criteria. The final logistic regression model included difficulty in swallowing, hypothyroidism, echogenicity, hypervascularity, margins, calcification, and cytology diagnosis as input parameters. The model was highly successful in determining the outcome (p value: 0.001) with a R2 (Nagelkerke) score of 0.93. The area under the curve as determined by receiver operating characteristics was 0.91. The accuracy of the model on the training dataset was 93% (sensitivity and specificity 92% and 96%, respectively) and, on the validation dataset, 80% (sensitivity and specificity 91% and 67%, respectively). CONCLUSIONS: We report a model for risk assessment of thyroid nodules that has the potential to significantly reduce indeterminates and surgical overtreatment. We illustrate its application via a straightforward nomogram, which integrates clinical, ultrasonographic, and cytologic data, and can be used to create clear, evidence-based management plans for patients.