Incidental Diagnosis of Parathyroid Lesions by Preoperative Use of Next-Generation Molecular Testing.

BACKGROUND: Parathyroid glands can mimic thyroid follicular lesions on fine-needle aspiration (FNA) cytology and can lead to unnecessary or incorrect surgery. Newer molecular panel tests using next-generation sequencing (NGS) include analysis of cell type-specific gene expression profiles such as parathyroid. The study aim is to determine the frequency and clinical implications of parathyroid tissue identification by molecular testing in cytologically indeterminate "thyroid" lesions. METHODS: Molecular analysis of indeterminate thyroid FNA specimens is obtained routinely and relies on amplification-based NGS inclusive of PTH-specific expression profiles. For this study, we retrospectively examined the clinical data and management of patients with molecular results positive for PTH expression from May 2014 until May 2016. RESULTS: Among 4765 consecutive patients with indeterminate cytology for a presumed thyroid nodule, NGS instead indicated a parathyroid lesion in 20 patients (0.42%). The clinical data of 15 patients were available, and the subsequent clinical management was altered in 93% (14/15 patients), including five (33%) eucalcemic patients who could avoid unnecessary surgery. Primary hyperparathyroidism was not suspected in seven patients until the molecular analysis results, and primary hyperparathyroidism was diagnosed in one (14%). During parathyroid exploration, most patients (6/8, 75%) required concurrent thyroidectomy or lobectomy, but thyroid preservation was still possible in two patients. A parathyroid gland was histologically confirmed in 89%. CONCLUSIONS: In 0.42% of patients with indeterminate cytology results, next-generation molecular results will indicate the presence of a parathyroid lesion. When this occurs, it is accurate and can robustly impact clinical management (93%).

Molecular-derived risk of malignancy and the related positive call rate of indeterminate thyroid cytology diagnoses as quality metrics for individual cytopathologists.

BACKGROUND: Indeterminate thyroid cytopathology diagnoses represent differing degrees of risk that are corroborated by follow-up studies. However, traditional cytologic-histologic correlation may overestimate the risk of malignancy (ROM) because only a subset of cases undergo resection. Alternatively, some molecular tests provide probability of malignancy data to calculate the molecular-derived risk of malignancy (MDROM) and the positive call rate (PCR). The authors investigated MDROMs and PCRs of indeterminate diagnoses for individual cytopathologists as quality metrics. METHODS: This study was approved by the Department of Pathology Quality Improvement Program. Thyroid cytopathology diagnoses and ThyroSeq v3 results were retrieved for each cytopathologist for a 2-year period with at least 3 years of follow-up for the atypia of undetermined significance (AUS), follicular neoplasia (FN), and follicular neoplasia, oncocytic-type (ONC) cytopathologic diagnoses. MDROMs and PCRs were compared with reference ROMs and cytologic-histologic correlation outcomes. RESULTS: The overall MDROMs (and ranges for cytopathologists) for the AUS, FN, and ONC categories were 13.4% (range, 5.8%-20.8%), 28.1% (range, 22.1%-36.7%), and 27.0% (range, 19.5%-41.5%), respectively, and most individual cytopathologists' MDROMs were within reference ROM ranges. However, PCRs more effectively parsed the differences in cytopathologists' ROM performance. Although the overall PCRs were not significantly different across cytopathologists (p = .06), the AUS PCRs were quite different (p = .002). By cytologic-histologic correlation, six of 55 resected cases (10.9%) were falsely negative, and there were no false-positive cases. CONCLUSIONS: MDROMs and PCRs evaluate concordance with reference ROMs and with one another and provide individual feedback, which potentially facilitates quality improvement.

Integration of KRAS testing in the diagnosis of pancreatic cystic lesions: a clinical experience of 618 pancreatic cysts.

