Tumor Size Differences Between Preoperative Endoscopic Ultrasound and Postoperative Pathology for Neoadjuvant-Treated Pancreatic Ductal Adenocarcinoma Predict Patient Outcome.

BACKGROUND & AIMS: The assessment of therapeutic response after neoadjuvant treatment and pancreatectomy for pancreatic ductal adenocarcinoma (PDAC) has been an ongoing challenge. Several limitations have been encountered when employing current grading systems for residual tumor. Considering endoscopic ultrasound (EUS) represents a sensitive imaging technique for PDAC, differences in tumor size between preoperative EUS and postoperative pathology after neoadjuvant therapy were hypothesized to represent an improved marker of treatment response. METHODS: For 340 treatment-naïve and 365 neoadjuvant-treated PDACs, EUS and pathologic findings were analyzed and correlated with patient overall survival (OS). A separate group of 200 neoadjuvant-treated PDACs served as a validation cohort for further analysis. RESULTS: Among treatment-naïve PDACs, there was a moderate concordance between EUS imaging and postoperative pathology for tumor size (r = 0.726, P < .001) and AJCC 8th edition T-stage (r = 0.586, P < .001). In the setting of neoadjuvant therapy, a decrease in T-stage correlated with improved 3-year OS rates (50% vs 31%, P < .001). Through recursive partitioning, a cutoff of ≥47% tumor size reduction was also found to be associated with improved OS (67% vs 32%, P < .001). Improved OS using a ≥47% threshold was validated using a separate cohort of neoadjuvant-treated PDACs (72% vs 36%, P < .001). By multivariate analysis, a reduction in tumor size by ≥47% was an independent prognostic factor for improved OS (P = .007). CONCLUSIONS: The difference in tumor size between preoperative EUS imaging and postoperative pathology among neoadjuvant-treated PDAC patients is an important prognostic indicator and may guide subsequent chemotherapeutic management.

Molecular alterations in Hürthle cell nodules and preoperative cancer risk.

Hürthle cell carcinoma (HCC) is a distinct type of thyroid cancer genetically characterized by DNA copy number alterations (CNA), typically of genome haploidization type (GH-type). However, whether CNA also occurs in benign Hürthle cell adenomas (HCA) or Hürthle cell hyperplastic nodules (HCHN), and have diagnostic impact in fine-needle aspiration (FNA) samples, remains unknown. To address these questions, we (1) analyzed 26 HCC, 24 HCA, and 8 HCHN tissues for CNA and other mutations using ThyroSeq v3 (TSv3) next-generation sequencing panel, and (2) determined cancer rate in 111 FNA samples with CNA and known surgical outcome. We identified CNA, more often of the GH-type, in 81% of HCC and in 38% HCA, but not in HCHN. Among four HCC with distant metastasis, all had CNA and three TERT mutations. Overall, positive TSv3 results were obtained in 24 (92%) HCC, including all with ATA high risk of recurrence or metastasis. Among 111 FNA cases with CNA, 38 (34%) were malignant and 73 (66%) benign. A significant correlation between cancer rate and nodule size was observed, particularly among cases with GH-type CNA, where every additional centimeter of nodule size increased the malignancy odds by 1.9 (95% CI 1.3-2.7; P = 0.001). In summary, the results of this study demonstrate that CNA characteristic of HCC also occur in HCA, although with lower frequency, and probability of cancer in nodules with CNA increases with nodule size. Detection of CNA, in conjunction with other mutations and nodule size, is helpful in predicting malignancy in thyroid nodules.

Impact of molecular testing on detecting mimics of oncocytic neoplasms in thyroid fine-needle aspirates diagnosed as follicular neoplasm of Hürthle cell (oncocytic) type.

