Search

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance.Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568–81. ©2018 AACR.See related commentary by Sullivan, p. 532.See related article by Romano et al., p. 556.This article is highlighted in the In This Issue feature, p. 517

Supplementary Figures

Materials and Methods

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the preferred method for diagnosing pancreatic masses. While the diagnostic success of EUS-FNA is widely accepted, the actual performance of EUS-FNA is not known. This study sought to define the EUS-FNA accuracy compared with the gold standard, surgically resected specimens. The study was a single institution, retrospective, and chart review of patients with surgically resected pancreatic specimens from 2005 to 2015 with a preoperative EUS-FNA or biliary brushing. Cytological reports were organized from least concerning (i.e., low chance of malignancy) to most concerning (high chance of malignancy) into eight cytologic categories. We identified 741 cytologic cases: 530 EUS-FNA and 211 endoscopic brushings. For EUS-FNA samples, 62.5% of "benign" samples proved to be "benign" on surgical pathology. A cytologic diagnosis of "suspicious for malignancy" or "positive for malignancy" were concordant with a cancer diagnosis on surgical pathology 93.3% and 98.0% of cases, respectively. EUS-FNA proved to be highly reliable at diagnosing malignancy for cytologic samples that were "suspicious" or "positive" for malignancy. Paired with supportive clinical data, these interpretations may be used to justify cancer treatment.

Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is a sensitive and specific method for diagnosing cancer in solid pancreatic masses. However, some cases receive indeterminate atypical diagnoses, which creates management dilemmas. In the 2014 Papanicolaou Society of Cytopathology (PSC) standardized guidelines for pancreatobiliary cytology, specimens in the "Atypical" category show a spectrum of architectural and/or cellular changes beyond normal or reactive, but, quantitatively or qualitatively, insufficient for classification as neoplastic (benign/other), suspicious or positive for malignancy. Implementation of the PSC system decreased atypical diagnoses, particularly for cystic lesions, and redistributed many cases into benign and neoplastic categories. Because no set cytologic criteria exist for the "Atypical" category there is wide variability in its use, and its frequency ranges from 0%-16% (mean 6%). It consists of a heterogeneous mix of cases that occur because of preanalytic, lesion-specific (low cellularity, necrosis, cystic, reactive and premalignant changes), to pathologist-dependent factors (experience, expertise, training and institutional case volume). Outcomes of atypical diagnoses in solid pancreatic masses range from benign to premalignant and malignant and include reactive atypia in pancreatitis, well differentiated adenocarcinoma, and non-ductal malignancies. The associated risk of malignancy (ROM) ranges from 28%-100%, with an overall intermediate ROM in large-scale studies. Cytopathologists and institutions should monitor and keep their personal and/or laboratory's atypical rates low by judiciously using rapid onsite evaluation, ancillary studies, consensus or expert review, as well as correlation with clinical and radiologic findings. Early repeat EUS-FNA is indicated for unresolved cases.

Cellular plasticity contributes to intra-tumoral heterogeneity and phenotype switching, which enable adaptation to metastatic microenvironments and resistance to therapies. Mechanisms underlying tumor cell plasticity remain poorly understood. SOX10, a neural crest lineage transcription factor, is heterogeneously expressed in melanomas. Loss of SOX10 reduces proliferation, leads to invasive properties, including the expression of mesenchymal genes and extracellular matrix, and promotes tolerance to BRAF and/or MEK inhibitors. We identify the class of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) inhibitors as inducing cell death selectively in SOX10-deficient cells. Targeted therapy selects for SOX10 knockout cells underscoring their drug tolerant properties. Combining cIAP1/2 inhibitor with BRAF/MEK inhibitors delays the onset of acquired resistance in melanomas in vivo. These data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma to produce a targeted inhibitor tolerant state that is likely a prelude to the acquisition of resistance. Furthermore, we provide a therapeutic strategy to selectively eliminate SOX10-deficient cells.

Background Basaloid salivary gland neoplasms (BSNs), which include benign primary tumors and primary or metastatic malignancies, show overlapping morphology in fine-needle aspiration (FNA). The Milan system recommends assigning a grade (low or high) to malignant salivary neoplasms because of the impact on surgical planning. This study investigated cytomorphologic features of BSNs on FNA that would help to favor a high-grade malignancy over a low-grade malignancy or a benign tumor. Methods Two pathologists performed a double-blinded cytologic evaluation of FNA cases diagnosed as BSNs that had corresponding surgical resections. The diagnosis made with the Milan system was correlated with the final surgical diagnosis and grade. Cytologic sensitivity, specificity, and predictive values were calculated. Results There were 132 BSN FNA cases; cytology slides were available for 77 of 87 patients who had undergone resection. The risk of malignancy for the benign neoplasm (BN), salivary gland neoplasm of uncertain malignant potential (SUMP), suspicious for malignancy (SFM), and malignant categories were 13.6%, 22%, 100%, and 100%, respectively. The sensitivity of the malignant/SFM category was 51.7%; another 37.9% of confirmed malignancies were diagnosed as SUMP. The specificity of the BN category was 86%. Favoring a high-grade malignancy on FNA had 100% accuracy (5 of 5). Favoring a low-grade malignancy on FNA had 75% accuracy (6 of 8). The most specific cytomorphologic clues for a high-grade malignancy were necrotic/apoptotic debris, mitoses, discohesion, and anisonucleosis. Conclusions BSNs encompass a broad spectrum of primary and metastatic tumors. Necrotic/apoptotic debris, mitotic activity, discohesion, and significant anisonucleosis, alone or especially in combination, should make a cytopathologist suspect a high-grade malignancy.