With improvements in abdominal imaging, detection of incidental pancreatic cysts are becoming increasingly common. Analysis of pancreatic cyst fluid from fine-needle aspiration is particularly important in identifying intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), which have significant implications in clinical intervention and follow-up. Previous controlled studies have shown that KRAS mutations in cyst fluid are highly specific for mucinous differentiation in pancreatic cysts; however, this has not been examined in the clinical setting. Over a 6-year study period, 618 pancreatic cyst fluids obtained by fine-needle aspiration at the time of endoscopic ultrasound were tested for KRAS mutations as part of routine evaluation for a cystic neoplasm. Of the 618 specimens, 603 (98%) from 546 patients were satisfactory for molecular analysis. Patients ranged in age from 17 to 90 years (mean, 63.9 years) and were predominantly female (68%). Pancreatic cysts were relatively evenly distributed throughout the pancreas and ranged in size from 0.6 to 11.0 cm (mean, 2.3 cm). Mutations in KRAS were detected in 232 of 603 (38%) aspirates. Although sufficient for molecular analysis, 320 of 603 (53%) specimens were either less than optimal (38%) or unsatisfactory (15%) for cytopathologic diagnosis. Surgical follow-up information was available for 142 (26%) patients and consisted of 53 KRAS-mutated and 89 KRAS-wild-type cysts. Overall, KRAS mutations had a specificity of 100%, but a sensitivity of 54% for mucinous differentiation. When stratified by cyst type, KRAS had a sensitivity of 67% and 14% for IPMNs and MCNs, respectively. In summary, KRAS mutations were highly specific for mucinous differentiation, but were inadequate in identifying MCNs. Future molecular studies and the combination of other fluid markers are required to improve the detection and classification of pancreatic mucinous neoplasms by endoscopic ultrasound fine-needle aspiration.

A thyroid EIF1AX story: how clinical, cytologic, and molecular surveillance led to appropriate management.

INTRODUCTION: Indeterminate thyroid cytology diagnoses are associated with intermediate risks of malignancy. Application of molecular testing (MT) to indeterminate specimens provides additional diagnostic and prognostic information. While a positive or suspicious MT result may prompt surgery, a negative MT result is associated with a low probability of cancer or noninvasive follicular thyroid neoplasm with papillary-like nuclear features and approximates that of a benign cytology diagnosis. Furthermore, ThyroSeq v3 MT has a "currently negative" result for findings with the probability of cancer or noninvasive follicular thyroid neoplasm with papillary-like nuclear feature that is slightly greater than that for the negative ThyroSeq v3 MT result but less than 10%, suggesting active surveillance. In this report, we discuss a case of a patient for whom clinical, cytologic, and molecular surveillance led to timely surgery and management. CLINICAL DETAILS: A 53-year-old man with a thyroid isthmus nodule had a fine-needle aspiration cytology diagnosis of atypia of undetermined significance and a subsequent ThyroSeq v3 MT, which revealed an EIF1AX mutation and a "currently negative" MT result. Surveillance with additional fine-needle aspiration samples demonstrated concerning genomic alterations (fluctuating EIF1AX allelic frequency and a non-V600E BRAF mutation), culminating in the conversion to a positive MT result 3 years later. Resection revealed an encapsulated noninvasive, oncocytic solid subtype of papillary thyroid carcinoma with increased mitotic activity. CONCLUSION: The case is notable for clinical, cytologic, and molecular surveillance demonstrating sequential pathologic alterations in an indeterminate thyroid nodule with EIF1AX mutation, leading to timely resection of the neoplasm before invasion manifested.

Postoperative infection and bone sarcoma survival: systematic review of the role of infection in bone sarcoma prognosis.