BACKGROUND: Some thyroid nodules cytologically presenting as follicular neoplasm, Hürthle cell (Oncocytic) type (FNHCT), are not oncocytic tumors and represent autonomously functioning thyroid nodules (AFTNs) with TSHR, GNAS, and EZH1 mutations or oncocytic metaplasia. A to be defined subset of FNHCT harbors genome haploidisation-type DNA copy number alterations (GH-CNA). Molecular profiling of FNHCT may distinguish oncocytic neoplasms from its mimics. METHODS: Consecutive fine-needle aspirates of 180 thyroid nodules over 37 months diagnosed as FNHCT and tested by ThyroSeq v3 were identified. Histologic follow-up was available for 79 of 180 nodules (44%). RESULTS: No molecular alterations were found in 76 of 180 nodules (42%), of which 15 were resected (oncocytic metaplasia, n = 7; follicular oncocytic adenoma, n = 8). Of nodules followed without surgery, 17 of 101 (17%) showed TSHR, EZH1, and GNAS mutations of AFTNs. Papillary thyroid carcinoma was identified by BRAF V600E (n = 2) and hyalinizing trabecular adenoma by PAX8-GLIS3 (n = 1). GH-CNA alone was detected in 42 of 180 FNHCT nodules (23%), of which 29 were resected and histologically diagnosed as follicular oncocytic neoplasms. All remaining resected nodules were histologically proven oncocytic neoplasms: 1) RAS-like alterations without GH-CNA (n = 25) and 2) TERT and/or TP53 mutations co-occurring with GH-CNA (n = 6), including anaplastic thyroid carcinoma arising from follicular oncocytic carcinoma with TP53, TERT mutations with GH-CNA (n = 2). CONCLUSIONS: A proportion of FNHCT nodules are AFTNs and oncocytic metaplasias, which can be suspected based on characteristic mutations or lack of alterations on molecular testing. Among resected FNHCTs, GH-CNAs characterize approximately half of histologically confirmed follicular oncocytic neoplasms.

Thyroid cytology smear slides: An untapped resource for ThyroSeq testing.

BACKGROUND: Molecular testing of thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology is commonly used to guide patient management and is typically performed on freshly collected FNA samples. In this study, the authors evaluated the performance of the ThyroSeq test in cytology smear slides. METHODS: Air-dried Diff-Quik (DQ)-stained and alcohol-fixed Papanicolaou (Pap)-stained smears were used to determine required cellularity and sensitivity of mutation detection and to compare ThyroSeq v3 Genomic Classifier (GC) results obtained in cytology smears and fresh FNA samples from the same nodules. RESULTS: ThyroSeq testing of 31 cytology smears revealed that 25 smears (81%) were adequate for ThyroSeq analysis, including 14 Pap-stained smears (100%) and 11 DQ-stained smears (65%), whereas 6 DQ-stained smears (35%) failed RNA sequencing. The overall accuracy for detecting molecular alterations was 98%, with 100% concordance for mutations and gene expression alterations, 96% concordance for fusions, and 94% concordance for copy number alterations. Cytology smears were adequate for ThyroSeq analysis when at least 200 to 300 cells were present in 1 to 3 slides. ThyroSeq detected all studied mutations down to 5% allele frequency and BRAF mutations down to 1% allele frequency. Testing of smears yielded a positive ThyroSeq GC result in all nodules originally classified as positive. CONCLUSIONS: Thyroid FNA cytology smear slides with adequate cellularity can be successfully used for ThyroSeq GC testing in approximately 80% of cases, with an even higher success rate in Pap-stained smears. Compared with FNA samples collected into preservative solution, 94% to 100% of different genetic alterations could be accurately detected in smears, validating cytology smears as an alternative for ThyroSeq testing in patients with indeterminate thyroid cytology.

Evaluation of NR4A3 immunohistochemistry (IHC) and fluorescence in situ hybridization and comparison with DOG1 IHC for FNA diagnosis of acinic cell carcinoma.

BACKGROUND: Acinic cell carcinoma (AcCC) is diagnostically challenging on fine-needle aspiration because it can mimic several other neoplasms or even normal acinar tissue. Immunopositivity for DOG1, especially circumferential membranous staining, can support the diagnosis of AcCC but is not entirely specific, and it is prone to technical and interpretive challenges on small specimens. NR4A3 (nuclear receptor subfamily 4 group A member 3) translocation and nuclear NR4A3 overexpression were recently described in the majority of AcCCs. Here, the authors evaluate the performance of NR4A3 immunohistochemistry (IHC) and NR4A3 break-apart fluorescence in situ hybridization (FISH) on cell block preparations and compare them with DOG1 IHC in distinguishing AcCC from other entities in the differential diagnosis. METHODS: The authors identified 34 cytology cell blocks with lesional cells, including 11 specimens of AcCC (2 of which derived from 1 patient and showed high-grade transformation) as well as 2 secretory carcinomas, 7 salivary duct carcinomas, 4 mucoepidermoid carcinomas, 3 oncocytomas, 3 renal cell carcinomas, and 6 specimens containing nonneoplastic salivary gland tissue. NR4A3 IHC, DOG1 IHC, and NR4A3 FISH were attempted for all cases. RESULTS: NR4A3 IHC had 81.8% sensitivity and 100% specificity for AcCC, whereas NR4A3 FISH had 36.4% sensitivity and 100% specificity, although 4 cases (3 mucoepidermoid carcinomas and 1 salivary gland tissue sample) could not be analyzed because of low cellularity. Notably, no normal acinar tissue specimens showed NR4A3 positivity by IHC or FISH. In addition, DOG1 IHC had 72.7% sensitivity and 92% specificity. CONCLUSIONS: NR4A3 IHC is highly specific for the diagnosis of AcCC and is more sensitive than DOG1 IHC and NR4A3 FISH. In addition, NR4A3 IHC performance is not improved by the inclusion of DOG1 IHC. Finally, NR4A3 positivity resolves the perennial problem of distinguishing AcCC from normal acinar tissue.

