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.

Performance of Molecular Lymphosonography for Detection and Quantification of Metastatic Involvement in Sentinel Lymph Nodes.

OBJECTIVES: To assess the performance of molecular lymphosonography with dual-targeted microbubbles in detecting and quantifying the metastatic involvement in sentinel lymph nodes (SLNs) using a swine melanoma model. METHODS: Targeted microbubbles were labeled with P-selectin and αV ß3 -integrin antibodies. Control microbubbles were labeled with immunoglobulin G antibodies. First lymphosonography with Sonazoid (GE Healthcare, Oslo, Norway) was used to identify SLNs. Then dual-targeted and control microbubbles were injected intravenously to detect and quantify metastatic disease in the SLNs. Distant non-SLNs were imaged as benign controls. All evaluated lymph nodes (LNs) were surgically removed, and metastatic involvement was characterized by a histopathologic analysis. Two radiologists blinded to histopathologic results assessed the baseline B-mode images of LNs, and the results were compared to the histologic reference standard. The mean intensities of targeted and control microbubbles within the examined LNs were measured and compared to the LN histologic results. RESULTS: Thirty-five SLNs and 34 non-SLNs from 13 Sinclair swine were included in this study. Twenty-one SLNs (62%) were malignant, whereas 100% of non-SLNs were benign. The sensitivity of B-mode imaging for metastatic LN diagnosis for both readers was relatively high (90% and 71%), but the specificity was very poor (50% and 58%). The sensitivity and specificity of molecular lymphosonography for metastatic LN detection were 91% and 67%, respectively. The mean intensities from dual-targeted microbubbles correlated well with the degree of metastatic LN involvement (r = 0.6; P < 0.001). CONCLUSIONS: Molecular lymphosonography can increase the specificity of metastatic LN detection and provide a measure to quantify the degree of metastatic involvement.

Contiguous verrucous proliferations in syringocystadenoma papilliferum: A retrospective analysis with additional evaluation via mutation-specific BRAFV600E immunohistochemistry.

BACKGROUND: Syringocystadenoma papilliferum (SCAP) is an uncommon cutaneous adnexal proliferation. There have been several reports describing collision lesions of SCAP and verruca, although little is known about the frequency of this association. Molecular testing has revealed the BRAFV600E mutation in a large proportion of SCAP cases, although its expression pattern has not been previously evaluated. METHODS: In this retrospective analysis, we explored the potential histopathological association between verruca and SCAP. We also evaluated mutation-specific BRAFV600E expression in these lesions by immunohistochemistry. Cases of SCAP diagnosed over a 7-year period were closely reviewed for the presence of contiguous verrucous proliferations. Additional sections were cut and stained using the BRAFV600E-specific clone VE1 antibody. RESULTS: Contiguous verrucous proliferations were identified in 9 out of 12 identified cases. Furthermore, expression of the BRAFV600E mutation was identified in 7 out of 12 cases. Interestingly, in SCAP associated with endophytic verrucous proliferations (n = 4), expression of BRAFV600E was found in both the glandular and the contiguous hyperplastic squamous epithelium. CONCLUSION: Overall, these findings suggest that contiguous verrucous proliferations in SCAP are common. Both components of the neoplasm may express the BRAFV600E mutation, which is suggestive of a common origin.

Space radiation-associated lung injury in a murine model.

Despite considerable progress in identifying health risks to crewmembers related to exposure to galactic/cosmic rays and solar particle events (SPE) during space travel, its long-term effects on the pulmonary system are unknown. We used a murine risk projection model to investigate the impact of exposure to space-relevant radiation (SR) on the lung. C3H mice were exposed to (137)Cs gamma rays, protons (acute, low-dose exposure mimicking the 1972 SPE), 600 MeV/u (56)Fe ions, or 350 MeV/u (28)Si ions at the NASA Space Radiation Laboratory at Brookhaven National Laboratory. Animals were irradiated at the age of 2.5 mo and evaluated 23.5 mo postirradiation, at 26 mo of age. Compared with age-matched nonirradiated mice, SR exposures led to significant air space enlargement and dose-dependent decreased systemic oxygenation levels. These were associated with late mild lung inflammation and prominent cellular injury, with significant oxidative stress and apoptosis (caspase-3 activation) in the lung parenchyma. SR, especially high-energy (56)Fe or (28)Si ions markedly decreased sphingosine-1-phosphate levels and Akt- and p38 MAPK phosphorylation, depleted anti-senescence sirtuin-1 and increased biochemical markers of autophagy. Exposure to SR caused dose-dependent, pronounced late lung pathological sequelae consistent with alveolar simplification and cellular signaling of increased injury and decreased repair. The associated systemic hypoxemia suggested that this previously uncharacterized space radiation-associated lung injury was functionally significant, indicating that further studies are needed to define the risk and to develop appropriate lung-protective countermeasures for manned deep space missions.