Background: Osteosarcoma (OS) and chondrosarcoma (CS) are primary bone malignancies whose prognoses have stagnated despite advancements in surgical management, chemotherapy, radiation therapy, and immunotherapy. The role of the immune system in generating anti-cancer physiologic responses is critical to prognosis. Prior studies have explored if immune system activation via infection enhances survival in bone sarcomas without a clear consensus. Methods: This study sought to (I) retrospectively examine the effect of postoperative infection on survival in OS and CS and (II) systematically review the effect of postoperative infection on survival in primary bone malignancies. We performed a retrospective case-control study of 192 patients treated between 1/2000-12/2015 at a single academic sarcoma referral center. Patients with OS or CS undergoing operative resection were included. Eligible patients were grouped by presence of metastasis, and survival was compared between patients with or without postoperative infection. Furthermore, we performed a systematic review following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines investigating the effect of infection on primary bone malignancy survival. Risk of bias assessment was performed utilizing the ROBINS-I (Risk of Bias in Non-randomized Studies-of Interventions) assessment tool. All presented studies included author information, study population, and overall or disease-free survival results. Results: One hundred and four patients were included, with 85 without infection (26 metastatic, 59 non-metastatic) and 19 with infection (10 metastatic, 9 non-metastatic). Five-year survival was greatest in patients without metastasis with a postoperative infection (100%), followed by patients without metastasis who were infection-free (80%). Five-year survival was comparatively lower in patients with metastasis who were infection-free (35%) and lowest in patients with metastasis with a postoperative infection (20%). No significant differences were present (P=0.17) on log-rank analysis. Our systematic review collected six studies exploring the impact of infection on primary bone malignancy survival, with two studies reporting significant findings of infection improving survival. Limitations of this review included risk of bias due to confounding, inconsistency comparing outcomes, and differences in patient populations. Conclusions: This retrospective study and systematic review suggests postoperative infection may play a role in modulating immune response to malignancy. Understanding the synergy between anti-pathogen and anti-cancer responses warrants further investigation as an alternative method of targeted cancer treatment.

Accuracy of definitive rapid onsite evaluation cytopathology diagnoses: Assessment of potentially critical diagnoses as a quality assurance measure.

INTRODUCTION: Intraprocedural rapid onsite evaluation (ROSE) of cytology specimens enhances cytopathology practice. More recently, ROSE diagnoses, like frozen section (FS) diagnoses, have guided immediate clinical decisions. In this study, we evaluated the diagnostic accuracy of definitive ROSE diagnoses in our quality assurance system over a 52-month period. MATERIALS AND METHODS: Cytopathology cases with ROSE from January 2017 to April 2021were retrieved from our laboratory information system. After excluding cases that were deferred or nondiagnostic/unsatisfactory, each definitive ROSE diagnosis (ie, negative for malignant cells or positive for malignant cells) was categorized as having agreement or disagreement with the final diagnosis. For comparison, concordance of FS diagnoses from the same time period were tabulated and compared to those of ROSE diagnoses by using χ2 testing with P < 0.05 considered statistically significant. RESULTS: Of the 1649 ROSE diagnoses, there were 15 disagreements (0.9%) with 1 final moderate interpretive disagreement (0.06%). By comparison, of the 17,469 FS diagnoses, there were 141 disagreements (0.8%) with 49 final moderate or major interpretive disagreements (0.3%). The remaining disagreements were minor. There were no statistically significant differences in the rates of final moderate and major interpretive disagreements. CONCLUSIONS: The final interpretive disagreement rates for definitive ROSE and FS diagnoses were similar in this study. Given the expanding role of ROSE and its use for immediate clinical decisions in some cases, monitoring the accuracy of definitive diagnoses may serve as an initial quality assurance measure.

Variability in the atypia of undetermined significance/follicular lesion of undetermined significance diagnosis in the Bethesda System for Reporting Thyroid Cytopathology: sources and recommendations.

OBJECTIVE: Of the 6 categories in the Bethesda System for Reporting Thyroid Cytopathology (BSRTC), the atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) category has received the most attention. The objectives of this study were to review the use of the AUS/FLUS category in recent studies, to search for likely sources of the variability in its use, and to address possible methods for improvement. STUDY DESIGN: A PubMed search was performed to retrieve peer-reviewed articles that have comprehensively detailed the incidence and outcome of AUS/FLUS and other BSRTC categories. Related thyroid cytology articles on the BSRTC were also included. RESULTS: Recent series that reported experiences with the BSRTC categories showed that the AUS/FLUS category exhibited a marked variability in incidence (0.7-18%) and malignant outcome (6-48%) in resection specimens. Review of the literature revealed institutional differences in technical aspects, interpretation and application of criteria, analysis of outcome data, and clinicopathologic interactions. CONCLUSIONS: A heightened awareness of technical issues, diagnostic borders of AUS/FLUS, and clinical management may aid in diagnostic refinement and help avoid overuse of this category.

DNA mutational differences in cytological specimens from pancreatic cancer and cholangiocarcinoma.