KRAS amplification in metastatic colon cancer is associated with a history of inflammatory bowel disease and may confer resistance to anti-EGFR therapy.

Mutations in RAS occur in 30-50% of metastatic colorectal carcinomas (mCRCs) and correlate with resistance to anti-EGFR therapy. Consequently, mCRC biomarker guidelines state RAS mutational testing should be performed when considering EGFR inhibitor treatment. However, a small subset of mCRCs are reported to harbor RAS amplification. In order to elucidate the clinicopathologic features and anti-EGFR treatment response associated with RAS amplification, we retrospectively reviewed a large cohort of mCRC patients that underwent targeted next-generation sequencing and copy number analysis for KRAS, NRAS, HRAS, BRAF, and PIK3CA. Molecular testing was performed on 1286 consecutive mCRC from 1271 patients as part of routine clinical care, and results were correlated with clinicopathologic findings, mismatch repair (MMR) status and follow-up. RAS amplification was detected in 22 (2%) mCRCs and included: KRAS, NRAS, and HRAS for 15, 5, and 2 cases, respectively (6-21 gene copies). Patients with a KRAS-amplified mCRC were more likely to report a history of inflammatory bowel disease (p < 0.001). In contrast, mutations in KRAS were associated with older patient age, right-sided colonic origin, low-grade differentiation, mucinous histology, and MMR proficiency (p ≤ 0.017). Four patients with a KRAS-amplified mCRC and no concomitant RAS/BRAF/PIK3CA mutations received EGFR inhibitor-based therapy, and none demonstrated a clinicoradiographic response. The therapeutic impact of RAS amplification was further evaluated using a separate, multi-institutional cohort of 23 patients. Eight of 23 patients with KRAS-amplified mCRC received anti-EGFR therapy and all 8 patients exhibited disease progression on treatment. Although the number of KRAS-amplified mCRCs is limited, our data suggest the clinicopathologic features associated with mCRC harboring a KRAS amplification are distinct from those associated with a KRAS mutation. However, both alterations seem to confer EGFR inhibitor resistance and, therefore, RAS testing to include copy number analyses may be of consideration in the treatment of mCRC.

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.

Comparison of the collection approaches of 2 large thyroid fine-needle aspiration practices reveals differing advantages for cytology and molecular testing adequacy rates.

INTRODUCTION: At our institution, almost all thyroid fine-needle aspiration (FNA) procedures are performed by either Endocrinology or Radiology personnel. In this study, we compared the cytology and molecular adequacy rates of these 2 thyroid FNA practices, which differ on several aspects of specimen procurement. MATERIALS AND METHODS: All thyroid FNA specimens from Endocrinology and Radiology practices between September 2008 and December 2016 were included. Over this time frame, the molecular testing modality transitioned from polymerase chain reaction (PCR)-based (7-gene panel era) to next generation sequencing (NGS)-based (ThyroSeq era). In measuring cytology adequacy, the Bethesda System unsatisfactory rate was determined. Molecular adequacy was categorized as Optimal, Limited Thyroid Epithelial Cells, Limited Nucleic Acids, or Failed. These parameters were compared for the 2 practices. RESULTS: The study cohorts comprised 5810 specimens from Endocrinology and 4597 from Radiology. More Endocrinology specimens were satisfactory for cytology diagnosis than those from Radiology (94.7% versus 90.0%, P < 0.001). For molecular adequacy, fewer Endocrinology specimens were optimal than specimens from Radiology for both the 7-gene panel era (76.2% versus 82.9%, P < 0.001) and the ThyroSeq era (88.1% versus 91.9%, P = 0.049). CONCLUSIONS: The 2 thyroid FNA practices varied inversely in their adequacy rates for cytology and molecular testing. Had one practice been superior for both cytology and molecular adequacy, a recommendation for the method of choice would have been straightforward. However, our results show that optimization of FNA practice for the current practice of thyroid cytology requires further investigation due to the complex nature of specimen procurement.

