Cytology of Benign Peritoneal Fluids and Pelvic Washing Specimens: Diagnostic Cytomorphologic Features and Pitfalls.

BACKGROUND: Pelvic washing and peritoneal fluid cytology specimens are used to detect peritoneal spread of malignancies. In most cases, identification of malignancy in these specimens is straightforward, but benign processes may occasionally mimic neoplasia and cause diagnostic difficulty. SUMMARY: In this article, we perform a focused review of common benign entities encountered in pelvic washing and peritoneal fluid specimens during routine practice which may cause difficulty and discuss helpful features for avoiding diagnostic pitfalls. KEY MESSAGES: Application of strict cytomorphologic criteria, along with judicious use of ancillary studies and correlation with clinical, intraoperative, radiologic, and other pathologic findings, can help resolve most problematic cases.

Role of Ancillary Techniques in Fluid Cytology.

The cytologic evaluation of serous effusions may be challenging for a number of reasons. Distinction of benign, reactive conditions from malignancy represents the main focus when examining these specimens. The morphologic diagnosis of malignancy may be difficult due to the relative paucity of abnormal cells. In other situations, cellularity is not an issue, but the ability to confidently identify a second, foreign (i.e., tumor) population within a background mesothelial cells on the basis of cytomorphologic features alone may pose problems. Cases with definitive morphologic evidence of malignancy may require additional studies in order to determine the tumor subtype and, in the case of carcinoma, the primary site of origin. Cases in which a definitive and precise diagnosis of malignancy is made may be optimal candidates for further molecular testing in order to gain prognostic information and guide personal therapeutic decisions. Finally, while an inflammatory or infectious condition can be suggested on the basis of cellular components and associated background elements, the identification of causative agent(s) may be difficult without additional studies. In all of these situations, the use of ancillary studies and techniques is critical; their utility and appropriate application are the subject of this review.

Measurement and immunophenotyping of pleural fluid EpCAM-positive cells and clusters for the management of non-small cell lung cancer patients.

OBJECTIVES: A malignant pleural effusion (MPE) is a common complication in non-small cell lung cancer (NSCLC) with important staging and prognostic information. Patients with MPEs are often candidates for advanced therapies, however, the current gold standard, cytological analysis of pleural fluid samples, has limited sensitivity. We aimed to demonstrate the feasibility of non-invasive enumeration and immunophenotyping of EpCAM-positive cells in pleural fluid samples for the diagnosis of a MPE in NSCLC patients. MATERIALS AND METHODS: Pleural fluid specimens were prospectively collected from patients with NSCLC and the CellSearch® technology was utilized for the enumeration of pleural EpCAM-positive cells (PECs) and determination of PD-L1 expression on PECs from pleural fluid samples. The diagnostic performance of the enumeration of single PECs and PEC clusters was assessed using receiver operating characteristic (ROC) curves. The Kaplan-Meier method and Cox proportional hazards model was used to assess the impact of PECs and PEC clusters on overall survival (OS). RESULTS: 101 NSCLC patients were enrolled. The median number of PECs was significantly greater in the malignant (n = 84) versus non-malignant group (n = 17) (730 PECs/mL vs 1.0 PEC/mL, p < 0.001). The area under the ROC curve was 0.91. A cutoff value of 105 PECs/mL had a sensitivity and specificity of 73% and 100% for the diagnosis of a MPE, respectively. Among 69 patients with a pathology-confirmed MPE and tissue immunohistochemistry (IHC) results, 15 (22%) had greater than 50% PD-L1+ PECs. Overall concordance between tissue and PEC PD-L1 expression was 76%. Higher numbers of pleural effusion single PECs were associated with inferior overall survival (Cox adjusted HR 1.8, 95% CI: 1.02-3.05 p = 0.043). CONCLUSION: Non-invasive measurement of PECs in NSCLC patients, using an automated, clinically available approach, may improve the diagnostic accuracy of a MPE, allow for immunophenotyping of PECs, and provide prognostic information.

Ultrahigh-speed line-scan SD-OCT for four-dimensional in vivo imaging of small animal models.