MicroRNA-663 regulates human vascular smooth muscle cell phenotypic switch and vascular neointimal formation.

RATIONALE: Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. MicroRNAs (miRNAs) have emerged as important regulators for VSMC function, and we recently identified miR-663 as critical for controlling human aortic smooth muscle cell proliferation. OBJECTIVE: To investigate whether miR-663 plays a role in human VSMC phenotypic switch and the development of neointima formation. METHODS AND RESULTS: By using quantitative reverse-transcription polymerase chain reaction, we found that miR-663 was significantly downregulated in human aortic VSMCs on platelet-derived growth factor treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that overexpression of miR-663 increased expression of VSMC differentiation marker genes, such as smooth muscle 22α, smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited platelet-derived growth factor-induced VSMC proliferation and migration. We identified the transcription factor JunB and myosin light chain 9 as downstream targets of miR-663 in human VSMCs, because overexpression of miR-663 markedly inhibited expression of JunB and its downstream molecules, such as myosin light chain 9 and matrix metalloproteinase 9. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by ≈50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. CONCLUSIONS: These results indicate that miR-663 is a novel modulator of human VSMC phenotypic switch by targeting JunB/myosin light chain 9 expression. These findings suggest that targeting miR-663 or its specific downstream targets in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

Comparison of Chromogenic In Situ Hybridization and Fluorescence In Situ Hybridization for the Evaluation of MDM2 Amplification in Adipocytic Tumors.

BACKGROUND: Atypical lipomatous tumor/well-differentiated liposarcoma (ALT-WDLPS) and dedifferentiated liposarcoma (DDLPS) are characterized cytogenetically by a 12q13-15 amplification involving the mouse double minute 2 (MDM2) oncogene. Fluorescence in situ hybridization (FISH) is used frequently to detect this amplification and aid with the diagnosis of these entities, which is difficult by morphology alone. Recently, bright-field in situ hybridization techniques such as chromogenic in situ hybridization (CISH) have been introduced for the determination of MDM2 amplification status. METHODS: The present study compared the results of FISH and CISH for detecting MDM2 amplification in 41 cases of adipocytic tumors. Amplification was defined in both techniques as a MDM2/CEN12 ratio of 2 or greater. RESULTS: Eleven cases showed amplification with both FISH and CISH, and 26 cases showed no amplification with both methods. Two cases had discordant results between CISH and FISH, and two cases were not interpretable by CISH. CONCLUSION: CISH is advantageous for allowing pathologists to evaluate the histologic and molecular alterations occurring simultaneously in a specimen. Moreover, CISH is found to be more cost- and time-efficient when used with automation, and the signals do not quench over time. CISH technique is a reliable alternative to FISH in the evaluation of adipocytic tumors for MDM2 amplification.

Radiation mitigating properties of the lignan component in flaxseed.