Artificial intelligence in cytopathology: a review of the literature and overview of commercial landscape.

Artificial intelligence (AI) has made impressive strides recently in interpreting complex images, thanks to improvements in deep learning techniques and increasing computational power. Researchers have started applying these advanced techniques to pathology images, although most efforts have been focused on histopathology. Cytopathology, however, remains the original field of pathology for which AI models for clinical use were successfully commercialized, to assist with automating Papanicolaou test screening. Recent AI efforts have focused on whole slide images of both gynecologic and non-gynecologic cytopathology. This review summarizes the literature and commercial landscape of AI as applied to cytopathology.

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.

A Model Based on Pathologic Features of Superficial Esophageal Adenocarcinoma Complements Clinical Node Staging in Determining Risk of Metastasis to Lymph Nodes.

BACKGROUND & AIMS: It is important to identify superficial (T1) gastroesophageal adenocarcinomas (EAC) that are most or least likely to metastasize to lymph nodes, to select appropriate therapy. We aimed to develop a risk stratification model for metastasis of superficial EAC to lymph nodes using pathologic features of the primary tumor. METHODS: We collected pathology data from 210 patients with T1 EAC who underwent esophagectomy from 1996 through 2012 on factors associated with metastasis to lymph nodes (tumor size, grade, angiolymphatic invasion, and submucosal invasion). Using these variables, we developed a multivariable logistic model to generate 4 categories for estimated risk of metastasis (<5% risk, 5%-10% risk, 15%-20% risk, or >20% risk). The model was validated in a separate cohort of 39 patients who underwent endoscopic resection of superficial EAC and subsequent esophagectomy, with node stage analysis. RESULTS: We developed a model based on 4 pathologic factors that determined risk of metastasis to range from 2.9% to 60% for patients in the first cohort. In the endoscopic resection validation cohort, higher risk scores were associated with increased detection of lymph node metastases at esophagectomy (P = .021). Among patients in the first cohort who did not have lymph node metastases detected before surgery (cN0), those with high risk scores (>20% risk) had 11-fold greater odds for having lymph node metastases at esophagectomy compared with patients with low risk scores (95% confidence interval, 2.3-52 fold). Increasing risk scores were associated with reduced patient survival time (P < .001) and shorter time to tumor recurrence (P < .001). Patients without lymph node metastases (pT1N0) but high risk scores had reduced times of survival (P < .001) and time to tumor recurrence (P = .001) after esophagectomy than patients with pT1N0 tumors and lower risk scores. CONCLUSIONS: Pathologic features of primary superficial EACs can be used, along with the conventional node staging system, to identify patients at low risk for metastasis, who can undergo endoscopic resection, or at high risk, who may benefit from induction or adjuvant therapy.

BRAF-mutated microsatellite stable colorectal carcinoma: an aggressive adenocarcinoma with reduced CDX2 and increased cytokeratin 7 immunohistochemical expression.

Reduced CDX2 and cytokeratin 20 (CK20) expression in colorectal carcinoma with BRAF mutation and high-level microsatellite instability (MSI-H) has been well documented. The immunophenotype of BRAF-mutated microsatellite stable (MSS) colorectal carcinoma has not been reported. We analyzed 205 colorectal carcinomas including 28 BRAF-mutated MSS, 53 BRAF-mutated MSI-H, and 124 BRAF wild-type MSS tumors for CDX2, cytokeratin 7 (CK7), and CK20 immunohistochemical expression. CDX2 was scored semiquantitatively for both staining intensity and percent of tumor cells staining and a modified CDX2 H-score was calculated. Patients with BRAF-mutated MSS colorectal carcinomas were more frequently stage IV at presentation compared to patients with BRAF-mutated MSI-H colorectal carcinomas and BRAF wild-type MSS colorectal carcinomas (32% versus 8% versus 15%, P < .001). BRAF-mutated MSS colorectal carcinoma displayed reduced CDX2 expression compared to BRAF wild-type MSS colorectal carcinoma (75% versus 94%; mean CDX2 H-score 98 versus 150, P < .001). CK7 expression was more often identified in BRAF-mutated MSS colorectal carcinoma compared to both BRAF-mutated MSI-H colorectal carcinoma and BRAF wild-type MSS colorectal carcinoma (39% versus 6% versus 6%, P = .0001). BRAF-mutated MSI-H colorectal carcinomas were less often CK20 positive compared to BRAF-mutated MSS and BRAF wild-type MSS tumors (70% versus 93% versus 90%, P = 0.001). In summary, BRAF-mutated MSS colorectal carcinoma often displays reduced CDX2 and increased CK7 expression. Knowledge of this altered immunophenotype is important as patients with BRAF-mutated MSS colorectal carcinoma often present with metastatic disease and the altered tumor immunophenotype may lead to the erroneous assumption that origin from the colon/rectum is unlikely.

