Chromogenic immunohistochemical quadruplex provides accurate diagnostic differentiation of non-small cell lung cancer.

Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung malignancies, the vast majority of which are non-small cell lung carcinomas (NSCLC). Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or by immunohistochemistry, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung. Each marker is represented by a distinct color that can be read by a pathologist, using standard, bright field microscopy. We evaluated the ability of pathologists to differentiate NSCLCs using the multiplexed assay as compared to standard, single marker per slide diaminobenzidine (DAB)-based IHC. All cases in a cohort of 264 NSCLCs showed concordance of information (including positivity of stain, intensity of stain and coverage) between single IHC stains and the multiplex assay. This new multiplex IHC offers the capability to accurately diagnose and sub-classify primary lung NSCLCs, while conserving precious tissue for additional testing.

Pilot study demonstrating metabolic and anti-proliferative effects of in vivo anti-oxidant supplementation with N-Acetylcysteine in Breast Cancer.

BACKGROUND: High oxidative stress as defined by hydroxyl and peroxyl activity is often found in the stroma of human breast cancers. Oxidative stress induces stromal catabolism, which promotes cancer aggressiveness. Stromal cells exposed to oxidative stress release catabolites such as lactate, which are up-taken by cancer cells to support mitochondrial oxidative phosphorylation. The transfer of catabolites between stromal and cancer cells leads to metabolic heterogeneity between these cells and increased cancer cell proliferation and reduced apoptosis in preclinical models. N-Acetylcysteine (NAC) is an antioxidant that reduces oxidative stress and reverses stromal catabolism and stromal-carcinoma cell metabolic heterogeneity, resulting in reduced proliferation and increased apoptosis of cancer cells in experimental models of breast cancer. The purpose of this clinical trial was to determine if NAC could reduce markers of stromal-cancer metabolic heterogeneity and markers of cancer cell aggressiveness in human breast cancer. METHODS: Subjects with newly diagnosed stage 0 and I breast cancer who were not going to receive neoadjuvant therapy prior to surgical resection were treated with NAC before definitive surgery to assess intra-tumoral metabolic markers. NAC was administered once a week intravenously at a dose of 150 mg/kg and 600 mg twice daily orally on the days not receiving intravenous NAC. Histochemistry for the stromal metabolic markers monocarboxylate transporter 4 (MCT4) and caveolin-1 (CAV1) and the Ki67 proliferation assay and TUNEL apoptosis assay in carcinoma cells were performed in pre- and post-NAC specimens. RESULTS: The range of days on NAC was 14-27 and the mean was 19 days. Post-treatment biopsies showed significant decrease in stromal MCT4 and reduced Ki67 in carcinoma cells. NAC did not significantly change stromal CAV1 and carcinoma TUNEL staining. NAC was well tolerated. CONCLUSIONS: NAC as a single agent reduces MCT4 stromal expression, which is a marker of glycolysis in breast cancer with reduced carcinoma cell proliferation. This study suggests that modulating metabolism in the tumor microenvironment has the potential to impact breast cancer proliferation.

Review of Cytology Practice at Thomas Jefferson University Hospital before and after High-Risk Human Papillomavirus Testing.

OBJECTIVE: We performed a retrospective review of Papanicolaou (Pap) testing to assess whether the cytology practice in our institution was affected by the introduction of high-risk (HR) human papillomavirus (HPV) assays over time. STUDY DESIGN: Cytology, HPV and histopathology records were retrieved from our laboratory information system from 2003 to 2015. Records for Digene Hybrid Capture 2®, Hologic Cervista® and Roche Cobas® HPV assays were obtained. A 3-month follow-up for HPV detected cases was performed, and results were correlated with cytology and biopsies. A 1-year follow-up of HPV 16/18 and other HR HPV detected cases was also performed. RESULTS: From 2008 to 2015, a noticeable decrease in Pap testing volume occurred, from 11,792 to 4,664, while the percentage of HPV testing increased from 19 to 59%. Similar HPV detection rates and follow-up results for both reflex and cotesting were observed in the 3 HPV assays. CONCLUSIONS: The decrease in Pap testing was due to the lengthening of the test interval when cotesting results were negative. Practitioners adhering to guidelines accounts for increased molecular testing volume. A trend towards higher-grade cervical intraepithelial neoplasia in the follow-up of detected HPV 16/18 was noted. So far there has been no demand for HPV as a stand-alone test.