BACKGROUND: Wholegrain flaxseed (FS), and its lignan component (FLC) consisting mainly of secoisolariciresinol diglucoside (SDG), have potent lung radioprotective properties while not abrogating the efficacy of radiotherapy. However, while the whole grain was recently shown to also have potent mitigating properties in a thoracic radiation pneumonopathy model, the bioactive component in the grain responsible for the mitigation of lung damage was never identified. Lungs may be exposed to radiation therapeutically for thoracic malignancies or incidentally following detonation of a radiological dispersion device. This could potentially lead to pulmonary inflammation, oxidative tissue injury, and fibrosis. This study aimed to evaluate the radiation mitigating effects of FLC in a mouse model of radiation pneumonopathy. METHODS: We evaluated FLC-supplemented diets containing SDG lignan levels comparable to those in 10% and 20% whole grain diets. 10% or 20% FLC diets as compared to an isocaloric control diet (0% FLC) were given to mice (C57/BL6) (n=15-30 mice/group) at 24, 48, or 72-hours after single-dose (13.5 Gy) thoracic x-ray treatment (XRT). Mice were evaluated 4 months post-XRT for blood oxygenation, lung inflammation, fibrosis, cytokine and oxidative damage levels, and survival. RESULTS: FLC significantly mitigated radiation-related animal death. Specifically, mice fed 0% FLC demonstrated 36.7% survival 4 months post-XRT compared to 60-73.3% survival in mice fed 10%-20% FLC initiated 24-72 hours post-XRT. FLC also mitigated radiation-induced lung fibrosis whereby 10% FLC initiated 24-hours post-XRT significantly decreased fibrosis as compared to mice fed control diet while the corresponding TGF-beta1 levels detected immunohistochemically were also decreased. Additionally, 10-20% FLC initiated at any time point post radiation exposure, mitigated radiation-induced lung injury evidenced by decreased bronchoalveolar lavage (BAL) protein and inflammatory cytokine/chemokine release at 16 weeks post-XRT. Importantly, neutrophilic and overall inflammatory cell infiltrate in airways and levels of nitrotyrosine and malondialdehyde (protein and lipid oxidation, respectively) were also mitigated by the lignan diet. CONCLUSIONS: Dietary FLC given early post-XRT mitigated radiation effects by decreasing inflammation, lung injury and eventual fibrosis while improving survival. FLC may be a useful agent, mitigating adverse effects of radiation in individuals exposed to incidental radiation, inhaled radioisotopes or even after the initiation of radiation therapy to treat malignancy.

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.

Decorin antagonizes IGF receptor I (IGF-IR) function by interfering with IGF-IR activity and attenuating downstream signaling.

We have recently discovered that the insulin-like growth factor receptor I (IGF-IR) is up-regulated in human invasive bladder cancer and promotes migration and invasion of transformed urothelial cells. The proteoglycan decorin, a key component of the tumor stroma, can positively regulate the IGF-IR system in normal cells. However, there are no available data on the role of decorin in modulating IGF-IR activity in transformed cells or in tumor models. Here we show that the expression of decorin inversely correlated with IGF-IR expression in low and high grade bladder cancers (n = 20 each). Decorin bound with high affinity IGF-IR and IGF-I at distinct sites and negatively regulated IGF-IR activity in urothelial cancer cells. Nanomolar concentrations of decorin promoted down-regulation of IRS-1, one of the critical proteins of the IGF-IR pathway, and attenuated IGF-I-dependent activation of Akt and MAPK. This led to decorin-evoked inhibition of migration and invasion upon IGF-I stimulation. Notably, decorin did not cause down-regulation of the IGF-IR in bladder, breast, and squamous carcinoma cells. This indicates that decorin action on the IGF-IR differs from its known activity on other receptor tyrosine kinases such as the EGF receptor and Met. Our results provide a novel mechanism for decorin in negatively modulating both IGF-I and its receptor. Thus, decorin loss may contribute to increased IGF-IR activity in the progression of bladder cancer and perhaps other forms of cancer where IGF-IR plays a role.

Assessment of fine needle aspiration specimen adequacy for high-risk HPV detection and genotyping in oropharyngeal squamous cell carcinoma.

OBJECTIVE: The aim of this study was to determine the adequacy of archived and fresh fine needle aspiration (FNA) specimens from metastatic head and neck squamous cell carcinoma (SCC) for the molecular detection and genotyping of high-risk (HR) HPV. STUDY DESIGN: Thirty-seven specimens from 26 patients diagnosed by FNA with metastatic SCC were included as retrospective specimens [19 slides stained with Papanicolaou (Pap) and 18 with Diff-Quik® (DQ)]. Twenty fresh FNA specimens from 18 patients were included as prospective specimens. These specimens were analyzed using the standard protocol for ThinPrep® cervical specimens, with a Cervista HR HPV detection kit. The positive specimens were tested for the HPV 16 and 18 genotypes. RESULTS: Forty-four of 57 specimens (77%) had sufficient cells to yield a valid HPV result. The adequacy rate for Pap-stained slides was 15/19 (79%), for DQ-stained slides it was 13/18 (72%), and for fresh needle aspirates it was 16/20 (80%). HR HPV was detected in 23/44 (52%) specimens. Among the 23 HPV-positive specimens, 19 were genotyped as HPV 16 and 1 as HPV 18. CONCLUSIONS: HR HPV detection and genotyping can be performed on FNA specimens of head and neck SCC prospectively collected in PreservCyt as well as on archival slides with either Pap or DQ stain.

Does mitosis-specific marker phosphohistone H3 help the grading of upper tract urothelial carcinomas in cell blocks?