Background Hepatocellular carcinoma (HCC) rarely involves the biliary tree and may be inadvertently sampled on bile duct brushings (BDBs). Methods The pathology archives of 5 institutions were searched for BDBs with HCC involvement. Results A total of 17 BDBs from 14 patients were obtained. There was a male:female ratio of 6:1; the median age of the patients was 59.5 years (range, 22-80 years). The median hepatic tumor size was 6.2 cm (range, 2.2-13.0 cm). HCC risk factors included viral hepatitis (5 patients), cirrhosis (5 patients), hemochromatosis (1 patient), and alcoholic steatohepatitis (1 patient). Jaundice with elevated bilirubin, liver enzymes, and α-fetoprotein was common. Endoscopic retrograde cholangiopancreatography demonstrated bile duct dilatation, polypoid intraductal masses (5 samples), clots/debris (2 samples), or strictures (4 samples). All BDBs had single and clustered large cells with naked atypical nuclei, granular cytoplasm, high nuclear/cytoplasmic ratios, and nuclei with prominent macronucleoli. Less common findings included clear/microvesicular cytoplasm (35%), papillae (29%), and anisonucleosis (35%). Classic HCC features (widened trabeculae [35%], endothelial wrapping [24%], multinucleation [24%], and cytoplasmic bile pigment [35%]) were uncommon. A total of 11 BDBs were diagnosed as malignant (10 with HCC and 1 with cholangiocarcinoma), 2 were diagnosed as atypical, and 1 BDB was diagnosed as negative; approximately two-thirds were found to have polysomy on fluorescence in situ hybridization. Approximately 71% of patients died of disease at a median of 3.5 months. Conclusions HCC may extend into the intrahepatic and/or extrahepatic biliary tree, causing masses and/or strictures that may be sampled on BDB. Although cytologically malignant, the classic features of HCC are uncommon, which can cause misdiagnosis. Cytopathologists should be mindful of this differential when evaluating BDBs, particularly when concomitant liver masses and/or HCC risk factors are present. Because of the associated high mortality and rapid rate of death, its presence should be conveyed clearly in pathology reports.

Introduction Fine-needle aspiration (FNA) of nodal metastases plays a key role in the diagnosis of oropharyngeal squamous cell carcinoma (OPSCC). Because of significant clinical implications of human papillomavirus (HPV)-related OPSCC, immunohistochemistry for p16 as a surrogate marker for high-risk HPV is an important ancillary test. After our laboratory switched from CytoLyt to formalin fixative for FNA needle rinses generating cell block (CB) material, we investigated the impact of this protocol change on the accuracy of p16 results. Materials and methods FNA specimens of head and neck lesions with p16 staining performed on CB, from 1 year before and after the implementation of formalin-fixed CB (FCB) were identified. Nuclear and cytoplasmic p16 expression was scored and compared to p16 status on corresponding surgical specimens. Results There were no false-positive results with either fixative. CytoLyt-fixed CB (CCB) had 47% (7 of 15) false-negative cases, whereas FCB had none, with 100% diagnostic accuracy for p16-negative (n = 6) and p16-positive (n = 15) results. False-negative CCB showed 0% to 10% nuclear and 0% to 65% weak cytoplasmic staining, whereas true-positive CCB showed 10% to 85% nuclear and 35% to 90% cytoplasmic staining. p16-negative FCB showed 0% nuclear and cytoplasmic staining, and p16-positive FCB showed 30% to 100% moderate-strong nuclear and cytoplasmic staining. Interobserver variability was greater with CCB. Conclusions In our laboratory, formalin fixation of CB material improved the accuracy of p16 interpretation. Staining in FCB was also more robust than CCB, which showed weaker cytoplasmic and more focal nuclear staining. Therefore, we advocate formalin fixation for head and neck cytology specimens that may require p16 testing.