BACKGROUND/AIMS: Preoperative distinction between pancreatic cancer (PC) and extrahepatic cholangiocarcinoma (CC) is desirable due to diverging management options, and to optimize enrollment into neoadjuvant trials. METHODS: A single-center retrospective study of patients with PC or CC was undertaken. Four blinded pathologists reviewed all cases and reached a consensus diagnosis (PC or CC). Microdissection-based multiple microsatellite loss analysis and direct sequencing of K-ras oncogene was performed and compared for PC and CC. RESULTS: Of 33 cases studied (17 males; 16 PC, 17 CC; 10 with primary sclerosing cholangitis), a K-ras mutation was present in 14/16 (87.5%) PC and 1/17 (5.9%) CC cases (p < 0.001), sensitivity and specificity were 87.5 and 94%, respectively. The mean fractional mutational rate was higher in PC (0.51; 95% CI 0.45-0.58) compared to CC (0.34; 95% CI 0.28-0.39, p < 0.001). CONCLUSIONS: The presence of a K-ras mutation in cytology specimens distinguishes PC from CC in this study. and IAP.

Molecular-derived estimation of risk of malignancy for indeterminate thyroid cytology diagnoses.

INTRODUCTION: One of the key features of the Bethesda System for Reporting Thyroid Cytopathology is the risk of malignancy (ROM), which guides management for each diagnostic category. However, calculation of the ROM can be challenging for indeterminate diagnoses because only a portion of cases will be resected for cytologic-histologic correlation (CHC) analyses. In the present study, we used the probability of cancer information from ThyroSeq, version 3, reports to calculate the molecular-derived (MD) ROM for indeterminate categories. MATERIALS AND METHODS: Cytology cases with indeterminate BSRTC diagnoses and adequate molecular test results were retrieved from our cytopathology laboratory for a 12-month period. The probability of cancer information from the ThyroSeq, version 3, molecular reports were tabulated, and the mean ROM was calculated for each diagnostic category. The MDROM included noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) as a "malignant" outcome because it is considered a surgical disease. RESULTS: A total of 361 cases had adequate material for molecular testing. The diagnostic distribution was as follows: atypia of undetermined significance/follicular lesion of undetermined significance, 271 cases (75.1%), follicular neoplasm/suspicious for a follicular neoplasm, 59 cases (16.3%), and Hürthle cell type/suspicious for a follicular neoplasm, Hürthle cell type, 31 cases (8.6%). The corresponding estimated MDROMs were 14.9%, 32.6%, and 34.4%. A comparison with the CHC data was performed, and the 95% confidence intervals of the MDROMs overlapped well with the 2 endpoint CHC values. CONCLUSIONS: Calculation of the MDROMs provides a new method to approximate the ROMs of indeterminate diagnoses and has the advantage of potentially evaluating all cases, not just those resected. Furthermore, for those using the same platform, interinstitutional comparisons will be possible.

Benign call rate and molecular test result distribution of ThyroSeq v3.

BACKGROUND: The benign call rate (BCR) is the percentage of cytomorphologically indeterminate cases with subsequent benign or negative molecular results. For rule-out tests, the BCR is an important parameter because these molecular "negative" cases may be managed similarly to those with a benign cytology diagnosis. Although earlier versions of ThyroSeq molecular tests were less effective in excluding malignancy, the extensively expanded v3 version with a high negative predictive value is considered to represent a rule-out test. In the current study, the authors evaluated the BCR and the overall molecular result distribution of ThyroSeq v3. METHODS: All indeterminate thyroid fine-needle aspiration cases (except those deemed suspicious for malignancy) with available ThyroSeq v3 results from November 2017 to June 2018 were retrieved from the cytopathology files. Because the ThyroSeq v3 genomic classifier was designed for thyroid follicular-derived lesions, cases in which parathyroid and medullary carcinoma molecular markers were detected and those that were inadequate or limited for molecular testing were excluded from the study. For the remaining cases, the indeterminate diagnoses were correlated with molecular and histologic results. RESULTS: Among the 224 indeterminate cases (except those deemed suspicious for malignancy), the overall ThyroSeq v3 molecular test BCR was 74.1% (166 of 224 cases). The category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) had higher BCRs compared with the other indeterminate diagnostic categories. RAS mutations were the most common positive results. CONCLUSIONS: The high BCR demonstrates that ThyroSeq v3 is potentially effective in sparing the large majority of indeterminate cases with "negative" results from surgery while offering risk assessment for the positive cases and guiding clinical management.

