Liquid-based rapid onsite evaluation of endobronchial ultrasound cytologies.

INTRODUCTION: Rapid onsite evaluation (ROSE) generally uses smears made at the site of the procedure ("smear-based ROSE"). It requires considerable time, generally 2 individuals, technical expertise, and it can be difficult to estimate material available for ancillary studies. We developed an alternative ROSE using liquid-based cytology ThinPrep with hematoxylin and eosin (H&E) stain ("liquid-based ROSE") and assessed its advantages. MATERIALS AND METHODS: Clinicians rinse the sample(s) into CytoRich Red and send to Pathology. A defined proportion of the needle rinse is removed for a ThinPrep stained with a rapid H&E. Adequacy and diagnosis were compared to final outcome. Total time was recorded. RESULTS: Among 52 liquid-based ROSE readings, 28 (53.8%) were interpreted as "adequate" with final as adequate; 17 (32.7%) were interpreted as "inadequate" with final as inadequate; 7 (13.5%) were interpreted as "inadequate" with final as adequate. Of 23 readings provided with onsite diagnosis, 15 (65.2%) were interpreted as definitive positive or negative diagnoses; 6 (26%) were interpreted as nondiagnostic; and 2 (8.7%) were interpreted as atypical. All definitive diagnoses were concordant with final diagnoses. The time for liquid ROSE performance ranges from 6 to 22 minutes (mean: 13 minutes) and required only 1 individual. CONCLUSIONS: Liquid-based ROSE allows accurate adequacy determination and diagnosis, takes about 15 minutes of cytologist time, and can be performed by just 1 person. The technique produces well-preserved and stained slides, it may allow a better estimation of the total amount of material in the specimen vial and may provide a better platform for telecytology.

Fluorescence Polarization Imaging of Methylene Blue Facilitates Quantitative Detection of Thyroid Cancer in Single Cells.

BACKGROUND: Diagnostic accuracy of the standard of care fine-needle aspiration cytology (FNAC) remains a significant problem in thyroid oncology. Therefore, a robust and accurate method for reducing uncertainty of cytopathological evaluation would be invaluable. METHODS: In this double-blind study, we employed fluorescence emission and quantitative fluorescence polarization (Fpol) confocal imaging for sorting thyroid cells into benign/malignant categories. Samples were collected from malignant tumors, benign nodules, and normal thyroid epithelial tissues. RESULTS: A total of 32 samples, including 12 from cytologically indeterminate categories, were stained using aqueous methylene blue (MB) solution, imaged, and analyzed. Fluorescence emission images yielded diagnostically relevant information on cytomorphology. Significantly higher MB Fpol was measured in thyroid cancer as compared to benign and normal cells. The results obtained from 12 indeterminate samples revealed that MB Fpol accurately differentiated benign and malignant thyroid nodules. CONCLUSIONS: The developed imaging approach holds the potential to provide an accurate and objective biomarker for thyroid cancer, improve diagnostic accuracy of cytopathology, and decrease the number of lobectomy and near-total thyroidectomy procedures.

Lenvatinib Targets PDGFR-ß Pericytes and Inhibits Synergy With Thyroid Carcinoma Cells: Novel Translational Insights.

CONTEXT: Pericyte populations abundantly express tyrosine kinases (eg, platelet-derived growth factor receptor-ß [PDGFR-ß]) and impact therapeutic response. Lenvatinib is a clinically available tyrosine kinase inhibitor that also targets PDGFR-ß. Duration of therapeutic response was shorter in patients with greater disease burden and metastasis. Patients may develop drug resistance and tumor progression. OBJECTIVES: Develop a gene signature of pericyte abundance to assess with tumor aggressiveness and determine both the response of thyroid-derived pericytes to lenvatinib and their synergies with thyroid carcinoma-derived cells. DESIGN: Using a new gene signature, we estimated the relative abundance of pericytes in papillary thyroid carcinoma (PTC) and normal thyroid (NT) TCGA samples. We also cocultured CD90+;PAX8- thyroid-derived pericytes and BRAFWT/V600E-PTC-derived cells to determine effects of coculture on paracrine communications and lenvatinib response. RESULTS: Pericyte abundance is significantly higher in BRAFV600E-PTC with hTERT mutations and copy number alterations compared with NT or BRAFWT-PTC samples, even when data are corrected for clinical-pathologic confounders. We have identified upregulated pathways important for tumor survival, immunomodulation, RNA transcription, cell-cycle regulation, and cholesterol metabolism. Pericyte growth is significantly increased by platelet-derived growth factor-BB, which activates phospho(p)-PDGFR-ß, pERK1/2, and pAKT. Lenvatinib strongly inhibits pericyte viability by down-regulating MAPK, pAKT, and p-p70S6-kinase downstream PDGFR-ß. Critically, lenvatinib significantly induces higher BRAFWT/V600E-PTC cell death when cocultured with pericytes, as a result of pericyte targeting via PDGFR-ß. CONCLUSIONS: This is the first thyroid-specific model of lenvatinib therapeutic efficacy against pericyte viability, which disadvantages BRAFWT/V600E-PTC growth. Assessing pericyte abundance in patients with PTC could be essential to selection rationales for appropriate targeted therapy with lenvatinib.