Tumor budding is associated with an increased risk of lymph node metastasis and poor prognosis in superficial esophageal adenocarcinoma.

The treatment approach for superficial (stage T1) esophageal adenocarcinoma critically depends on the pre-operative assessment of metastatic risk. Part of that assessment involves evaluation of the primary tumor for pathologic characteristics known to predict nodal metastasis: depth of invasion (intramucosal vs submucosal), angiolymphatic invasion, tumor grade, and tumor size. Tumor budding is a histologic pattern that is associated with poor prognosis in early-stage colorectal adenocarcinoma and a predictor of nodal metastasis in T1 colorectal adenocarcinoma. In a retrospective study, we used a semi-quantitative histologic scoring system to categorize 210 surgically resected, superficial (stage T1) esophageal adenocarcinomas according to the extent of tumor budding (none, focal, and extensive) and also evaluated other known risk factors for nodal metastasis, including depth of invasion, angiolymphatic invasion, tumor grade, and tumor size. We assessed the risk of nodal metastasis associated with tumor budding in univariate analyses and controlled for other risk factors in a multivariate logistic regression model. In all, 41% (24 out of 59) of tumors with extensive tumor budding (tumor budding in ≥3 20X microscopic fields) were metastatic to regional lymph nodes, compared with 10% (12 out of 117) of tumors with no tumor budding, and 15% (5 out of 34) of tumors with focal tumor budding (P<0.001). When controlling for all pathologic risk factors in a multivariate analysis, extensive tumor budding remains an independent risk factor for lymph node metastasis in superficial esophageal adenocarcinoma associated with a 2.5-fold increase (95% CI=1.1-6.3, P=0.039) in the risk of nodal metastasis. Extensive tumor budding is also a poor prognostic factor with respect to overall survival and time to recurrence in univariate and multivariate analyses. As an independent risk factor for nodal metastasis and poor prognosis after esophagectomy, tumor budding should be evaluated in superficial (T1) esophageal adenocarcinoma as a part of a comprehensive pathologic risk assessment.

FSH-receptor isoforms and FSH-dependent gene transcription in human monocytes and osteoclasts.

Cells of the monocyte series respond to follicle stimulating hormone (FSH) by poorly characterized mechanisms. We studied FSH-receptors (FSH-R) and FSH response in nontransformed human monocytes and in osteoclasts differentiated from these cells. Western blot and PCR confirmed FSH-R expression on monocytes or osteoclasts, although at low levels relative to ovarian controls. Monocyte and osteoclast FSH-Rs differed from FSH-R from ovarian cells, reflecting variable splicing in exons 8-10. Monocytes produced no cAMP, the major signal in ovarian cells, in response to FSH. However, monocytes and osteoclasts transcribed TNFalpha in response to the FSH. No relation of expression of osteoclast FSH-R to the sex of cell donors or to exposure to sex hormones was apparent. Controls for FSH purity and endotoxin contamination were negative. Unamplified cRNA screening in adherent CD14 cells after 2h in 25ng/ml FSH showed increased transcription of RANKL signalling proteins. Transcription of key proteins that stimulate bone turnover, TNFalpha and TSG-6, increased 2- to 3-fold after FSH treatment. Smaller but significant changes occurred in transcripts of selected signalling, adhesion, and cytoskeletal proteins. We conclude that monocyte and osteoclast FSH response diverges from that of ovarian cells, reflecting, at least in part, varying FSH-R isoforms.