Suppressing the activation of protein kinase A as a DNA damage-independent mechanistic lead for dihydromethysticin prophylaxis of NNK-induced lung carcinogenesis.

Our earlier work demonstrated varying potency of dihydromethysticin (DHM) as the active kava phytochemical for prophylaxis of tobacco carcinogen nicotine-derived nitrosamine ketone (NNK)-induced mouse lung carcinogenesis. Efficacy was dependent on timing of DHM gavage ahead of NNK insult. In addition to DNA adducts in the lung tissues mitigated by DHM in a time-dependent manner, our in vivo data strongly implicated the existence of DNA damage-independent mechanism(s) in NNK-induced lung carcinogenesis targeted by DHM to fully exert its anti-initiation efficacy. In the present work, RNA seq transcriptomic profiling of NNK-exposed (2 h) lung tissues with/without a DHM (8 h) pretreatment revealed a snap shot of canonical acute phase tissue damage and stress response signaling pathways as well as an activation of protein kinase A (PKA) pathway induced by NNK and the restraining effects of DHM. The activation of the PKA pathway by NNK active metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) at a concentration incapable of promoting DNA adduct was confirmed in a lung cancer cell culture model, potentially through NNAL binding to and activation of the ß-adrenergic receptor. Our in vitro and in vivo data overall support the hypothesis that DHM suppresses PKA activation as a key DNA damage-independent mechanistic lead, contributing to its effective prophylaxis of NNK-induced lung carcinogenesis. Systems biology approaches with a detailed temporal dissection of timing of DHM intake versus NNK exposure are warranted to fill the knowledge gaps concerning the DNA damage-driven mechanisms and DNA damage-independent mechanisms to optimize the implementation strategy for DHM to achieve maximal lung cancer chemoprevention.

Characterization of adductomic totality of NNK, (R)-NNAL and (S)-NNAL in A/J mice, and their correlations with distinct lung carcinogenicity.

Lung cancer is the leading cause of cancer-related deaths. While tobacco use is the main cause, only 10-20% of smokers eventually develop clinical lung cancer. Thus, the ability of lung cancer risk prediction among smokers could transform lung cancer management with early preventive interventions. Given that DNA damage by tobacco carcinogens is the potential root cause of lung carcinogenesis, we characterized the adductomic totality of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a potent lung carcinogen in tobacco, commonly known as NNK) in the target lung tissues, the liver tissues and the peripheral serum samples in a single-dose NNK-induced lung carcinogenesis A/J mouse model. We also characterized these adductomic totalities from the two enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major in vivo metabolite of NNK) given their distinct carcinogenicity in A/J mice. With these adductomic data, we demonstrated that tissue protein adductomics have the highest abundance. We also identified that the adductomic levels at the 8 h time point after carcinogen exposure were among the highest. More importantly, the relationships among these adductomics were characterized with overall strong positive linear correlations, demonstrating the potential of using peripheral serum protein adductomics to reflect DNA adductomics in the target lung tissues. Lastly, we explored the relationships of these adductomics with lung tumor status in A/J mice, providing preliminary but promising evidence of the feasibility of lung cancer risk prediction using peripheral adductomic profiling.

Cytological appearance of pancreatic cystosis on fine-needle aspiration.

A 22-year-old Caucasian male with cystic fibrosis and recently diagnosed insulin-dependent diabetes mellitus underwent magnetic resonance imaging (MRI) and was found to have multiple cystic lesions in the pancreas. Endoscopic ultrasound evaluation revealed multiple macro- and microcystic components without mural nodules. One of the cysts in the body of the pancreas was in clear direct communication with the nondilated main pancreatic duct. Fine-needle aspiration (FNA) of two cysts was performed and showed foamy macrophages and rare ductal as well as acinar cells. Cell blocks showed nonpolarizable pink crystalloid material and small nonlaminated concretions consistent with inspissated secretions. Special stains for chymotrypsin and trypsin highlighted the acinar cells. Periodic acid Schiff, with and without diastase, was negative. Biopsy of the cyst wall showed ductal epithelial cells with underlying fibrotic stroma. This is the first description of the FNA appearance of pancreatic cystosis. We discuss the cytological differential diagnosis of cystic lesions of the pancreas and the biochemical as well as imaging findings used to arrive at the diagnosis.

Validation of immunohistochemical tests performed on cytology cell block material: Practical application of the College of American Pathologists' guidelines.

The advent of fiberoptic endoscopy with biopsy has revolutionized procurement of specimens from deep sites. This has translated into more cytologic specimens whereby the material is limited and best handled by cytology laboratory staff. While the diagnosis of the pathologic process is of utmost importance, there is increasing expectation that the diagnosis be specific and accurate as not to require additional biopsy for initiation of treatment. This expectation has driven demand in immunohistochemical (IHC) and molecular studies conducted specifically on material processed as cytology specimens. The Clinical Laboratory Improvement Amendments of 1988 requires laboratories in the United States of America to verify the performance of patient tests. Due to varying laboratory practices with respect to validation of IHC assays, the College of American Pathologists introduced guidelines for analytic validation of IHC tests. These guidelines address how to perform validation by recommending the number of cases in the validation set, comparator concordance, and when to revalidate. The main thrust of the guidelines is based on formalin-fixed paraffin-embedded tissue with only one expert consensus opinion referring to validation of IHC tests on cytology specimens which delegates to the medical director, the determination of number of positive and negative cases to be tested. This article will outline how an academic center approaches validation of IHC studies performed on cytology cell block specimens using the College of American Pathologists guidelines. A stepwise approach from selection of antibodies to validate followed by building the validation panel and evaluating the stain results for concordance against the gold standard of histology tissue specimen will be described. A rationale for dealing with discordant results and future innovations will conclude the report.