Diagnostic value of a comprehensive, urothelial carcinoma-specific next-generation sequencing panel in urine cytology and bladder tumor specimens.

BACKGROUND: Urine cytology can reliably diagnose high-grade urothelial carcinoma (HGUC) but not low-grade urothelial carcinoma (LGUC), and a more sensitive test is needed. Previously, a pilot study highlighted the possible diagnostic utility of next-generation sequencing (NGS) in identifying both LGUC and HGUC in urine cytology specimens. METHODS: Twenty-eight urine ThinPrep cytology specimens and preceding or subsequent bladder tumor biopsy/resection specimens obtained within 3 months were included in the study (LGUC, n = 15; HGUC, n = 13). A customized, bladder-specific NGS panel was performed; it covered 69 frequently mutated or altered genes in urothelial carcinoma (UC) that were reported by The Cancer Genome Atlas and the Catalogue of Somatic Mutations in Cancer. RESULTS: The sequencing results were compared between the urine cytology specimens and the corresponding bladder tumor biopsies/resections. TP53 was the most frequently identified mutation in HGUC cases (11 of 13 [85%]). PIK3CA and KDM6A were the most frequently identified mutations in LGUC: they occurred in 7 of 15 cases (47%) and in 6 of 15 cases (40%), respectively. Additional frequent mutations identified in the panel included ARID1A (n = 5), EP300 (n = 4), LRP1B (n = 3), ERBB2 (n = 2), STAG2 (n = 2), FGFR3 (n = 3), MLL (n = 2), MLL3 (n = 2), CREBBP1 (n = 1), RB1 (n = 1), and FAT4 (n = 1). Overall, the concordance between the cytology and surgical specimens was 75%. The sensitivity and specificity for identifying mutations in urine cytology specimens were 84% and 100%, respectively. CONCLUSIONS: A bladder-specific NGS panel increases the sensitivity and specificity of urine cytology's diagnostic utility in both low- and high-grade tumors and may serve as a noninvasive surveillance method in the follow-up of patients with UC harboring known mutations.

The Utility of MYB Immunohistochemistry (IHC) in Fine Needle Aspiration (FNA) Diagnosis of Adenoid Cystic Carcinoma (AdCC).

Differentiating adenoid cystic carcinoma (AdCC) from other basaloid neoplasm in a fine needle aspiration (FNA) sample can be challenging. Activation of MYB in AdCC by the fusion transcript MYB-NFIB has been recently demonstrated in salivary gland and other organs. The aim of this study is to evaluate the utility of MYB immunohistochemistry (IHC) in distinguishing AdCCs and other basaloid neoplasm in cytology specimens. Eighteen FNA cases, from salivary gland and other sites, and their subsequent surgical resection specimens were included in the study. Eight cases were confirmed AdCC on resection. MYB IHC was performed on slides made from cytology cell block and surgical resection paraffin blocks. Percentage and intensity of nuclear staining in tumor cells was scored as 0 to 3. The staining results were concordant between cytology specimens and their corresponding surgical resection tumors. Strong diffuse nuclear staining (score 3, N = 5) was exclusively observed in AdCC, both in cytology and surgical specimens. Only one pleomorphic adenoma and one poorly differentiated basaloid carcinoma were positive for MYB staining (score 1 to 2). Any degree of nuclear MYB labeling was seen in 100% AdCC cases (N = 8/8) compared with of 20% (N = 2/10) of all other non-AdCC cases (P = < 0.001). The sensitivity and specificity of any degree MYB positivity for AdCC in cytology specimen is 100% and 78%. The sensitivity and specificity of strong diffuse MYB labeling (score 2 to 3) for AdCC is 83% and 100% in cytology specimen. Strong diffuse nuclear staining of MYB is valuable in supporting a cytologic diagnosis of AdCC. However, weak and focal labeling of MYB should be interpreted with caution as it can be seen in benign and other malignant basaloid lesions.