Introduction Reported upgrade rate to malignancy of radial scars (RS) ranges widely (0%-40%) making management controversial. Methods A retrospective chart review was performed on patients with RS on core needle biopsy (CNB). Upgrade rates to malignancy and atypia on surgical excision were evaluated. Results Of 127 patients with RS on CNB, 53 were excluded due to malignancy or missing records. Of 74 patients reviewed, 52 (70.3%) had surgical excision with four (7.7%) upgraded to malignancy. Eight patients (10.8%) had atypia with RS on CNB with two (25%) upgraded to malignancy. When isolated RS was on CNB, 2 of 44 (4.5%) upgraded to malignancy while 15 of 44 (34%) had atypia on excision. Of 22 patients (29.7%) who did not have excision, zero developed cancer. Conclusion We found higher than expected upgrade rates of isolated RS to atypia which can alter management. Additionally, 25% of RS with atypia upgraded to malignancy suggesting these patients are at higher risk.

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance. Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568–81. ©2018 AACR. See related commentary by Sullivan, p. 532. See related article by Romano et al., p. 556. This article is highlighted in the In This Issue feature, p. 517

Therapy for non-small cell lung carcinoma (NSCLC) is currently determined by histologic subtype and the presence or absence of actionable mutations. Accurate subclassification is therefore essential for appropriate selection of cases for molecular studies and guiding treatment. The gold standard for subclassification of NSCLC is identification of differentiating morphologic features in correlation with diagnostic immunohistochemistry (IHC) in cases of poorly differentiated carcinoma. Whereas Napsin A, TTF1, and p40 antibodies have been used individually for the subtyping of NSCLC, few studies have examined the 3 in cocktail form. Using a novel triple IHC antibody cocktail (TNP) composed of TTF1 (brown nuclear), Napsin A (red granular cytoplasmic), and p40 (red nuclear), a randomized, double-blinded subclassification was performed on a representative histologic section of 32 previously resected primary NSCLCs. TNP results were then compared with the gold-standard diagnosis. TNP accurately identified all (100%, 10/10) squamous cell carcinomas (SCCs) (p40+/TTF1-/Napsin A-) and 89% (16/18) of adenocarcinomas (ADCs) (p40-/TTF1+/Napsin A+). TNP was negative in 7 (20%) tumors (p40-/TTF1-/Napsin A-), including 2 mucinous ADCs. TNP showed no overlapping or discordant immunostaining. Using traditional IHC with p63, CK5/6, and TTF1, all TNP (-) cases remained unclassifiable. With the exception of mucinous ADC, which was TNP negative, all TNP cases correlated with gold-standard diagnosis; 78% of tumors were also definitively classified as either ADC or SCC and required only a single slide for classification.

Intratumoral heterogeneity and cellular plasticity enable tumors to alter phenotypes and adapt to foreign microenvironments and resist targeted inhibitors. While the ability to switch between phenotypic states has been broadly characterized, the key mechanisms that underlie tumor plasticity remain poorly understood. We studied the neural crest lineage transcription factor, SOX10, in the context of cutaneous melanoma and resistance to targeted therapies. SOX10 is heterogeneously expressed in melanoma samples. Using bio-informatics as well as in vivo and 3D in vitro melanoma models, SOX10 loss was sufficient to induce an invasive but slow proliferating phenotype in vitro and in vivo that was associated with expression of a mesenchymal gene set. Interestingly, while SOX10 knockout initially induced a targeted inhibitor tolerant state, longer exposure of co-mixed populations of SOX10 proficient and SOX10 deficient to targeted therapy drives the clonal selection of SOX10 knockout cells. Furthermore, cell lines generated from xenograft tumors that have acquired resistance to either vemurafenib, paradox-breaking BRAFi or the combination of BRAFi + MEKi showed dramatically reduced SOX10 expression compared to their parental counterparts. Altogether these data suggest that acquired resistant clones may arise from drug tolerant persister cells. As a strategy to selectively target this invasive, drug-tolerant SOX10-deficient sub-population, we screened a drug compound library and identified a class of cIAP1/2 inhibitors to be synthetically lethal for SOX10-deficient cells. Our preliminary data suggest that birinapant can delay or prevent resistance to BRAFi/MEKi in vivo. Together, these data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma and enables tumor adaptation to altered microenvironments and drug treatments which could be targeted using cIAP1/2 inhibitors. Citation Format: Claudia Capparelli, Timothy J. Purwin, Manoela Tiago, Nicole Wilski, Danielle Pomante, McKenna Glasheen, Sheera Rosenbaum, Mai Q. Nguyen, Weijia Cai, Richard Zheng, Gaurav Kumar, Inna Chervoneva, Ayako Shimada, Adam E. Snook, Paolo Fortina, Xiaowei Xu, Kim Hookim, Edna Cukierman, Michael A. Davies, Meenhard Herlyn, Andrew E. Aplin. Targeting SOX10-deficient cells to reduce resistance to targeted therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 40.