The diagnostic and cellularity yield of reverse bevel versus fork-tip fine needle biopsy.

INTRODUCTION: Two new systems with a novel tip (Procore™ and SharkCore™) have been introduced for ultrasound-guided fine needle biopsy (US-FNB). Direct comparison of the diagnostic yield of these needles in the evaluation of pancreatic lesions is currently under investigation. This study aims to compare the diagnostic and cellular yields of the two needle systems. METHODS: Consecutive patients with upper gastrointestinal lesions undergoing EUS-FNB using 22 gauge Procore™ (reverse bevel) or SharkCore™ (fork-tip) needles were included in the study. Cytological rapid on-site evaluation (ROSE) slides were scored on a numerical scale of diagnostic yield relative to the number of passes. Similarly, histology of biopsy material was assessed on diagnostic quality using a numeric score. The final diagnosis was based on resection specimens and/or follow-up of clinical and imaging data of the subject. RESULTS: The diagnostic yield was similar between the fork-tip and reverse bevel needles (125/163; 77% vs 103/139;74% with P = .60). Sub-analysis for solid pancreatic masses demonstrated similar results (69/88; 78% vs. 83/107; 78% with P = .88). The fork-tip needle had a lower mean number of passes (2.5 vs 3.1; P = .04) and ROSE was utilized in significantly less cases than in the reverse bevel needle group (77% versus 98.3%). CONCLUSIONS: Although we observed no difference in the diagnostic yield using either the fork-tip or the reverse bevel needle, the fork-tip needle had significantly better performance with regards to achieving more adequate cytologic specimen in fewer number of passes while at the same time requiring fewer episodes of ROSE.

The First Reported Case of Recurrent Carcinoid Tumor in the External Auditory Canal.

OBJECTIVE: Cutaneous neuroendocrine lesions of the external auditory canal (EAC) are exceptionally rare, with only five cases reported in the literature. In this case report, we present a patient with a recurrent carcinoid tumor in the EAC, which has yet to be described. PATIENTS: A 38-year-old woman presenting with otalgia, aural fullness, and decreased hearing was found to have a recurrent EAC carcinoid tumor, 8-years after initial resection at an outside facility. INTERVENTIONS: The recurrent tumor involved much of the proximal, anterior bony ear canal and was trans-tympanic, extending to the middle ear and epitympanum; therefore, a lateral temporal bone resection was performed to ensure complete resection. MAIN OUTCOME MEASURES: Surgical pathology confirmed the presence a recurrent carcinoid tumor in the EAC, with immunohistochemistry positive for pancytokeratin (MAK6), CD56, and synaptophysin, with chromogranin showing rare cells positive for cytoplasmic granules. There was no evidence of metastasis. RESULTS: Lateral temporal bone resection was successful and the patient is being followed with annual imaging. The patient is considering future hearing rehabilitation with a bone anchored hearing device. CONCLUSION: This case report highlights the first known case of recurrent carcinoid tumor in the EAC, treated with lateral temporal bone resection. Clinical presentation, imaging, treatment, and pathology are reviewed along with a review of the literature.

Cytologic diagnosis of coccidioidomycosis: Spectrum of findings in Southern Arizona patients over a 10 year period.

BACKGROUND: The largest series examining the cytological diagnosis of coccidioidomycosis was reported more than 20 years ago and only considered fine needle aspiration (FNA) specimens from pulmonary nodules. Since then, there has been a substantial increase in the incidence of the disease in endemic regions. The aims of this study were to examine the spectrum of Coccidioides in all cytologic specimens and detail their diagnostic and clinical features. The prevalence of infection is also examined against temperature and precipitation data. METHODS: The Department of Pathology database was retrospectively searched for cases diagnosed as coccidioidomycosis. Climate report was obtained from National Weather Service Forecast Office. Statistical analysis was carried out using JMP version 11.2.0. RESULTS: The presence of Coccidiodes was microscopically diverse with immature spherules, variably sized spherules, endosporulating spherules, empty spherules, and ruptured spherules being observed. Bronchoalveolar lavages demonstrated the greatest number of different forms of Coccidioides and were associated with acute inflammatory response. Granulomas were rare and mostly seen in lymph nodes and extra pulmonary sites. A necrotic background with paucity of inflammation was frequently seen in lung FNA. Immune competent patients outnumbered immunocompromised patients, illustrating the importance of considering the diagnosis in all patients. The reported association between rainfall and peak prevalence was not observed in our study. CONCLUSION: Coccidioides has diverse cytological appearance and varied host response depending on the site and type of preparation examined. In endemic areas, the disease should be considered in all patients, independent of immune status, and throughout the year.