Colposcopic endocervical brushing cytology appears to be more sensitive than histologic endocervical curettage for detecting endocervical adenocarcinoma.

INTRODUCTION: Colposcopic endocervical brushing cytology (CEB) is more sensitive than endocervical curettage (ECC) for detecting squamous intraepithelial lesions. There are no data on performance of CEB for detecting endocervical adenocarcinoma. MATERIALS AND METHODS: A total of 151 patients were identified in a word search for "endocervical adenocarcinoma" in surgical pathology reports from January 2007 to June 2019. To measure sensitivity, reports of CEB or ECC samples within 1 year preceding the first surgical pathology diagnosis of at least endocervical adenocarcinoma in situ (AIS+) were examined. Specificity was measured in a cohort in which at least atypical glandular cells (AGC+) was reported in CEB or ECC. RESULTS: Seven CEB preceding diagnosis of AIS were identified: 6 of 7 were positive or suspicious for AIS+. One of 7 was negative and it was negative on re-review. Three of 6 positive CEB cases used cell blocks with immunohistochemistry. Seventy ECC samples preceding diagnosis of AIS were identified: 40 of 70 were diagnosed as AGC+. The sensitivities of CEB and ECC for detecting AIS+ at a threshold of AGC+ are 86% and 57% (too few patients for statistics), respectively. For specificity, 12 of 18 CEB and 9 of 25 ECC reports with AGC+ were false positive by follow-up surgical pathology. The specificities of CEB and ECC are 99.4% and 99.9%, respectively. CONCLUSION: Sensitivity of CEB for detecting AIS+ (86%) is at least as high as ECC (57%). Specificity of CEB is similar to ECC. Addition of a cell block to CEB may be useful. CEB appears to be an appropriate test for follow-up of atypical glandular cells reported on Papanicolaou tests.

Cytologically targeted next-generation sequencing: a synergy for diagnosing urothelial carcinoma.

INTRODUCTION: Cytology and cystoscopy are used to detect urothelial carcinoma (UC), but together they still fail to detect some UC cases and are not suitable for screening asymptomatic individuals. Mutations are present in more than 98% of UC, mutations have therapeutic significance, and they can be detected by next generation sequencing (NGS) in urine samples. We review the role of NGS in UC detection. MATERIALS AND METHODS: Comprehensive literature review on UC genetics, economics of NGS, and previous reports of UC detection by NGS. RESULTS: The raw costs of NGS have decreased to about 14,000 base pairs per penny, making it appear economically feasible to use NGS widely. Reported NGS assays fall short of predicted sensitivity. Decreased sensitivity is attributed to a low frequency of mutant alleles in many urine samples. Attempts to increase the percentage of mutant alleles, by using cell-free urinary DNA, or by using cell sorting and microfluidics, have been unsuccessful or remain unproven. However, cytologic examination can immediately enable NGS: Urine cytologies with sufficient proportions of abnormal cells could be directly triaged to NGS with high sensitivity for UC detection. For cases with a low proportion of abnormal cells, cytologically targeted microdissection of cells for NGS should maintain sensitivity and decrease sequencing costs. Cytologically targeted urothelial cells for NGS could allow a screening test for low grade UC. CONCLUSIONS: Cytology is immediately poised to allow NGS to improve the diagnosis of UC, allowing NGS to be an ancillary test for atypical cytologies, and potentially allowing a screening test for low-grade UC.

Cytologic processing of ureteral microbiopsies is associated with higher sensitivity for detection of urothelial carcinoma compared with conventional biopsy processing.