The influence of the noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) resection diagnosis on the false-positive thyroid cytology rate relates to quality assurance thresholds and the application of NIFTP criteria.

BACKGROUND: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is a newly defined entity and recent studies have suggested a decrease of a few percentage points in the rate of malignancy (ROM) for the positive-for-malignancy (PFM) cytology category as a result of NIFTP implementation. However, the distinction between a diagnosis of PFM and one of suspicious for malignancy (SFM) may depend on a variety of factors. In the current study, the authors investigated the ROM for the PFM and SFM diagnoses before and after histologic NIFTP reclassification. METHODS: Cytology cases with PFM and SFM diagnoses and subsequent surgical resection specimens were searched in the files of the study institution from September 2008 to September 2016. The surgical pathology cases of noninvasive encapsulated follicular variant of papillary thyroid carcinoma were reexamined to determine whether they qualified for NIFTP. The distinct ROMs for the PFM and SFM cases were calculated accordingly. RESULTS: The authors' search identified 338 cases of PFM and 139 cases of SFM with a resection outcome. Before NIFTP reclassification, the PFM cases had a ROM of 99.4%; after NIFTP reclassification, the ROM was 99.1% (P = .6861). The ROM of the SFM cases decreased from 75.5% to 66.9% with NIFTP reclassification (P = .1402). One case in the PFM group and 6 cases in the SFM group could not be verified due to insufficient sampling. CONCLUSIONS: In the current large series, NIFTP reclassification did not appear to significantly alter the high ROM for the PFM diagnosis. The authors attribute this finding to a strict quality assurance policy, an emphasis on key cytologic criteria, and systematic application of the NIFTP criteria to follicular-patterned lesions. Cancer Cytopathol 2017;125:692-700. © 2017 American Cancer Society.

K-Ras and microsatellite marker analysis of fine-needle aspirates from intraductal papillary mucinous neoplasms of the pancreas.

Preoperative diagnosis of pancreatic cystic lesions is difficult despite the combination of cytomorphology, radiographic imaging characteristics, and fluid tumor markers such as carcinoembryonic antigen. Intraductal papillary mucinous neoplasms (IPMNs) represent a subset of preinvasive pancreatic cystic neoplasms and are associated with accumulated genetic mutations, especially K-ras and tumor suppressor genes such as p53. Application of molecular techniques to cyst fluid obtained by endoscopic ultrasound guided fine-needle aspiration (EUSFNA) may contribute to preoperative assessment. Sixteen patients with pancreatic cystic lesions had cyst fluid obtained by preoperative pancreatic EUSFNA or intraoperative aspiration. All patients subsequently underwent surgical resection of the pancreas and IPMN was documented in all (6 adenomas, 6 borderline tumors, and 4 carcinomas). DNA was extracted from the cyst fluids and mutational analysis for K-ras point mutations and loss of heterozygosity (LOH) analysis using a preselected panel of genomic loci were performed. LOH was observed in 3 of 4 carcinomas as compared to 4 of 11 adenomas and borderline lesions (1 was QNS). LOH and K-ras mutations were both acquired in 2 of 4 carcinomas and in 1 of 12 adenoma/borderline lesions. Although the study is small, molecular analysis for LOH and K-ras mutations is useful in the preoperative evaluation of cystic pancreatic lesions. Increasing degree of neoplasia appears to correlate with increased genetic abnormality using a panel of selected genomic markers.

Changes in Resident Graduate Characteristics in a Large Pathology Training Program, 1994 to 2013.