Background The cytologic features of undifferentiated pancreatic carcinoma with osteoclastic giant cells (UOC) are rarely described. Methods Cytologic and clinicopathologic characteristics in 15 UOC fine-needle aspiration (FNA) specimens were analyzed. Results FNA specimens were obtained from 6 men and 8 women with a mean age of 65 years who had UOCs (head, n = 7; body, n = 3; and tail, n = 4) with a mean radiologic size 7.3 cm, and some had a cystic component (n = 9). Three cell types (osteoclastic giant cells, pleomorphic tumor giant cells, and spindled/histiocytoid cells) were observed in 12 of 15 specimens (80%); and pancreatic ductal adenocarcinoma (PDAC) was present in 11 specimens. FNA diagnoses were UOC (n = 6), PDAC (n = 5), poorly differentiated carcinoma (n = 2), "suspicious for neoplasm" (n = 1), and "negative" (n = 1). Five of 5 specimens with osteoclastic giant cells were positive for cluster of differentiation 68 (CD68) (a glycoprotein that binds to low-density lipoprotein). Pleomorphic tumor giant cells and spindled/histiocytoid cells were positive for pancytokeratin (6 of 7 specimens), CAM5.2 (2 of 3 specimens), and epithelial membrane antigen (2 of 2 specimens). INI-1 protein expression was retained in 3 of 3 specimens. The Ki-67 labeling index was assessed in 3 specimens and was 12%, 18%, and 40%; 4 of 12 resected UOCs were pure, and 8 were mixed with PDAC. One resection specimen had intraductal papillary mucinous neoplasm, and 2 had mucinous cystic neoplasms. The median overall survival (OS) of patients who had UOCs identified on FNA was 8 months (6 died [OS, 8 months; range, 2-22 months], and 8 remained alive [OS, 3 months; range, 1-27 months]), which was similar to the survival of 74 patients who had PDACs identified on FNA (OS, 15 months; P = .279) but worse than that of the 27 patients with UOCs who did not undergo FNA (OS, 92 months; P = .0135). Conclusions The 3 classical UOC cell types are identifiable on FNA, making cytologic diagnosis possible if considered in the differential. A PDAC component is often also observed. The survival advantage of UOC over pure PDAC appears to be negated by FNA and requires further investigation. Cancer Cytopathol 2017;125:563-75. © 2017 American Cancer Society.

A correctly taken cervical smear or liquid-based cytology preparation contains a variety of epithelial cells from the ectocervical squamous epithelium, transformation zone, and endocervical canal. A benign sample could also contain exfoliated endometrial cells, histiocytes, leukocytes, red blood cells, and normal vaginal flora. The type of epithelial cells seen on slides is determined by factors like the degree of maturation of the cervical epithelium, the location of the squamocolumnar junction which depends on the hormonal status at the time of sampling, the presence of metaplastic changes in the transformation zone, or the presence of inflammation or infection. Knowing the spectrum of benign findings is of paramount importance for the correct interpretation of cervical cytology specimens.

Objectives: The objective of this study was to determine if spatial distribution of multiparametric magnetic resonance imaging–transrectal ultrasound (mpMRI-TRUS) fusion biopsy cores to the index lesion reveals trends in the detection of intra-lesion Gleason heterogeneity and a more optimal prostate biopsy strategy. Methods: Index lesion was the lesion with longest diameter on T2-weighted (T2W)-MRI. In cohort 1, fusion biopsy cores biopsies were taken in areas in the center of the target as well as 1 cm laterally on each side. For cohort 2, targeted biopsies were taken from the center of the lesion only. Heterogeneity was defined as difference in maximum Gleason score obtained from fusion cores in the center of the index lesion versus cores obtained from the periphery (cohort 1), or any difference in maximum Gleason score obtained from fusion cores targeted to the index lesion (cohort 2) compared with systematic 12 cores TRUS biopsy. Results: Ninety-nine consecutive patients (35 and 64 in cohorts 1 and 2, respectively) with median age (SD) and prostate-specific antigen (PSA) of 66.9 (±5.9) and 9.7 (±8.2) respectively, were included. Age, PSA, Prostate Imaging Reporting and Data System (PI-RADS) score, and preoperative MRI lesion size were not significantly different between cohorts. Gleason heterogeneity was observed at a significantly higher rate in cohort 1 versus cohort 2 (58% versus 24%; p = 0.041). In cohort 1, cores obtained from the center of the lesion had higher Gleason score than cores obtained from the periphery of the targeted lesion in 57% of cases. Conclusions: We demonstrate that there is observable tumor heterogeneity in biopsy specimens, and that increased number of cores, as well as cores focused on the center and periphery of the largest lesion in the prostate, provide more comprehensive diagnostic information about the patient’s clinical risk category than taking nonspecific cores targeted within the tumor.

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor, AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance.