INTRODUCTION: Because of the high rates of false-negative or nondiagnostic ureteral Piranha microbiopsies associated with low cellularity, we assessed the effect of processing these using cytology. MATERIALS AND METHODS: We included 2 groups of 44 consecutive microbiopsies processed from formalin as a standard surgical biopsy and 22 processed by cytology. All samples were from the ureter or renal pelvis or calyx. The cytology samples were collected in alcohol-based media and were prepared with a Cellient cell block only (n = 9) or with a Cellient cell block for the visible particles, together with ThinPrep, to capture the remaining desquamated cells (n = 13). RESULTS: Malignancy was diagnosed in 5 of 44 conventionally processed microbiopsies (11%) compared with 14 of 22 cytologically processed microbiopsies (64%; P < 0.001), including 1 case with invasion. Nineteen site-matched biopsies from 2 patients had undergone both cytologic and surgical processing, with 8 of 8 cytologically processed biopsies diagnosed as malignant. None of the 11 surgically processed biopsies from the same patients matched for site were diagnosed as malignant. Of the 11, 2 (18%) were suspicious for high-grade urothelial carcinoma and 6 (55%) were considered atypical. Increased sensitivity from cytologic processing appears related to increased cell recovery; large numbers of well-preserved urothelial cells were identified in the ThinPrep (range, 1000-25,000 cells/slide), and a nonsignificant trend was found toward increased urothelium (defined as >200 cells/profile) in the Cellient cell blocks (14 of 22 [64%]) compared with the histologic biopsies (17 of 44 [39%]; P = 0.070). CONCLUSIONS: Cytologic processing of ureteral microbiopsies showed superior sensitivity for detecting high-grade urothelial carcinoma, apparently owing to the increased cellular recovery.

An Automatable Method for Determining Adequacy of Thyroid Fine-Needle Aspiration Samples.

CONTEXT.­: Thyroid nodules are a common clinical problem. Cytologic evaluation via fine-needle aspiration is often employed in the diagnostic workup, and rapid on-site assessment of adequacy can help ensure an adequate sample is obtained. However, rapid on-site assessment of adequacy only examines part of the sample, a part that may not then be available for ancillary testing. Moreover, the procedure is time-consuming and poorly reimbursed. OBJECTIVE.­: To develop an automatable fluorescence-based image analysis system for assessing the adequacy of thyroid fine-needle aspirations that uses the entire aspirated sample. DESIGN.­: There were 12 previously diagnosed cases that served as a training set, and 11 cases were used for validation of an image analysis algorithm. The samples were fluorescently stained and imaged using a fluorescent microscope. The images were assessed for adequacy by an image analysis algorithm. Following image analysis, a ThinPrep slide was prepared and blindly scored by a cytopathologist. The standard and computer-derived results were then compared. RESULTS.­: The algorithm was optimized using the 12 cases in the training set and then applied to the 11 test cases. A total of 8 of 8 adequate samples in the test group were correctly scored as adequate, and 2 of 3 cases that were inadequate were correctly scored as inadequate by the algorithm. One case was erroneously designated as not adequate by the algorithm. CONCLUSIONS.­: Our results demonstrate the feasibility of automating thyroid adequacy assessment using a fluorescent labeling technique followed by computer image analysis.

Causes of false-negative for high-grade urothelial carcinoma in urine cytology.

INTRODUCTION: The Paris System for classifying urine cytology emphasizes identification of high-grade urothelial carcinoma (HGUC). The causes of false-negative urine cytologies (UC) within this system are not well described. MATERIAL AND METHODS: We identified 660 cases between 2005 and 2013 with both UC and subsequent cystoscopic biopsies. UC were classified as either Negative for HGUC or "Abnormal" ("Atypical", "Suspicious", and "Malignant"). Apparent false-negative cases were reviewed in a nonblinded fashion by two cytopathologists and two subspecialized genitourinary pathologists. RESULTS: A total of 199 of the 660 cases (30%) were histologically diagnosed as HGUC. The UC were "Abnormal" in 170/199 cases (sensitivity/specificity of 86%/71%). Twenty four apparent false negative cases were available for retrospective review. Five of 24 (21%) cystoscopic biopsies were found not to be HGUC on review (one false positive and four low-grade urothelial carcinoma (LGUC on review). Of the remaining 19 UC, 7 (29%) cytology samples were found to be truly negative on review, 11 (46%) were found to be Atypical, and 1 (4%) suspicious. Of the 12 UC that were at least "Atypical" with histologic HGUC on review: six misses (half) were attributed to obscuring inflammation/blood, four to poor preservation, eight to paucity of abnormal cells, and 1 case to interpretive error; many cases demonstrated overlapping reasons. CONCLUSION: About one fifth of apparent false negative diagnoses for HGUC can be because of overdiagnosis of HGUC by surgical pathologists. If poor preservation or obscured samples are called nondiagnostic, the sensitivity/specificity of UC for HGUC can be as high as 94%/71%. Diagn. Cytopathol. 2016;44:994-999. © 2016 Wiley Periodicals, Inc.