We report an ultrahigh-speed and high-resolution line-scan spectral-domain optical coherence tomography (SD-OCT) system that integrates a number of mechanisms for improving image quality. The illumination uniformity is significantly improved by the use of a Powell lens; Phase stepping and differential reconstruction are combined to suppress autocorrelation artifacts; Nonlocal means (NLM) is employed to enhance the signal to noise ratio while minimizing motion artifacts. The system is capable of acquiring cross-sectional images at more than 3,500 B-scans per second with sensitivities between 70dB and 90dB. The high B-scan rate enables image post-processing with nonlocal means, an advanced noise reduction algorithm that affords enhanced morphological details and reduced motion artifacts. The achieved axial and lateral resolutions are 2.0 and 6.2 microns, respectively. We have used this system to acquire four-dimensional (three-dimensional space and one-dimensional time) imaging data from live chicken embryos at up to 40 volumes per second. Dynamic cardiac tissue deformation and blood flow could be clearly visualized at high temporal and spatial resolutions, providing valuable information for understanding the mechanical and fluid dynamic properties of the developing cardiac system.

Concordance between fine-needle aspiration and core biopsies for osseous lesions by lesion imaging appearance and CT attenuation.

OBJECTIVES: To compare the concordance between fine-needle aspiration and core biopsies for osseous lesions by lesion imaging appearance and CT attenuation. MATERIALS AND METHODS: Retrospective review of 215 FNAs of osseous lesions performed in conjunction with core biopsy at our institution over a 6-year period (2011-2016). FNAs were interpreted independently of core biopsies. We assessed if FNA in conjunction with core biopsy increased diagnostic accuracy compared to core biopsy alone. We also calculated the concordance between FNA and core biopsy by lesion appearance, lesion CT attenuation, lesion histology, lesion location and FNA needle gauge size. RESULTS: Core biopsy alone provided the diagnosis in 207/215 cases (96.3%), however, the FNA provided the diagnosis in the remaining 8/215 cases (3.7%) where the core biopsy was non-diagnostic. There were 154 (71.6%) lytic lesions, 21 (9.8%) blastic lesions, 25 (11.6%) mixed lytic and blastic lesions and 15 (7.0%) lesions that were neither lytic nor blastic. The concordance between FNA and core biopsy for lytic osseous lesions (136/154 cases, 88.3%) was statistically significantly higher than that for blastic osseous lesions (13/21 cases, 61.9%) [P = 4.2 × 10-3; 95% CI (0.02, 0.50)]. The concordance between FNA and core biopsy was higher for low-attenuation- (110/126) than high-attenuation (58/77) lesions (P = 0.028). The concordance between FNA and core biopsy was also higher for metastases (102/119 cases, 85.7%) than non-metastases (78/96, 81.3%) [P = 0.487; 95% CI (- 0.15, 0.065)]. There was no difference in the rate of concordance between FNA and core biopsy by lesion location or FNA needle gauge size (P > 0.05). CONCLUSION: FNA with core biopsy increases diagnostic rate compared to core biopsy alone or FNA alone. The concordance between FNA and core biopsy is higher for lytic lesions than for blastic lesions; and higher for low-attenuation lesions than for high-attenuation lesions.

Diagnostic utility of fine-needle aspiration cytology of lesions involving bone.

BACKGROUND: Fine-needle aspiration (FNA) is utilized in the diagnostic work-up of bone lesions in a number of institutions, either in isolation or in conjunction with core biopsy. We report our experience with FNA of bone-based lesions, including comparison of this approach with concurrent core biopsy specimens. METHODS: Retrospective review over a 5-year period (2011-2015) revealed 233 cases of bone FNAs. RESULTS: The most commonly encountered diagnosis was malignant neoplasm (160 cases, 68.7%); within this group of malignancies, 103 cases (64.4%) represented metastatic carcinoma. Benign lesions were encountered infrequently (9 cases, 3.9%). While 37 cases (15.9%) revealed "no evidence of malignancy," 12 cases (5.2%) showed atypical findings, 3 cases (1.3%) demonstrated inflammatory conditions, and 12 aspiration biopsies were deemed nondiagnostic (5.2%). In 202 cases, concurrent core biopsies were performed following FNA and rapid on-site evaluation (ROSE). Comparison of the FNA and core biopsy diagnoses among malignant neoplasms revealed 19 diagnostic discrepancies, including 16 cases with a false-negative FNA (7.9% of all FNAs with concurrent core biopsy) and 3 cases with a false-negative core biopsy (1.5% of all cases with corresponding FNA). CONCLUSION: Our findings indicate that FNA of bone lesions is a useful diagnostic technique with high sensitivity, particularly when the cytologic findings are interpreted in conjunction with the core biopsy and pertinent clinical and radiologic findings. In addition, ROSE followed by open, dynamic communication with the performing radiologist leads to an extremely low rate of inadequate core biopsy specimens, resulting in optimal patient diagnosis and management. Diagn. Cytopathol. 2017;45:608-613. © 2017 Wiley Periodicals, Inc.