BACKGROUND This study describes the cytologic features of 26 angiosarcomas diagnosed on fine-needle aspiration. METHODS Twenty-six angiosarcomas from 20 patients were confirmed by cytomorphology and immunocytochemical (immunohistochemistry) positivity for at least 2 of 3 vascular markers. Specimens were examined for spindled/epithelioid/plasmacytoid single cells, 3-dimensional clusters, multiple prominent/bar-shaped nucleoli (5 times longer than their width), chromatin strands, abnormal mitoses, necrosis, and vasoformative features. RESULTS Eight males and 12 females with a mean age of 52 years (range, 2-94 years) underwent aspiration of tumors in the following: soft tissue or skin/subcutis (n = 10), bone (n = 4), nodes (n = 5), lung (n = 2), liver (n = 2), heart (n = 1), parotid gland (n = 1), and pleural fluid (n = 1). An angiosarcoma diagnosis was rendered for 24 of the 26 cases (92%); 1 was diagnosed as “atypical cells, cannot exclude angiosarcoma,” and another was diagnosed as a malignant vascular neoplasm. Abnormal mitoses were most frequent (85%), and they were followed by single malignant cells (81%: epithelioid [69%], spindled [62%], and plasmacytoid [19%]), 3-dimensional clusters (54%), multiple prominent (62%) or bar-shaped nucleoli (54%), and chromatin strands (31%). Vasoformative features, including hemophagocytosis (54%), cytoplasmic lumina/vacuoles (69%) containing red blood cells (54%)/neutrophils (31%), and endothelial wrapping (69%), were seen in 88%; 23% had all vasoformative features, 88% had at least 1, and 12% had none. CONCLUSIONS Angiosarcomas show a range of cytomorphologic features that make them potentially recognizable on cytology. Although vasoformative features are highly suggestive, they are not specific for angiosarcoma and may be seen in some nonvascular neoplasms. Immunohistochemistry and a high index of suspicion are required for an accurate diagnosis. Cancer Cytopathol 2016. © 2016 American Cancer Society.

Metastatic cancer remains a clinical challenge; however, patients diagnosed prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion steps within the metastatic cascade, but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome of melanoma cells. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Increased expression of CADM1 resulted in significant inhibition of motility and invasiveness of melanoma cells. In addition, elevated CADM1 elicited caspase-independent cell death in non-adherent conditions. Expression array analysis suggests that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Importantly, tissue microarray analysis and clinical data from TCGA indicate that CADM1 expression is inversely associated with melanoma progression and positively correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be considered a biomarker for favorable prognosis.

Background Fine-needle aspiration (FNA) is routinely used to evaluate primary thyroid lesions (PTLs), however, its role in diagnosing secondary thyroid neoplasms (STNs) has not been extensively studied. The goal was to examine the clinical and cytopathologic features of STNs diagnosed on FNA. Methods The clinico-pathologic features of 28 STNs were analyzed. All PTLs, lymphomas, and locally invasive tumors were excluded. Results There were 28 STNs (0.18% incidence) out of 15,800 thyroid FNAs (12 males, 16 females, 32 − 85 years), all occurring metachronously (3 weeks−20 years, average 78.3 months) comprising 24 (85.7%) metastatic carcinomas (14 [50%] renal; 4 [14.3%] head and neck squamous cell carcinomas, 3 [10.7%] breast, and 1 [3.6%] colorectal, uterine serous carcinoma, and lung adenosquamous carcinoma, respectively), 3 sarcomas (10.7%) and 1 melanoma (3.6%). Conclusions STNs are rare and diverse tumors which may occur decades after primary malignancy. Renal carcinomas are the most common. Prior history of malignancy, high index of suspicion, and attention to key distinguishing cytologic clues are critical for accurate diagnosis. Diagn. Cytopathol. 2015;43:904–911. © 2015 Wiley Periodicals, Inc.

Objective The Hybrid Capture II high-risk HPV test (HC II hrHPVT) improves early detection of cervical neoplasia in Pap tests. However, weakly positive HC II results may be reported as indeterminate or "equivocal," for which there is little clinical guidance. This study is designed to evaluate the clinical outcome of equivocal HC II hrHPVTs and concurrent atypical squamous cells of undetermined significance (ASC-US) on ThinPrep Pap specimens through correlation with 2-year follow-up cervical biopsies. Materials and methods Over a 5-year period, ThinPrep Pap tests diagnosed as ASC-US were grouped according to their hrHPVT results (i.e., positive, negative, or equivocal) and correlated with histologic follow-up. All equivocal and representative positive and negative hrHPVTs were included. Biopsies showing high-grade dysplasia were reviewed by two pathologists. Results Of 9,012 ASCUS Pap tests, 945 had corresponding hrHPVTs and follow-up cervical biopsies. High-grade squamous intraepithelial lesion (HSIL-cervical intraepithelial neoplasia grades 2/3, CIN2/3) was identified in 20.3% (14/69) of biopsies after equivocal hrHPVTs (CIN2-5.8%, CIN3-14.5% (p=.0261); 16.7% (25/150) after positive hrHPVT (CIN2-12%, CIN3-4.7%); and 5.4% (5/93) of biopsies after negative hrHPVT (CIN2-4.3%, CIN3-1.1%). Conclusion ASC-US in association with equivocal and positive HC II results respectively shows similar incidences of CIN2/3 on 2-year follow-up cervical biopsy. Additionally, a significant proportion of CIN3 biopsies are in the equivocal HC II cohort. As clinical decision making would be impacted by this finding, laboratories should consider evaluating the clinical performance of their HC II assay via correlation with subsequent cervical biopsies.