The field of pathology has changed dramatically over the recent decades and has become more complex with emphasis toward subspecialization. These changes potentially influence resident training as programs and trainees search for cutting-edge skills in the evolving field. Over the last 20 years, our institution's residency education was modified profoundly to emphasize subspecialty practice. Furthermore, efforts were made to search for and recruit candidates who desired such training. In this study, we examined a 20-year time period to determine how these changes may have influenced the characteristics of our resident graduates. For each trainee who graduated from our pathology residency program (1994-2013), the following parameters were evaluated: highest academic degree, gender, graduating medical school, type of training, number of publications during residency, enrollment in fellowships, and type of career position. The data collected were divided into 4 time periods. Fisher exact test and 2-tailed t test were used for statistical analyses comparing the first half (1994-2003) to the latter half (2004-2013) of the study. In the second half, there were more graduates who pursued single track pathology training-anatomic pathology or clinical pathology versus combined anatomic/clinical pathology training (P = .035), more first author and total publications per graduate during residency (P < .001), more graduates who enrolled in fellowships (P < .001), and a greater tendency toward an academic career position than all other types combined (P = .034). In parallel to the subspecialization trends in our department, we witnessed changes in the characteristics of our resident graduates whose interests and career choices have become more focused.

Significance of what is not sampled: Characteristics of thyroid nonmicrocarcinomas (>1.0 cm) that were not targeted.

BACKGROUND: Although most unsuspected thyroid carcinomas qualify as microcarcinomas (≤1 cm), larger, nontargeted carcinomas may be found also. This study evaluated the significance of these nonmicrocarcinomas (>1 cm) in the setting of a large-volume thyroid practice. METHODS: Thyroid resection specimens from May 2007 to December 2012 were reviewed. For these cases, the pathologic characteristics of nontargeted carcinomas larger than 1.0 cm were evaluated. Those interpreted as intrathyroidal metastases were not included in this study. Specifically, the histologic classification, size, and molecular features were documented. RESULTS: From a total of 4815 thyroid resections and 9279 thyroid fine-needle aspiration procedures that were performed during the study period, 27 nontargeted nonmicrocarcinomas were identified (0.6% of resection cases) in 26 patients. The histologic classifications were as follows: follicular variant of papillary carcinoma (n = 19), classic papillary carcinoma (n = 3), papillary carcinoma with oncocytic features (n = 1), tall-cell variant of papillary carcinoma (n = 2), and follicular carcinoma (n = 2). The size parameters were as follows: mean, 1.9 cm; median, 1.4 cm; and range, 1.1 to 7.0 cm. RAS and BRAF mutations were identified in 8 and 7 cases, respectively (71% of the cases tested with a 7-gene panel), whereas 6 cases showed no mutation. Molecular information was not available for 6 cases. CONCLUSIONS: In the authors' experience, nontargeted thyroid nonmicrocarcinomas (>1 cm) are rare (0.6%), and the majority are low-grade carcinomas. The likelihood of finding one of the common mutations (71%) is comparable to the likelihood for thyroid carcinomas in general (∼70%).

Cytologic-histologic correlation of nongynecologic cytopathology cases: separation of determinate from indeterminate cytologic diagnoses for analysis and monitoring of laboratory performance.

Much of the literature on the quality-assurance aspect of cytologic-histologic correlation (CHC) has focused on gynecologic cytology. For nongynecologic cytopathology, the process is complicated by the use of determinate (positive for malignant cells, negative for malignant cells) and indeterminate (atypical, suspicious, or follicular lesion) diagnostic categories. Here, we illustrate our routine methodology for analyzing CHC data on nongynecologic cytopathology cases by separating determinate from indeterminate cases. A focused list of determinate and indeterminate cytopathology cases with surgical pathology correlation is generated each week. The determinate cases are ascertained as true positive (TP), true negative (TN), false positive (FP), or false negative (TN). The discrepant cases (FP and FN) are investigated to determine the cause (sampling, interpretation, or screening). For indeterminate cases, the surgical pathology outcome (benign, malignant) and suitability of the cytopathology category utilized are reviewed. For the focused period of 4 mo, sensitivity was 70% and specificity was 100%. The most common reason for false-negative diagnoses was a sampling problem in the cytologic specimen; there were no false-positive diagnoses. Malignant outcomes for follicular lesion, atypical, and suspicious diagnoses were 29%, 40%, and 76%, respectively. Data derived from regularly performed CHC are useful in reviewing the diagnostic performance of the laboratory.

Molecular profiling of primary and metastatic neoplasms in the lung using cytologic material obtained by fine-needle aspiration: report of two cases.