Diagnostic Yield of Endoscopic Ultrasound-Guided Fine-Needle Aspiration Cytology of Porta Hepatis Lesions: A Retrospective Study.

OBJECTIVE: Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has recently been used for the evaluation of various lesions arising in the porta hepatis. The purpose of this study is to evaluate the diagnostic yield of this increasingly utilized approach to porta hepatis lesions. STUDY DESIGN: A retrospective study of 72 consecutive samples of porta hepatis lesions obtained via EUS-FNA between 2004 and 2015 was conducted. Clinical histories and endoscopic findings were available prior to the diagnostic interpretation. The diagnosis of each lesion was based on its cytologic features on smears, its histologic features on cell block, a comparison with any relevant prior specimens, immunohistochemistry and flow cytometric studies when applicable. RESULTS: A total of 72 lesions (59 lymph nodes, 2 cysts and 11 masses) were biopsied in 70 patients. Adequate specimens were obtained in 65/72 cases (90%). Most of the lymph nodes were benign (n = 40, 67%) and most of the masses were malignant or suspicious (n = 8, 73%). A variety of diagnoses, primary and metastatic, were made, including hepatocellular carcinoma, cholangiocarcinoma and lymphoma. In addition, we have noted a significant increase in the number of EUS-FNAs in recent years. CONCLUSION: EUS-FNA is an effective and increasingly utilized diagnostic approach for the evaluation of multiple types of lesions in the porta hepatis.

Parathyroid Hormone-Related Peptide-Linked Hypercalcemia in a Melanoma Patient Treated With Ipilimumab: Hormone Source and Clinical and Metabolic Correlates.

A patient diagnosed with metastatic melanoma developed the paraneoplastic syndrome of humoral hypercalcemia of malignancy and cachexia after receiving ipilumumab. The cause of the hypercalcemia was thought to be secondary to parathyroid hormone-related peptide (PTHrP) as plasma levels were found to be elevated. The patient underwent two tumor biopsies: at diagnosis (when calcium levels were normal) and upon development of hypercalcemia and cachexia. PTHrP expression was higher in melanoma cells when hypercalcemia had occurred than prior to its onset. Metabolic characterization of melanoma cells revealed that, with development of hypercalcemia, there was high expression of monocarboxylate transporter 1 (MCT1), which is the main importer of lactate and ketone bodies into cells. MCT1 is associated with high mitochondrial metabolism. Beta-galactosidase (ß-GAL), a marker of senescence, had reduced expression in melanoma cells upon development of hypercalcemia compared to pre-hypercalcemia. In conclusion, PTHrP expression in melanoma is associated with cachexia, increased cancer cell lactate and ketone body import, high mitochondrial metabolism, and reduced senescence. Further studies are required to determine if PTHrP regulates cachexia, lactate and ketone body import, mitochondrial metabolism, and senescence in cancer cells.

Arginase-1 and HepPar-1 expression in fine-needle aspiration specimens of primary lung adenocarcinoma.