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model.

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF(V600E)-positive PTC.

Prior high-risk human papillomavirus testing and Papanicolaou test results of 70 invasive cervical carcinomas diagnosed in 2012: results of a retrospective multicenter study.

CONTEXT: Persistent high-risk human papillomavirus (hrHPV) infection is essential for the development of cervical cancer and its precursor lesions. High-risk HPV testing has a higher sensitivity than cytology does for detecting cervical epithelial lesions. However, a large study from a single institution showed 31% of patients with invasive cervical cancer had negative baseline hrHPV testing within 5 years preceding the diagnosis. OBJECTIVE: To investigate the limitation of hrHPV testing in detecting invasive cervical cancer. DESIGN: Cases from 2012 with a histologic diagnosis of invasive cervical carcinoma were retrieved from multiple institutions. From those records, prior hrHPV testing and Papanicolaou test results in the 5 years before the cancer diagnosis were recorded. RESULTS: Seventy patients with cervical carcinoma were included in the study. Negative HPV test result rates were 9% (5 of 53), 23% (6 of 26), and 25% (2 of 8) during the periods of less than 1 year, 1 to 3 years, and 3 to 5 years before the histologic diagnoses, respectively. Negative Papanicolaou testing results in the same time intervals were 3.4% (2 of 59), 33% (10 of 30), and 40% (6 of 15). Although the HPV(-) rate seemed to be different among different HPV test methods, no statistical significance was detected because of small sample size. Negative hrHPV rates in patients with adenocarcinoma were similar to those in patients with squamous cell carcinoma. CONCLUSIONS: These data expose limitations for the potential use of primary HPV testing. In addition, current screening guidelines recommending cotesting at 5-year intervals should be evaluated further with additional historic data collection because there are women with negative results for both Papanicolaou tests and hrHPV testing within the period of 3 to 5 years before an invasive carcinoma diagnosis.

Utility of liquid-based cytologic examination of distal esophageal brushings in the management of Barrett esophagus: a prospective study of 45 cases.

INTRODUCTION: The goal of Barrett esophagus surveillance is to identify high-grade dysplasia (HGD) for eradication. Surveillance programs currently rely on limited histologic sampling; however, the role of cytology in this setting is not well studied. MATERIALS AND METHODS: From December 1, 2011 to March 30, 2014, 45 patients underwent 4 circumferential brushings of the distal tubular esophagus followed by standard 4-quadrant biopsies. One ThinPrep slide and 1 Cellient cellblock (Hologic, Boxborough, Mass) were prepared. Six cytopathologists evaluated each for adequacy, intestinal metaplasia (IM) and dysplasia. Findings were classified using the traditional 5-tier system used for biopsies. A prospectively modified 3-tier cytologic classification was also tested: negative for HGD, indeterminate for HGD, and HGD. Sensitivity, specificity, and kappa values (interobserver agreement) for cytology were calculated. RESULTS: Ten of 45 patients had nondiagnostic cytologies; none of whom had dysplasia on biopsy. Cytology had good sensitivity (82%) and specificity (88%) for identifying IM compared with biopsy with moderate interobserver agreement (pairwise average of Fleiss and Krippendorf kappa value = 0.589, 79% agreement). One case had IM on cytology not detected on histology. Six of 45 patients had dysplasia on biopsy including 1 intramucosal adenocarcinoma, 1 indeterminate for dysplasia, 2 high-grade dysplasias, and 2 low-grade dysplasias. A non-negative adequate cytology sample had a sensitivity of 100% and a specificity of 88% and 94% for the 5-tier and the 3-tier classification, respectively. CONCLUSIONS: Cytology appears to have good sensitivity and specificity for diagnosis of HGD, and cytology may be poised to synergize with advances in other techniques for management of patients with Barrett esophagus. Improvements in brushing devices may help to decrease the nondiagnostic rate.