BACKGROUND: Grading upper tract urothelial carcinomas (UTUC) in cell blocks with small distorted tissue fragments can be challenging; interobserver agreement is poor among pathologists. Mitotic figure (MF) counting along with nuclear features is important in grading these tumors. We evaluated the use of the mitotic-specific marker phosphohistone H3 (PHH3) as an adjunct to hematoxylin and eosin (H&E) stain for grading UTUC in cell blocks. METHODS: Formalin-fixed, paraffin-embedded tissues from the cell blocks of 61 UTUC were stained with H&E and PHH3 antibody. The grading of tumors was performed independently by 3 pathologists, on both H&E-stained and PHH3 plus H&E-stained slides. The grading system used was the 1973 WHO 3-point grading system. Gradings were compared by all the pathologists for H&E staining versus PHH3 plus H&E staining with the Stuart-Maxwell test of marginal homogeneity that accounts for the matched data. RESULTS: The average pairwise agreement by H&E alone was 55%, and 80% by PHH3 plus H&E. CONCLUSION: By adding PHH3 immunostain to the H&E, the agreement in grading the carcinomas among the 3 pathologists improved dramatically. PHH3 immunostain may play an important role in grading UTUC in small cell block samples.

MicroRNA profiling in lung cancer reveals new molecular markers for diagnosis.

OBJECTIVE: To identify new molecular diagnostic markers for non-small cell lung carcinoma (NSCLC) by analyzing microRNA (miRNA) expression profile differences in samples from NSCLC patients and adults with nonneoplastic diseases. STUDY DESIGN: miRNA expression was studied in archival formalin-fixed, paraffin-embedded tissues by microarray and confirmed by real-time PCR analysis of NSCLC and normal lung tissues. An algorithm for discriminating normal, squamous cell carcinoma (SQCC), and adenocarcinoma (ADC) tissue was derived from miRNA expression studies and applied towards characterization of poorly differentiated NSCLC samples. RESULTS: Microarray data from a genome-wide scan revealed 34 differentially expressed miRNAs, 5 of which enabled algorithmic discrimination of normal tissue from carcinoma (SQCC or ADC), as well as SQCC from ADC. Expression of miR-21 was significantly increased in both tumor types, whereas levels of miR-451 and miR-486-5p were reduced. SQCC was distinguished from normal tissue and ADC by high-level miR-205 expression and decreased miR-26b. Comparison of miRNA profiles to histological and immunohistochemical findings in 19 poorly differentiated specimens demonstrated the potential clinical utility of miRNA profiling to provide important insights into the classification of SQCC and ADC. CONCLUSION: This study presents a novel algorithm for specimen classification in cases of poorly differentiated NSCLC.

Human abdominal aortic aneurysm (AAA): Evidence for an autoimmune antigen-driven disease.

Abdominal aortic aneurism (AAA) is a complex immunological disease with a strong genetic component, and one of the ten leading causes of death of individuals 55-74 years old worldwide. Strong evidence has been accumulated suggesting that AAA is an autoimmune specific antigen-driven disease. Mononuclear cells infiltrating AAA lesions comprised of T and B lymphocytes and other cells expressing early-, intermediate- and late-activation antigens, and the presence of antigen-presenting cells have been documented, demonstrating an ongoing immune response. The three components of the trimolecular complex, T-cell receptor (TCR)/peptide (antigen)/HLA have been identified in AAA, and specifically: (i) clonal expansions of T-cell clones in AAA lesions; (ii) the association of AAA with particular HLA Class I and Class II; and (iii) self or nonself putative AAA-associated antigens. IgG autoantibodies recognizing proteins present in normal aortic tissue have been reported in patients with AAA. Molecular mimicry, defined as the sharing of antigenic epitopes between microorganisms (bacteria, viruses) and self antigens, maybe is responsible for T-cell responses and antibody production in AAA. Also, the frequency and the suppressor activity of CD4+ CD25+ FOXP3+ Tregs and the expression of FOXP3 transcripts and protein have been reported to be significantly impaired in AAA patients vs normal donors.

Dietary flaxseed administered post thoracic radiation treatment improves survival and mitigates radiation-induced pneumonopathy in mice.