Fine-needle aspiration cytology of endometriosis.

Endometriosis commonly involves the pelvis, but may also present as a palpable mass in extrapelvic sites, such as the abdominal wall or inguinal region, where it can be evaluated by fine needle aspiration (FNA). In this report, we illustrate the findings seen in seven cases of endometriosis diagnosed by FNA in patients with a chief complaint of pain associated with an abdominal wall or pelvic mass, occurring in a setting of prior pelvic surgery. The most common previous surgery was Cesarean section (n = 6), followed by hysterectomy (n = 2), and hernia repair (n = 1). In all cases, cytologic examination revealed a glandular component composed largely of orderly fragments of cohesive epithelial cells, a spindle cell stromal component presenting either as loosely organized tissue fragments or single cells, and rare hemosiderin-laden macrophages. Four cases showed focal cytologic atypia in the glandular component with extreme nuclear atypia identified in two of these cases. Atypical features included nuclear crowding and disorganization, nuclear enlargement, hyperchromasia with irregular chromatin distribution and anisonucleosis, raising the possibility of a coexistent malignancy and recommendation for excision. Although malignancy was not identified in follow-up surgical excision specimens, the wide range of cytomorphologic changes that can be seen in FNA specimens of endometriosis should be recognized. Diagn. Cytopathol. 2017;45:359-363. © 2016 Wiley Periodicals, Inc.

A method to study the hemodynamics of chicken embryo's aortic arches using optical coherence tomography.

Congenital cardiovascular defects are the leading cause of birth defect related death. It has been hypothesized that fluid mechanical forces of embryonic blood flow affect cardiovascular development and play a role in congenital malformations. Studies in small animal embryos can improve our understanding of congenital malformations and can lead to better treatment. We present a feasibility study in which high-resolution optical coherence tomography (OCT) and computational fluid dynamics (CFD) are combined to provide quantitative analysis of the embryonic flow mechanics and the associated anatomy in a small animal model.

Pleural effusion, pneumothorax, and lung entrapment in rheumatoid arthritis.

Rheumatoid arthritis (RA)-associated pleural effusions are usually small and asymptomatic with no need for intervention, but complex and symptomatic rheumatoid pleural effusions may be seen and are associated with significant morbidity and mortality. Pleural effusions may develop before, concurrently with, or after the joint manifestations of RA. The classic features of RA-associated pleural effusions include high cell counts and protein, lipid, and lactate dehydrogenase levels and very low glucose levels, along with distinctive cytopathologic findings: slender spindle-shaped cells, multinucleated giant cells, eosinophilic granular debris, and the absence of mesothelial cells. Rarely, rheumatoid pleural involvement can include pneumothorax or can be severe enough to progress to lung entrapment, which may cause significant restrictive lung disease and require surgical therapy. Rheumatoid pleural involvement may not always correlate with joint activity but can be a significant cause of shortness of breath for patients with RA.

Fine-needle aspiration of superficial myxoid neurofibroma in the region of the breast.

Myxoid neurofibromas are benign spindle cell tumors of perineural cell origin with a broad pathologic differential diagnosis, which includes myxoma, myxoid liposarcoma, myxoid dermatofibrosarcoma protuberans, and low-grade fibromyxoid sarcoma. We present an unusual case of superficial myxoid neurofibroma in the region of the breast that underwent pre-operative fine-needle aspiration (FNA). The differential diagnosis for a myxoid subcutaneous lesion should include myxoid neurofibroma when myxoid material is encountered in an otherwise hypocellular FNA.