Significance of cytopathologist's review of Pap tests screened as negative for intraepithelial lesion or malignancy that are positive for high-risk human papillomavirus.

INTRODUCTION: Cytopathologist's review of Papanicolaou tests (PTs) screened by cytotechnologists as negative for intraepithelial lesion or malignancy (NILM) that are positive for high-risk human papillomavirus (hrHPV+) may be a useful quality control measure. MATERIALS AND METHODS: From January 1, 2012 to December 31, 2012 all NILM/hrHPV+ PTs underwent cytopathologist's review before report issuance as per routine quality control procedures. HrHPV status was known at the time of screening and at final review. The rate of upgraded diagnoses resulting from the cytopathologist's review were examined. Two-year follow-up was obtained. RESULTS: Cytopathologist's review upgraded 250 of 1282 PTs (19.5%) by 1 step to atypical squamous cells of undetermined significance and 13 (1%) were upgraded by 2 steps or more to low-grade squamous intraepithelial lesion or higher. During the same period, significantly fewer NILM PTs (of unknown hrHPV status) were upgraded by 2 steps or more as a result of random 10% rescreening by cytotechnologists (0.2%, P < 0.001). Follow-up was available in 740 of 1282 patients (57.7%). The upgraded group was significantly more likely to be referred for colposcopy (68.3% versus 30.5%, P < 0.001) and cervical intraepithelial neoplasia (CIN) 2 or higher (CIN2+) was diagnosed in more upgraded patients (8.9% versus 3.0%, P < 0.01) than in those not upgraded. There was no significant difference in the percentage of colposcopy patients diagnosed with CIN2+ in the 2 groups, respectively (13.1% versus 9.8%, P = 0.47). CONCLUSIONS: cytopathologist's review of NILM/hrHPV+ PTs identified more 2-step discrepancies than routine 10% rescreening. Significantly more patients in the upgraded group were found to harbor CIN2+; however, this could be related to the higher rate of referral to colposcopy in this group.

Immunohistochemistry practices of cytopathology laboratories: a survey of participants in the College of American Pathologists Nongynecologic Cytopathology Education Program.

CONTEXT: Immunohistochemistry (IHC) is important for cytology but poses special challenges because preanalytic conditions may differ from the conditions of IHC-positive controls. OBJECTIVE: To broadly survey cytology laboratories to quantify preanalytic platforms for cytology IHC and identify problems with particular platforms or antigens. To discover how validation guidelines for HER2 testing have affected cytology. DESIGN: A voluntary survey of cytology IHC practices was sent to 1899 cytology laboratories participating in the College of American Pathologists Nongynecologic Cytopathology Education Program in the fall of 2009. RESULTS: A total of 818 laboratories (43%) responded to the survey by April 2010. Three hundred fourty-five of 791 respondents (44%) performed IHC on cytology specimens. Seventeen different fixation and processing platforms prior to antibody reaction were reported. A total of 59.2% of laboratories reported differences between the platforms for cytology specimens and positive controls, but most (155 of 184; 84%) did not alter antibody dilutions or antigen retrieval for cytology IHC. When asked to name 2 antibodies for which staining conditions differed between cytology and surgical samples, there were 18 responses listing 14 antibodies. A total of 30.6% of laboratories performing IHC offered HER2 testing before publication of the 2007 College of American Pathologists/American Society of Clinical Oncologists guidelines, compared with 33.6% afterward, with increased performance of testing by reference laboratories. Three laboratories validated a nonformalin HER2 platform. CONCLUSIONS: The platforms for cytology IHC and positive controls differ for most laboratories, yet conditions are uncommonly adjusted for cytology specimens. Except for the unsuitability of air-dried smears for HER2 testing, the survey did not reveal evidence of systematic problems with any antibody or platform.

Cytologic diagnosis of metastatic alveolar rhabdomyosarcoma to the thyroid gland by fine-needle aspiration.