In the original version of [this article][1] ([1][2]), an error in Fig. 6C was introduced by the compositor during the production of the paper. Specifically, the Raptor Western blot was improperly shifted. The figure has been corrected in the latest online HTML and PDF versions of the article. The

The cytologic features of undifferentiated pancreatic carcinoma with osteoclastic giant cells (UOC) are rarely described.Cytologic and clinicopathologic characteristics in 15 UOC fine-needle aspiration (FNA) specimens were analyzed.FNA specimens were obtained from 6 men and 8 women with a mean age of 65 years who had UOCs (head, n = 7; body, n = 3; and tail, n = 4) with a mean radiologic size 7.3 cm, and some had a cystic component (n = 9). Three cell types (osteoclastic giant cells, pleomorphic tumor giant cells, and spindled/histiocytoid cells) were observed in 12 of 15 specimens (80%); and pancreatic ductal adenocarcinoma (PDAC) was present in 11 specimens. FNA diagnoses were UOC (n = 6), PDAC (n = 5), poorly differentiated carcinoma (n = 2), "suspicious for neoplasm" (n = 1), and "negative" (n = 1). Five of 5 specimens with osteoclastic giant cells were positive for cluster of differentiation 68 (CD68) (a glycoprotein that binds to low-density lipoprotein). Pleomorphic tumor giant cells and spindled/histiocytoid cells were positive for pancytokeratin (6 of 7 specimens), CAM5.2 (2 of 3 specimens), and epithelial membrane antigen (2 of 2 specimens). INI-1 protein expression was retained in 3 of 3 specimens. The Ki-67 labeling index was assessed in 3 specimens and was 12%, 18%, and 40%; 4 of 12 resected UOCs were pure, and 8 were mixed with PDAC. One resection specimen had intraductal papillary mucinous neoplasm, and 2 had mucinous cystic neoplasms. The median overall survival (OS) of patients who had UOCs identified on FNA was 8 months (6 died [OS, 8 months; range, 2-22 months], and 8 remained alive [OS, 3 months; range, 1-27 months]), which was similar to the survival of 74 patients who had PDACs identified on FNA (OS, 15 months; P = .279) but worse than that of the 27 patients with UOCs who did not undergo FNA (OS, 92 months; P = .0135).The 3 classical UOC cell types are identifiable on FNA, making cytologic diagnosis possible if considered in the differential. A PDAC component is often also observed. The survival advantage of UOC over pure PDAC appears to be negated by FNA and requires further investigation. Cancer Cytopathol 2017;125:563-75. © 2017 American Cancer Society.

This study describes the cytologic features of 26 angiosarcomas diagnosed on fine-needle aspiration.Twenty-six angiosarcomas from 20 patients were confirmed by cytomorphology and immunocytochemical (immunohistochemistry) positivity for at least 2 of 3 vascular markers. Specimens were examined for spindled/epithelioid/plasmacytoid single cells, 3-dimensional clusters, multiple prominent/bar-shaped nucleoli (5 times longer than their width), chromatin strands, abnormal mitoses, necrosis, and vasoformative features.Eight males and 12 females with a mean age of 52 years (range, 2-94 years) underwent aspiration of tumors in the following: soft tissue or skin/subcutis (n = 10), bone (n = 4), nodes (n = 5), lung (n = 2), liver (n = 2), heart (n = 1), parotid gland (n = 1), and pleural fluid (n = 1). An angiosarcoma diagnosis was rendered for 24 of the 26 cases (92%); 1 was diagnosed as "atypical cells, cannot exclude angiosarcoma," and another was diagnosed as a malignant vascular neoplasm. Abnormal mitoses were most frequent (85%), and they were followed by single malignant cells (81%: epithelioid [69%], spindled [62%], and plasmacytoid [19%]), 3-dimensional clusters (54%), multiple prominent (62%) or bar-shaped nucleoli (54%), and chromatin strands (31%). Vasoformative features, including hemophagocytosis (54%), cytoplasmic lumina/vacuoles (69%) containing red blood cells (54%)/neutrophils (31%), and endothelial wrapping (69%), were seen in 88%; 23% had all vasoformative features, 88% had at least 1, and 12% had none.Angiosarcomas show a range of cytomorphologic features that make them potentially recognizable on cytology. Although vasoformative features are highly suggestive, they are not specific for angiosarcoma and may be seen in some nonvascular neoplasms. Immunohistochemistry and a high index of suspicion are required for an accurate diagnosis. Cancer Cytopathol 2016;124:659-68. © 2016 American Cancer Society.