Use of flow cytometry in the diagnosis of lymphoproliferative disorders in fluid specimens.

The diagnostic evaluation of fluid specimens, including serous effusions and cerebrospinal fluids (CSFs), can be challenging for a number of reasons. The evaluation of lymphoid proliferations in these specimens can be particularly problematic, given the frequent presence of coexisting inflammatory conditions and the manner in which these specimens are processed. As a result, immunophenotypic analysis of hematopoietic cell populations by flow cytometry has emerged as a useful ancillary study in the diagnosis of these specimens, both in patients with and without a previous history of a lymphoproliferative disorder. In this study, we review our experience with flow cytometry in fluid specimens over a four-year period. Flow cytometry was performed in 184 of 6,925 total cases (2.7% of all fluids). Flow cytometry was performed in 4.8% of pleural fluids (positive findings in 38%, negative in 40%, and atypical in 18%), 1.1% of peritoneal fluids (positive in 40%, negative in 50%, and atypical in 10%), 1.9% of pericardial fluids (positive in 67%, negative in 33%), and 1.9% of CSFs (positive in 23%, negative in 55%, atypical in 3%). The specimen submitted was inadequate for analysis in 9.2% of cases, most commonly with CSF specimens, but was not related to the volume of fluid submitted. Atypical flow cytometry findings and atypical morphologic findings in the context of negative flow cytometry results led to the definitive diagnosis of a lymphoproliferative disorder in a significant number of cases when repeat procedures and ancillary studies were performed.

Use of circulating tumor cell technology (CELLSEARCH) for the diagnosis of malignant pleural effusions.

RATIONALE: Cytological analysis of pleural effusions (PEs) has a sensitivity of approximately 60%. We hypothesized that the CELLSEARCH technology (Janssen Research and Development, Huntingdon Valley, PA) currently used to detect circulating tumor cells could be adapted for the identification of tumor cells in PEs. METHODS: This was a single-center, prospective, observational study. Pleural fluid from subjects with undiagnosed PEs were analyzed by CELLSEARCH technology, which uses an epithelial cell adhesion molecule antibody-based capture system/cytokeratin antibodies to identify tumor cells. Subjects were prospectively monitored by periodic chart review to determine the etiology of the PE. MEASUREMENTS AND MAIN RESULTS: One hundred thirty-two subjects were analyzed. A malignant etiology was established in 81 subjects. The median number of "positive" pleural epithelial cells (PECs) detected per milliliter of pleural fluid was 6 in the benign group. The number of PECs was 52 in the malignant nonepithelial group (NS) and 526 in the malignant epithelial group (P < 0.001). Unlike blood, there was a baseline number of "positive" cells in benign pleural fluids; however, any cutoff greater than 852 positive cells/ml had 100% specificity. The area under the receiver operating characteristic curve was 0.86. Nine percent of our cancer cases had high numbers of PECs (>280/ml) but a negative or nondefinitive cancer diagnosis by cytology. CONCLUSIONS: The pleural CELLSEARCH assay may serve as a valuable addition to traditional cytology and provide useful information regarding the diagnosis of malignant effusions. Major advantages include that it is well standardized, relatively inexpensive, has a rapid turnaround, and is easily available. Our data support the conduct of additional studies of this approach to assist in the diagnosis of malignant PEs.

Folate receptor alpha expression in lung cancer: diagnostic and prognostic significance.

With the advent of targeted therapies directed towards folate receptor alpha, with several such agents in late stage clinical development, the sensitive and robust detection of folate receptor alpha in tissues is of importance relative to patient selection and perhaps prognosis and prediction of response. The goal of the present study was to evaluate the expression of folate receptor alpha in non-small cell lung cancer specimens to determine its frequency of expression and its potential for prognosis. The distribution of folate receptor alpha expression in normal tissues as well as its expression and relationship to non-small cell lung cancer subtypes was assessed by immunohistochemistry using tissue microarrays and fine needle aspirates and an optimized manual staining method using the recently developed monoclonal antibody 26B3. The association between folate receptor alpha expression and clinical outcome was also evaluated on a tissue microarray created from formalin fixed paraffin embedded specimens from patients with surgically resected lung adenocarcinoma. Folate receptor alpha expression was shown to have a high discriminatory capacity for lung adenocarcinomas versus squamous cell carcinomas. While 74% of adenocarcinomas were positive for folate receptor alpha expression, our results found that only 13% of squamous cell carcinomas were FRA positive (p<0.0001). In patients with adenocarcinoma that underwent surgical resection, increased folate receptor alpha expression was associated with improved overall survival (Hazard Ratio 0.39, 95% CI 0.18-0.85). These data demonstrate the diagnostic relevance of folate receptor alpha expression in non-small cell lung cancer as determined by immunohistochemistry and suggest that determination of folate receptor alpha expression provides prognostic information in patients with lung adenocarcinoma.