BACKGROUND: Flaxseed (FS) is a dietary supplement known for its antioxidant and anti-inflammatory properties. Radiation exposure of lung tissues occurs either when given therapeutically to treat intrathoracic malignancies or incidentally, such as in the case of exposure from inhaled radioisotopes released after the detonation of a radiological dispersion devise (RDD). Such exposure is associated with pulmonary inflammation, oxidative tissue damage and irreversible lung fibrosis. We previously reported that dietary FS prevents pneumonopathy in a rodent model of thoracic X-ray radiation therapy (XRT). However, flaxseed's therapeutic usefulness in mitigating radiation effects post-exposure has never been evaluated. METHODS: We evaluated the effects of a 10%FS or isocaloric control diet given to mice (C57/BL6) in 2 separate experiments (n = 15-25 mice/group) on 0, 2, 4, 6 weeks post a single dose 13.5 Gy thoracic XRT and compared it to an established radiation-protective diet given preventively, starting at 3 weeks prior to XRT. Lungs were evaluated four months post-XRT for blood oxygenation levels, inflammation and fibrosis. RESULTS: Irradiated mice fed a 0%FS diet had a 4-month survival rate of 40% as compared to 70-88% survival in irradiated FS-fed mouse groups. Additionally, all irradiated FS-fed mice had decreased fibrosis compared to those fed 0%FS. Lung OH-Proline content ranged from 96.5 ± 7.1 to 110.2 ± 7.7 µg/ml (Mean ± SEM) in all irradiated FS-fed mouse groups, as compared to 138 ± 10.8 µg/ml for mice on 0%FS. Concomitantly, bronchoalveolar lavage (BAL) protein and weight loss associated with radiation cachexia was significantly decreased in all FS-fed groups. Inflammatory cell influx to lungs also decreased significantly except when FS diet was delayed by 4 and 6 weeks post XRT. All FS-fed mice (irradiated or not), maintained a higher blood oxygenation level as compared to mice on 0%FS. Similarly, multiplex cytokine analysis in the BAL fluid revealed a significant decrease of specific inflammatory cytokines in FS-fed mice. CONCLUSIONS: Dietary FS given post-XRT mitigates radiation effects by decreasing pulmonary fibrosis, inflammation, cytokine secretion and lung damage while enhancing mouse survival. Dietary supplementation of FS may be a useful adjuvant treatment mitigating adverse effects of radiation in individuals exposed to inhaled radioisotopes or incidental radiation.

Cytological interpretation of p16 immunohistochemistry in head and neck carcinomas: does the choice of fixative matter?

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

Small airway pathology and bronchoreversibility in advanced emphysema.

BACKGROUND: Poorly reversible airflow obstruction is a hallmark feature of chronic obstructive pulmonary disease (COPD). However, some COPD patients demonstrate significant bronchodilator reversibility (BDR). The pathologic features associated with the presence or absence of this phenomenon are not known. METHODS: We analyzed 67 patients with advanced upper lobe predominant emphysema who underwent lung volume reduction surgery and divided them into 2 groups: the reversible group [BD(+)] had a >12% and >200 mL increase in FEV(1) or FVC with bronchodilator; the irreversible group [BD(-)] had a

Cytohistologic correlation of basaloid salivary gland neoplasms: Can cytomorphologic classification be used to diagnose and grade these tumors?

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

Targeted detoxification of selected reactive oxygen species in the vascular endothelium.

Oxidative stress underlies diverse vascular diseases, but its management remains elusive, in part because of our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites and our limited understanding which species need to be eliminated. The antioxidant enzymes (AOEs) superoxide dismutase (SOD) and catalase (which decompose and H(2)O(2), respectively), conjugated with an antibody to platelet-endothelial cell adhesion molecule-1 (PECAM-1), bind to endothelial cells and alleviate oxidative stress in cell culture models. Here, we studied the effects of these antioxidant conjugates in mouse models of vascular oxidative stress. Anti-PECAM/catalase and anti-PECAM/SOD conjugates, in contrast to control IgG/AOE conjugates, accumulated in the lungs and vascularized organs after intravenous injection in wild-type, but not PECAM KO mice. Anti-PECAM/catalase, but not anti-PECAM/SOD, protected mice from lung injury induced by H(2)O(2) produced by glucose oxidase deposited in the pulmonary vasculature. Anti-PECAM/catalase also reduced alveolar edema and attenuated decline in arterial oxygen in mice that underwent unilateral lung ischemia/reperfusion, whereas anti-PECAM/SOD was not effective, implying the key role of H(2)O(2) in tissue damage in this pathology. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase prevented oxidation of tetrahydrobiopterin and normalized vasoreactivity in the vessels of mice rendered hypertensive by pretreatment with angiotensin-II. This outcome agrees with reports implicating superoxide and peroxynitrite in altered endothelium-dependent vasodilatation in hypertension. Therefore, the use of endothelial cell-targeted antioxidants identifies the key specific species of ROS involved in various forms of vascular disease and holds promise for the mechanistically tailored treatment of these pathologies.