INTRODUCTION: Arginase-1 is a novel immunohistochemical (IHC) marker for hepatocellular differentiation. The purpose of this study was to evaluate the expression of Arginase-1 and HepPar-1 in lung adenocarcinoma to assess the potential value of these markers for diagnosing metastatic lung tumors, especially to the liver in fine-needle aspiration specimens. MATERIALS AND METHODS: Forty-four cytology specimens of lung adenocarcinoma, obtained by endobronchial ultrasound-guided fine-needle aspiration were retrospectively reviewed. IHC stains for Arginase-1 and HepPar-1 were performed on formalin-fixed paraffin-embedded cell blocks. Tissue from confirmed hepatocellular carcinoma was used as the positive control. TTF-1 IHC stain was performed in all cases. RESULTS: All 44 lung adenocarcinoma cases (100%) were negative for Arginase-1, whereas HepPar-1 expression was detected in 3 (7%) of lung adenocarcinomas and negative in 41 (93%). The 3 HepPar-1-positive lung adenocarcinoma cases demonstrated positive TTF-1 IHC stain performed on the same cell block. Although both Arginase-1 and HepPar-1 are useful diagnostic IHC markers to differentiate metastatic lung adenocarcinoma from hepatocellular carcinoma, Arginase-1 IHC stain shows better specificity than HepPar-1 does (Arginase-1 specificity 100% and HepPar-1 specificity 93%). CONCLUSIONS: Arginase-1 IHC can be used in combination with other markers in the workup of metastatic lung adenocarcinoma, especially to the liver.

Overview of nongynecological samples prepared with liquid-based cytology medium.

OBJECTIVE: Liquid-based cytology of nongynecological specimens is commonly used in cytology laboratories throughout the world and various processing methods, such as ThinPrep and SurePath, have been reported. The cytological features and performance of liquid-based cytology for various cytology specimens, including body cavity fluids, urine, brushing specimens and fine-needle aspiration of various lesions, were reviewed and compared with the experience of our laboratory and the literature published in PubMed. STUDY DESIGN: The parameters for the evaluation of liquid-based cytology and conventional smears were described in the various types of specimens. Criteria for the interpretation of nongynecological liquid-based cytology were highlighted to show differences in cell morphology, background and artifacts. RESULTS: The interpretation requires familiarity with the appearance of liquid-based cytology in the various types of preparations to avoid misdiagnosis. CONCLUSIONS: Cell blocks can be prepared with specimens preserved in a liquid-based cytology medium and immunocytochemical stains and molecular testing can be successfully performed. These are important adjuncts in order to reach a definitive diagnosis.

USP22 regulates oncogenic signaling pathways to drive lethal cancer progression.

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease.

Thyroglobulin wash testing in the surveillance of patients with thyroid carcinoma: proposal for a reflex test.

OBJECTIVE: Fine needle aspiration (FNA) cytology with thyroglobulin wash (TG-W) testing is recommended for follow-up of patients with differentiated thyroid carcinoma (DTC). The goal of this retrospective study was to determine if TG-W results contributed to the management of cases with positive FNA cytology. STUDY DESIGN: We reviewed data on patients with positive and suspicious cytology results, undergoing lymph node or thyroid bed FNA with TG-W testing as part of the preoperative or follow-up investigation of histologically proven DTC in our institution and from the literature. RESULTS: Of 30 positive/suspicious lymph node and thyroid bed FNAs in our institution, 22 (73%) had an elevated (>1 ng/ml) TG-W level. Seven of 8 TG-W-negative cases had DTC on follow-up. Of 577 cytology-positive/suspicious FNAs in the literature, 557 (97%) showed TG-W-positive results. Fourteen of 20 TG-W-negative cases had DTC on follow-up. All patients in retrospective and literature review groups with positive and suspicious FNA cytology and available follow-up were treated for recurrent or metastatic disease regardless of TG-W results. CONCLUSION: Observations of both our and other institutions support a recommendation of reflex FNA TG-W testing only for cases with negative or indeterminate cytology results.

The potential value of phosphohistone-h3 mitotic index determined by digital image analysis in the assessment of pancreatic endocrine tumors in fine-needle aspiration cytology specimens.