Functional profiling of live melanoma samples using a novel automated platform.

AIMS: This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform. METHODS: A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma. RESULTS: Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy. CONCLUSION: Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients.

Goals and guidelines for residency training in cytopathology.

The time available to train residents in cytopathology has been impacted by the contraction of the training period, in the face of growing expectations for training in other facets of anatomic pathology and laboratory medicine (i.e. molecular genetic pathology, laboratory management and administration). Guidelines for appropriate levels of achievement in cytopathology have been proposed by those within the field, under the guidance of the Accreditation Council for Graduate Medical Education (ACGME). Presented here is a discussion of recommendations for training programs, in order to effectively train residents to an acceptable level of competence in cytopathology, within the training time allotted.

Value of P63 and CK5/6 in distinguishing squamous cell carcinoma from adenocarcinoma in lung fine-needle aspiration specimens.

The current FDA-approved standard of care for nonsmall cell lung cancer is Carboplastin/Taxol/Avastin based upon an impressive survival benefit; however, patients with squamous carcinoma (SQCC) cannot receive Avastin because of a 30% mortality rate due to fatal hemoptysis. In this study we evaluated the role of cytomorphology and immunohistochemistry in differentiating SQCC from adenocarcinoma (ADC) in lung FNA specimens. The case cohort included 53 FNA cases of nonsmall cell lung carcinoma with surgical pathology follow-up. All FNA specimens were reviewed independently by a panel of cytopathologists to differentiate between SQCC and ADC. The cell block material was available in 23 cases (11 ADC and 12 SQCC) to perform immunohistochemical stains for TTF-1, CK7, CK20, P63, and CK5/6. On surgical resection, 35/53 (66%) cases were diagnosed as ADC and 18/53 (34%) as SQCC. The number of cases classified correctly on the basis of cytomorphology was 66% for ADC and 53% for SQCC (combined accuracy 60%). By immunohistochemical staining, 14/23 (61%) cases expressed TTF-1. Nine cases were TTF-1 negative; eight of the TTF-1 negative cases (89%) were SQCC. Twenty-three cases expressed CK7 (87%); one ADC case (4%) showed focal CK20 positivity. Both P63 and CK5/6 expression was seen in 9/12 (75%) SQCC cases; none of the ADC cases showed this dual expression. Cytomorphology alone may not be able to stratify all cases of nonsmall cell lung carcinoma into ADC and SQCC in FNA specimens. The immune-panel of TTF-1, CK7, CK20, P63, and CK5/6 is useful in differentiating SQCC from ADC.

Lymphoepithelial cysts of the pancreas: endosonography-guided fine needle aspiration.

Lymphoepithelial cyst (LEC) of the pancreas is a rare lesion. Here, we report three cases that we encountered during 2007 in our institution, which were evaluated by endosonography (EUS) and fine needle aspiration (FNA). All three patients were male with ages ranging from 47 to 77 years. Each patient had a single, cystic mass of the body/tail of the pancreas that was found incidentally on CT scan. The size of the masses ranged from 3 to 6.5 cm radiologically. For each patient, EUS showed a well-circumscribed, lobulated mass with smooth contours; the remainder of the pancreas appeared normal without ductal dilation. FNA of all three masses demonstrated predominantly anucleated squamous cells, amorphous keratinous debris, and lymphocytes. Cholesterol crystals were present in one case. One patient underwent surgical resection and the diagnosis of LEC was confirmed histologically. In conclusion, pancreatic LECs are extremely rare, benign cystic lesions. FNA study may help to render a correct diagnosis and reduce unnecessary surgery.