Small airway mucous metaplasia and inflammation in chronic obstructive pulmonary disease.

Mucous metaplasia is an important determinant of small airway obstruction in COPD. Its relationship to small airway inflammation is poorly defined. We analyzed 4 to 6 small airways in 19 COPD patients, GOLD stages 0-4, from lobectomy or lung volume reduction surgery tissue samples. To identify intracellular mucin, periodic acid fluorescent Schiff's (PAFS) stained slides were imaged by fluorescence microscopy. PAFS+ staining area, basement membrane length (L(BM)), epithelial height and area were measured. Mucin was expressed as a percentage of epithelial area. Mucin volume density (MVD) was calculated as PAFS+ area divided by the product of L(BM) and 4/pi. Airways were Giemsa stained for eosinophils and immunostained with antibodies against CD3, CD4, CD8, CD68, and neutrophil elastase (NE), and the number of positively stained cells/mm(2) was quantified in the airway wall. Mucin percent correlated with CD3(+) cell density (r = 0.553, P < 0.0001), and MVD correlated with CD3(+) (r = 0.570, P < 0.0001) and CD8(+) cell density (r = 0.279, P = 0.016). There were weak negative correlations between mucin percent as well as MVD and CD68(+) cell density (r = -0.270, P = 0.02 and r = -0.245, P = 0.036). There was no relationship between epithelial mucin content and CD4(+), NE(+), or eosinophil cell density. CD3(+) and CD8(+) lymphocytic inflammation is related to small airway mucous metaplasia in COPD and may play a causative role in its development.

Sentinel Lymph Node Characterization with a Dual-Targeted Molecular Ultrasound Contrast Agent.

PURPOSE: The purpose of this study was to assess the performance of molecular ultrasound with dual-targeted microbubbles to detect metastatic disease in the sentinel lymph nodes (SLNs) in swine model of naturally occurring melanoma. The SLN is the first lymph node in the lymphatic chain draining primary tumor, and early detection of metastatic SLN involvement is critical in the appropriate management of melanoma. PROCEDURE: Nine Sinclair swine (weight 3-7 kg; Sinclair BioResources, Columbia, MO, USA) with naturally occurring melanoma were examined. Siemens S3000 scanner with a 9L4 probe was used for imaging (Siemens Healthineers, Mountain View, CA). Dual-targeted contrast agent was created using Targestar SA microbubbles (Targeson, San Diego, CA, USA) labeled with ανß3-integrin and P-selectin antibodies. Targestar SA microbubbles labeled with IgG-labeled were used as control. First, peritumoral injection of Sonazoid contrast agent (GE Healthcare, Oslo, Norway) was performed to detect SLNs. After that, dual-targeted and IGG control Targestar SA microbubbles were injected intravenously with a 30-min interval between injections. Labeled Targestar SA microbubbles were allowed to circulate for 4 min to enable binding. After that, two sets of image clips were acquired several seconds before and after a high-power destruction sequence. The mean intensity difference pre- to post-bubble destruction within the region of interest placed over SLN was calculated as a relative measure of targeted microbubble contrast agent retention. This process was repeated for non-SLNs as controls. All lymph nodes evaluated on imaging were surgically removed and histologically examined for presence of metastatic involvement. RESULTS: A total of 43 lymph nodes (25 SLNs and 18 non-SLNs) were included in the analysis with 18 SLNs demonstrating metastatic involvement greater than 5 % on histology. All non-SLNs were benign. The mean intensity (± SD) of the dual-targeted microbubbles for metastatic SLNs was significantly higher than that of benign LNs (18.05 ± 19.11 vs. 3.30 ± 6.65 AU; p = 0.0008), while IgG-labeled control microbubbles demonstrated no difference in retained contrast intensity between metastatic and benign lymph nodes (0.39 ± 1.14 vs. 0.03 ± 0.24 AU; p = 0.14). CONCLUSIONS: The results indicate that dual-targeted microbubbles labeled with P-selectin and ανß3-integrin antibodies may aid in detecting metastatic involvement in SLNs of melanoma.