Two cases are presented in which molecular analyses of cytologic material obtained by fine-needle aspiration were helpful in establishing relationships between morphologically similar neoplasms in the same patient. For appropriate clinical management, it is important to ascertain whether the tumors represent independent primaries or metastases. Alcohol-fixed cytologic material prepared as cell blocks and formalin-fixed paraffin-embedded tissue were microdissected and analyzed for allelic loss of heterozygosity at multiple preselected genetic loci. The first case illustrates a 69-yr-old man with multiple intrapulmonary nodules involving the upper and lower lobes of the left lung. Genomic analysis showed that the neoplasms in the left upper and lower lung lobes were independent primaries, because the loss of heterozygosity (LOH) patterns were substantially different. By contrast, the second case is that of a 58-yr-old man with a right thyroid nodule and multiple pulmonary tumors. LOH analysis confirmed that a sampled pulmonary tumor represented a metastasis from the thyroid primary, as similar LOH patterns involving locus D9S252 were observed on comparison of the thyroid and pulmonary neoplasms. These cases illustrate the practical diagnostic utility of genomic analysis using cytologic material in the assessment of primary and metastatic malignancies.

Ancillary Studies in Thyroid Cytopathology.

Recent advances in thyroid imaging, clinical evaluation, cytopathology, surgical pathology, and molecular diagnostics have contributed toward greater understanding of thyroid nodules. In particular, the development of the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) has brought standardization to the field and the system dovetails well with the implementation of immunohistochemistry and molecular testing to diagnostic practice. Among the molecular strategies available, the application of the molecular panel of common genetic alterations can stratify indeterminate BSRTC diagnoses into low-risk and high-risk groups. The molecular panel markers have a high positive predictive value and therefore, the panel is considered to be a "rule-in" test. In contrast, the Afirma gene expression classifier by Veracyte Corporation is a test that has been reported to have a high negative predictive value, and therefore, considered to be a "rule-out" test. With further advances, refinements are expected to be made. In particular, the application of next-generation sequencing technology holds promise in bringing thyroid cytopathology to the next level.

Thyroid nodules with KRAS mutations are different from nodules with NRAS and HRAS mutations with regard to cytopathologic and histopathologic outcome characteristics.

BACKGROUND: Mutations in the RAS gene in the thyroid gland result in the activation of signaling pathways and are associated with a follicular growth pattern and the probability of a carcinoma outcome ranging from 74% to 87%. In the current study, the authors investigated the cytopathologic and histopathologic features of common RAS mutation subtypes. METHODS: Malignant, indeterminate, and selected benign thyroid cytology cases were tested prospectively for the presence of NRAS61, HRAS61, and KRAS12/13 mutations. For each case, the Bethesda System for thyroid cytopathology diagnosis, additional cytologic descriptors, and surgical pathology outcomes were documented. The Fisher exact test and Wilcoxon 2-sample test were used for statistical comparison between the groups. RESULTS: A total of 204 thyroid fine-needle aspiration cases with RAS mutations (93.6% of which were associated with indeterminate cytopathology diagnoses) and corresponding surgical pathology resection specimens were identified. The KRAS12/13 mutation was associated with a significantly lower carcinoma outcome (41.7%) when compared with HRAS61 (95.5%) and NRAS61 (86.8%) mutations (P<.0001). Furthermore, oncocytic change was observed in a significantly higher percentage of cytology and resection specimens with KRAS12/13 mutations (66.7% and 75.0%, respectively) in comparison with those with HRAS61 (4.5% and 4.5%, respectively) and NRAS61 (15.4% and 14.7%, respectively) mutations (P<.0001). RAS mutations also were identified in cases of poorly differentiated carcinoma (NRAS61), anaplastic carcinoma (HRAS61), and medullary thyroid carcinoma (HRAS61 and KRAS12/13). CONCLUSIONS: Subclassification of RAS mutations in conjunction with cytopathologic evaluation improves presurgical risk stratification, provides better insight into lesional characteristics, and may influence patient management. In particular, KRAS12/13-mutated thyroid nodules were found to be different from HRAS61-mutated and NRAS61-mutated nodules with regard to cytopathologic and surgical outcome characteristics.