Merkel cell carcinoma represents a highly aggressive cutaneous malignancy characterized by regional recurrences, lymph node metastases, distant metastases, and high mortality. As the cytomorphology of Merkel cell carcinoma can be mimicked by other malignancies, especially lymphoma and pulmonary small cell carcinoma, immunocytochemistry is often useful in confirming the diagnosis. Cell blocks, which are traditionally utilized for immunocytochemistry, occasionally exhibit insufficient cellularity. Hence, we prospectively investigated the application of CK20 immunocytochemistry to air-dried, unstained direct smears in the diagnosis of Merkel cell carcinoma fine needle aspirates (FNAs). Eight consecutive FNAs of Merkel cell carcinoma were prospectively examined in this series; seven (88%) cases exhibited immunoreactivity for CK20 in the tumor cells. The one CK20-negative Merkel cell carcinoma was immunoreactive for synaptophysin and CD56. This immunophenotype was identical to that of the original primary tumor. For comparison, air-dried direct smears prepared from three pulmonary small cell carcinoma FNAs were examined by CK20 immunocytochemistry. In all cases, no CK20 immunoreactivity was seen in any of the tumor cells. In conclusion, direct smears represent a feasible and robust source of cellular material for immunocytochemical studies to diagnose Merkel cell carcinoma. This methodology allows the cytologist to confirm on site that material for diagnostic immunocytochemistry is present thereby serving as a safeguard in instances where insufficient cell block cellularity is anticipated or encountered.

We conducted a group consensus review of thyroid aspirates that were previously interpreted as “atypia of undetermined significance/follicular lesion of undetermined significance” (AUS/FLUS) and followed by surgical interventions. The study aimed to investigate if consensus review would minimize the diagnosis of AUS/FLUS with an optimal interobserver agreement and also promote a better cytohistologic concordance. A group of reviewers who were blinded to the corresponding histologic findings simultaneously evaluated a total of 50 aspirates at a multiheaded light microscope. Using the Bethesda System for Reporting Thyroid Cytopathology as a guideline, a consensus interpretation was reached upon review of each aspirate. Interobserver agreement was calculated and recorded. The cytohistologic correlation was then performed between the consensus interpretation and the corresponding histologic diagnosis. The consensus review reclassified 26 (52%) aspirates as non-neoplasia/benign, 10 (20%) as follicular neoplasm/suspicious for a follicular neoplasm, 1 (2%) as papillary thyroid carcinoma, and 2 (4%) as nondiagnostic. Eleven (22%) aspirates remained AUS/FLUS. The interobserver agreement across the five diagnostic categories ranged from 71.6% to 100% with an average level of 88.8%. Cytohistologic concordance was achieved in 24 of 26 (92.3%) and 9 of 11 (81.8%) aspirates that were reclassified as non-neoplasia/benign and neoplasia/malignancy, respectively. A diagnostic accuracy of 89.2% (33/37) was obtained in reclassified cases. In conclusion, the group consensus review minimized AUS/FLUS, offered an optimal level of interobserver agreement, and most importantly, promoted excellent cytohistologic concordance in reclassified cases and, therefore, could play a substantial role in the future in reducing reaspiration and/or unnecessary surgeries. Diagn. Cytopathol. 2012. © 2011 Wiley-Liss, Inc

Fine-needle aspiration (FNA) is routinely used to evaluate primary thyroid lesions (PTLs), however, its role in diagnosing secondary thyroid neoplasms (STNs) has not been extensively studied. The goal was to examine the clinical and cytopathologic features of STNs diagnosed on FNA.The clinico-pathologic features of 28 STNs were analyzed. All PTLs, lymphomas, and locally invasive tumors were excluded.There were 28 STNs (0.18% incidence) out of 15,800 thyroid FNAs (12 males, 16 females, 32 - 85 years), all occurring metachronously (3 weeks-20 years, average 78.3 months) comprising 24 (85.7%) metastatic carcinomas (14 [50%] renal; 4 [14.3%] head and neck squamous cell carcinomas, 3 [10.7%] breast, and 1 [3.6%] colorectal, uterine serous carcinoma, and lung adenosquamous carcinoma, respectively), 3 sarcomas (10.7%) and 1 melanoma (3.6%).STNs are rare and diverse tumors which may occur decades after primary malignancy. Renal carcinomas are the most common. Prior history of malignancy, high index of suspicion, and attention to key distinguishing cytologic clues are critical for accurate diagnosis.