OBJECTIVE: According to the World Health Organization, pancreatic endocrine tumors are graded by assessment of the Ki67 proliferation index and/or mitotic count. The objective was to find comparable grading on the basis of the novel mitotic marker phosphohistone-H3 (PHH3). STUDY DESIGN: A computer-assisted system was used to assess 23 cell blocks stained with PHH3 and Ki67 antibodies. We investigated possible cut-points for PHH3 and computed percent agreement between the PHH3- and Ki67-based grading. RESULTS: The Spearman correlation between percent Ki67 positive and percent PHH3 positive was 0.76 (p = 0.001). A value of 0.3% for the lower cut-point ('cut-point 1', differentiating between grades 1 and 2) and values of about 1.8-1.9% for the higher cut-point ('cut-point 2', differentiating between grades 2 and 3) shows optimal agreement between PHH3 and Ki67 grading. The percentage of positive cells was much higher for Ki67 than for PHH3 (mean 10.6 vs. 3.0%). CONCLUSIONS: PHH3 has good correlation with Ki67, but the range of PHH3 positivity is much narrower than that of Ki67 (range 0-4% for PHH3 vs. 0-50% for Ki67). Therefore, to be as accurate, grading on the basis of PHH3 requires evaluation of a larger number of tumor cells for a precise determination of percent PHH3-positive nuclei.

Relationship of pancreatic mass size and diagnostic yield of endoscopic ultrasound-guided fine needle aspiration.

BACKGROUND: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is central to discerning the diagnosis of solid pancreatic tumors through tissue acquisition. Test performance is affected by a number of factors including location of mass within the pancreas, presence of onsite cytology technologist, and number of passes with the needle. The influence of tumor size has not been well studied. AIM: The objective of the current study was to determine whether the size of mass affects the diagnostic accuracy for solid pancreatic lesions aspirated under EUS guidance. METHODS: Data were collected retrospectively on all patients with solid pancreatic masses undergoing EUS-FNA from June 2003 to August 2010. The cytology samples were reported as positive, suspicious for malignancy, atypical, negative, or nondiagnostic. The gold standard for a cytological diagnosis was histological confirmation or clinical follow-up of more than 6 months with repeat imaging. Patients were divided into five groups based upon lesion size as follows: (a) less than 1 cm, (b) 1-2 cm, (c) 2-3 cm, (d) 3-4 cm, and (e) greater than 4 cm. Performance characteristics of EUS-FNA including sensitivity, specificity, and accuracy were compared for each group. Accuracy was defined as the ratio of the sum of true-positive and true-negative values divided by the number of lesions. RESULTS: We identified 583 patients with solid pancreatic lesions in which EUS-FNA was performed and adequate cellularity was obtained (47% men, mean age 65 ± 1.4 (SE) years). Overall, 486 (83%) of lesions were pancreatic adenocarcinoma, 18 (3%) were neuroendocrine tumors, 12 (2%) were lymphomas, and 67 (12%) were benign lesions. The median size of the mass was 3 cm (range, 0.5-7 cm). A mean of 4.9 passes (range, 1-9 passes) was needed to obtain adequate samples from lesions. The overall yield of obtaining adequate samples for diagnosis was 85%. When stratified by size, the EUS-FNA sensitivity for lesions with size <1, 1-2, 2-3, 3-4, and >4 cm was 40, 75.9, 86.9, 93.2, and 91.6%, respectively; EUS-FNA sensitivity strongly correlate with tumor size (p < 0.001). Similarly, the accuracy of EUS-FNA increased as lesion size increased, ranging from 47% for tumors less than 1 cm to 88% for tumors greater than 4 cm (p < 0.05). Location of tumor and number of needle passes did not significantly influence EUS-FNA performance characteristics. CONCLUSIONS: The sensitivity and diagnostic accuracy of EUS-FNA for solid pancreatic lesions is strongly correlated with tumor size. Sensitivity and accuracy decrease significantly for tumors that are smaller than 1 cm.

Large cell neuroendocrine carcinoma of the ovary associated with serous carcinoma with mucin production: a case report and literature review.

Large cell neuroendocrine carcinoma (LCNEC) of the ovary is a rare entity and is frequently associated with ovarian surface epithelial tumors. However, its association with serous carcinoma has only been described recently in one case. We report another case of such with mucin production in a 68-year old woman. Immunohistochemistry and mucicarmine stain confirmed the diagnosis. Its clinicopathologic association is discussed and the literature is reviewed.