BACKGROUND: Metastases to the thyroid gland, although rare, are important entities to consider when evaluating malignant cells on a thyroid fine-needle aspiration (TFNA) specimen. Cellular TFNA specimens with small round blue cells should prompt a broad differential: florid lymphocytic thyroiditis, lymphoma, metastases, as well as primary thyroid malignancies with similar morphologies such as poorly differentiated (insular) and medullary carcinomas. Age, clinical presentation and prior history must be considered in every case. CASE REPORT: We report, to the best of our knowledge, the first case of metastatic alveolar rhabdomyosarcoma (ARMS) to the thyroid gland, definitively diagnosed by TFNA. A 21-year-old female patient presented with a large mass in the right lobe of the thyroid. Her past history was significant for ARMS diagnosed 24 months earlier, currently in remission after successfully completing 40 weeks of chemoradiation therapy. The diagnosis of metastatic ARMS in the TFNA prompted a more thorough examination revealing previously unknown additional sites of metastases. CONCLUSION: Metastases to the thyroid gland are uncommon but should be considered in cases where atypical morphology is encountered. Small round blue cell tumors can metastasize to the thyroid gland, and clinical presentation, morphology, immunohistochemistry and molecular studies are helpful in differentiating between them.

The diagnostic pathology of the nuclear envelope in human cancers.

Cancer is still diagnosed on the basis of altered tissue and cellular morphology. The criteria that pathologists use for diagnosis include many morphologically distinctive alterations in the nuclear envelope (NE). With the expectation that diagnostic NE changes will have biological relevance to cancer, a classification of the various types of NE structural changes into three groups is proposed. The first group predicts chromosomal instability. The changes in this group include pleomorphism of lamina size and shape, as if constraints to maintain a spherical shape were lost. Also characteristic of chromosomal instability are the presence of micronuclei, a specific structural feature likely related to the newly described physiology of chromothripsis. The second group is predicted to be functionally important during clonal evolution, because the NE changes in this group are conserved during the clonal evolution of genetically unstable tumors. Two examples of this group include increased ratio of nuclear volume to cytoplasmic volume and the relatively fragile nuclei of small-cell carcinomas. The third and most interesting group develops in a near-diploid, genetically stable background. Many of these (perhaps ultimately all) are directly related to the activation of particular oncogenes. The changes in this group so far include long inward folds of the NE and spherical invaginations of cytoplasm projecting partially into the nucleus ("intranuclear cytoplasmic inclusions"). This group is exemplified by papillary thyroid carcinoma in which RET and TRK tyrosine kinases, and probably B-Raf mutations, directly lead to diagnostic longitudinal folds of the lamina ("nuclear grooves") and intranuclear cytoplasmic inclusions. B-Raf activation may also be linked to intranuclear cytoplasmic inclusions in melanoma and to nuclear grooves in Langerhans cell histiocytosis. Nuclear grooves in granulosa cell tumor may be related to mutations in the FOXL2 oncogene. Uncovering the precise mechanistic basis for any of these lamina alterations would provide a valuable objective means for improving diagnosis, and will likely reflect new types of functional changes, relevant to particular forms of cancer.

Melanoma spheroid formation involves laminin-associated vasculogenic mimicry.

Melanoma is a tumor where virulence is conferred on transition from flat (radial) to three-dimensional (tumorigenic) growth. Virulence of tumorigenic growth is governed by numerous attributes, including presence of self-renewing stem-like cells and related formation of patterned networks associated with the melanoma mitogen, laminin, a phenomenon known as vasculogenic mimicry. Vasculogenic mimicry is posited to contribute to melanoma perfusion and nutrition in vivo; we hypothesized that it may also play a role in stem cell-driven spheroid formation in vitro. Using a model of melanoma in vitro tumorigenesis, laminin-associated networks developed in association with three-dimensional melanoma spheroids. Real-time PCR analysis of laminin subunits showed that spheroids formed from anchorage-independent melanoma cells expressed increased α4 and ß1 laminin chains and α4 laminin expression was confirmed by in situ hybridization. Association of laminin networks with melanoma stem cell-associated nestin and vascular endothelial growth factor receptor-1 also was documented. Moreover, knockdown of nestin gene expression impaired laminin expression and network formation within spheroids. Laminin networks were remarkably similar to those observed in melanoma xenografts in mice and to those seen in patient melanomas. These data indicate that vasculogenic mimicry-like laminin networks, in addition to their genesis in vivo, are integral to the extracellular architecture of melanoma spheroids in vitro, where they may serve as stimulatory scaffolds to support three-dimensional growth.