Aims Erythroblast transformation specific related gene ( ERG ), a proto-oncogene member of the erythroblast transformation specific transcription factor family, is a sensitive marker of endothelial differentiation and is expressed in vascular tumours, including angiosarcomas (AS). Immunohistochemistry is necessary for the diagnosis of AS in fine needle aspirates where low cellularity and lack of preserved tissue architecture impedes diagnosis. The aim of this study was to assess the utility of an ERG-enriched immunohistochemistry panel in the cytological diagnosis of AS. Methods 25 AS diagnosed on fine needle aspirates were stained for ERG, CD31, CD34, and AE1/AE3. Staining intensity and percentage tumour cell positivity were evaluated. Spearman’s correlation was assessed for significant correlations between antibodies. Results Sensitivities for ERG, CD31, CD34 and AE1/AE3 were 100%, 100%, 60% and 21%, respectively. Spearman’s analysis revealed that ERG and CD31 staining correlated significantly; there was no significant correlation between CD31 and CD34 staining. Conclusions With equal sensitivity to, and strong correlation with CD31, ERG staining is highly suitable for the cytological diagnosis of AS. ERG and CD31 are more sensitive vascular markers than CD34. ERG, a nuclear stain, complements the cytoplasmic/membranous staining of CD31. Used in conjunction with CD31, ERG can corroborate the diagnosis of AS.

Introduction Napsin A is a diagnostic marker for pulmonary adenocarcinoma and a useful alternative to thyroid transcription factor 1 (TTF-1). TTF-1 also stains pulmonary small cell carcinoma (SCCA). Napsin A expression in SCCAs is not as established as it is in non-SCCAs. We analyzed napsin A and TTF-1 expression in 36 previously confirmed cytologic cases of pulmonary SCCA. Ours is currently the largest cytologic series of such cases examined for napsin A expression. Materials and Methods Thirty-six patients, (20 men, 16 women), age 43-87 years, mean 57 years, had primary or metastatic pulmonary SCCA diagnosed by fine-needle aspiration biopsies of mediastinum (n = 5); liver (n = 3); subcutaneous nodule (n = 1); lung (n = 6); and axillary, cervical, and mediastinal lymph nodes (n = 20), as well as a pleural effusion (n = 1). Napsin A and TTF-1 expression was tested. Also, previous expression (or lack thereof) with immunocytochemical stains pancytokeratin and neuroendocrine markers (synaptophysin, chromogranin, and cluster of differentiation marker CD56) were noted. Results All cases of pulmonary SCCA were positive for pancytokeratin. TTF-1 was positive in 35 of 36 cases (97%), and napsin A was negative in all 36 cases (100%). All 36 cases expressed ≥ 1 neuroendocrine marker, including the TTF-1 negative case. Conclusions This study showed napsin A was negative in all pulmonary SCCAs. This stain may prove to be a useful exclusionary marker in distinguishing pulmonary SCCA from other poorly differentiated lung carcinomas with similar morphologic features, especially those with concomitant TTF-1 expression.

BACKGROUND: The cytodiagnosis of melanoma in fine-needle aspiration (FNA) specimens can be challenging, often requiring the use of immunocytochemistry. As constitutively activating mutations in the BRAF oncogene are present in at least 40% of melanomas, the use of FNA material to interrogate the BRAF mutational status is likely to increase. Because cell blocks, traditionally used for these studies, can occasionally exhibit insufficient tumor cellularity, the authors investigated the utility of direct smears for immunocytochemistry and BRAF mutational analysis. METHODS: Immunocytochemistry for S-100, HMB-45, and Mart-1 was prospectively performed on direct smears in 17 FNAs of metastatic melanoma. Next, BRAF sequencing was performed using DNA isolated from archived Diff-Quik–stained direct smears for 15 cases. In parallel, sequencing was performed using DNA obtained from corresponding cell blocks. RESULTS: S-100 positivity in the tumor cells was observed in all 17 cases. HMB-45 and Mart-1 positivity was noted in 81% and 88% of cases, respectively. All 3 markers were positive in 76% of cases. Next, of the 15 archived melanoma FNAs tested, BRAF mutations were observed in 8 (53%); 5 and 3 melanomas harbored the V600E and V600K mutation, respectively. Corresponding cell blocks were also tested for all 15 cases, yielding concordant BRAF results in 14 (93%); 1 cell block yielded a false-negative result. CONCLUSIONS: Cytologic direct smears represent a robust and valuable source of cellular material for immunocytochemistry and molecular studies, especially in instances in which inadequate cell block cellularity is anticipated or encountered. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.

Giant cell myocarditis, a rare, fatal, and poorly understood cause of myocarditis, requires pathological examination for diagnosis. It is considered to be an autoimmune disease and is frequently associated with other conditions, in particular thymoma and myasthenia gravis. The typical patient with giant cell myocarditis is young and has severe, progressive congestive cardiac failure that is unresponsive to standard medical therapy and ultimately requires cardiac transplantation. Hence giant cell myocarditis is the most dangerous form of myocarditis. Here we report an unusual presentation of giant cell myocarditis, which mimicked acute myocardial infarction in an elderly woman with myasthenia gravis and a previous diagnosis of thymoma. This patient had evidence of anti-myocyte antibodies, consistent with an autoimmune mechanism.