Nuclear expression and gain-of-function ß-catenin mutation in glomangiopericytoma (sinonasal-type hemangiopericytoma): insight into pathogenesis and a diagnostic marker.

Glomangiopericytoma (sinonasal-type hemangiopericytoma) is a rare mesenchymal neoplasm with myoid phenotype (smooth muscle actin-positive), which distinguishes this tumor from soft tissue hemangiopericytoma/solitary fibrous tumor. Molecular genetic changes underlying the pathogenesis of glomangiopericytoma are not known. In this study, 13 well-characterized glomangiopericytomas were immunohistochemically evaluated for ß-catenin expression. All analyzed tumors showed strong expression and nuclear accumulation of ß-catenin. Following this observation, ß-catenin glycogen serine kinase-3 beta phosphorylation region, encoded by exon 3, was PCR amplified in all cases and evaluated for mutations using Sanger sequencing. Heterozygous mutations were identified in 12 of 13 tumors. All mutations consisted of single-nucleotide substitutions: three in codon 32 (c.94G>C (n=2) and c.95A>T), four in codon 33 (two each c.98C>G and c.98C>T), two in codon 37 (c.109T>G), one in codon 41 (c.121A>G), and two in codon 45 (c.133T>C). At the protein level, these substitutions would lead to p.D32H, p.D32V, p.S33C, p.S33F, p.S37A, p.T41A, and p.S45L mutations, respectively. Previously, similar mutations have been reported in different types of cancers and shown to trigger activation of ß-catenin signaling. All analyzed glomangiopericytomas showed prominent nuclear expression of cyclin D1, as previously shown for tumors with nuclear expression of ß-catenin as a sign of oncogenic activation. These results demonstrate that mutational activation of ß-catenin and associated cyclin D1 overexpression may be central events in the pathogenesis of glomangiopericytoma. In additon, nuclear accumulation of ß-catenin is a diagnostic marker for glomangiopericytoma.

Low-grade sinonasal sarcoma with neural and myogenic features--diagnostic challenge and pathogenic insight.

A low-grade sinonasal sarcoma with neural and myogenic features has recently been defined and characterized. We present a case of this morphologic entity and discuss the differential diagnostic considerations, immunophenotypic character, electron microscopy (EM) findings and positron emission tomography (PET) appearance. We propose an alternative hypothesis of its origin on the basis of immunophenotypic and EM features. A 59-year-old Caucasian male was found to have a mass filling the right ethmoid sinus. On PET, the lesion had a maximum standardized uptake value of 2.9, which is of borderline intensity for sarcoma. Pathologic examination showed morphologic features similar to those reported for low-grade sinonasal sarcoma with neural and myogenic features. The tumor was positive for S100, ß-catenin, caldesmon, and vimentin and negative for smooth muscle actin, muscle-specific actin, desmin, myogenin, and pankeratin. Positivity for ß-catenin raises the alternative possibility of fibroblastic differentiation instead of the proposed myogenic differentiation. EM findings were also consistent with fibroblastic cells.

Utility of molecular tests in cytopathology.

With the popularity of interventional radiology, diagnostic material obtained can be limited requiring critical decisions on making the best use of it. Molecular testing using nanogram amounts of tissue can add useful diagnostic information by improving sensitivity and/or specificity of the diagnosis. This review examines the use of molecular tests in cervical cytology, "indeterminate" thyroid cytology specimens, pancreatic cyst fluid, urinary tract and pulmonary adenocarcinoma cytologic material. Molecular human papillomavirus (HPV) testing combined with cervical cytology increases sensitivity of detection of high grade lesions. In cytologically negative cases, the HPV negative predictive value endorses longer screening intervals. With the high prevalence of benign thyroid nodules, cytology plays a vital role in screening. However, 10-40% of the specimens obtained are cytologically indeterminate. Molecular analysis of these specimens can predict the malignant risk in these cases. Increased detection of pancreatic cysts has necessitated accurate pre-operative diagnosis delineating non-mucinous from mucinous cysts, which have a potential for progression to adenocarcinoma. Multimodal diagnosis of pancreatic cysts and molecular analysis help to clarify neoplastic risk; and in cases of limited fluid, may be the only available diagnostic information. Urothelial carcinoma (UC) of the bladder, a common cancer with frequent recurrences, requires lifelong surveillance. The UroVysion ™ test kit can increase the sensitivity of detection of UC especially in cases of residual/recurrent carcinoma after therapy. Subsets of lung adenocarcinomas are now commonly targeted by therapies based on molecular mutation results of epidermal growth factor receptor, KRAS or echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase re-arrangements. The move toward standardization of reporting of cytology specimens commencing with cervical smears and more recently, thyroid cytology specimens is also changing the practice of cytopathology. Combining the stringent cytology criteria with ancillary molecular testing is expected to yield more discrete and diagnostic categories for research and reporting. The profession is at an exciting point of implementing novel molecular markers to refine diagnostic criteria and create clinically relevant classification systems.