Pre-treatment CD8+ tumour-infiltrating lymphocyte density predicts distant metastasis after definitive treatment in patients with stage III/IV hypopharyngeal squamous cell carcinoma.

OBJECTIVES: Although inflammatory markers, such as the neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and local immune markers have been shown to have prognostic utility, limited information is available regarding inflammatory and pre-existing tumour-infiltrating lymphocyte density and their association with prognosis in patients with hypopharyngeal squamous cell carcinoma. We investigated the prognostic ability of inflammatory markers and tumour-infiltrating lymphocyte density in stage III and stage IV hypopharyngeal squamous cell carcinoma patients receiving definitive treatment. DESIGN: Retrospective cohort study. SETTING: Kurume University Hospital. PARTICIPANTS: Ninety-six stage III or stage IV hypopharyngeal squamous cell carcinoma patients treated at the Kurume University Hospital between 2000 and 2014. MAIN OUTCOME MEASURES: Inflammatory markers and pre-treatment tumour-infiltrating lymphocyte density were examined from recorded haematologic data and immunohistochemical analysis. RESULTS: Multivariate analyses showed that the CD8+ tumour-infiltrating lymphocyte density was an independent predictive factor for distant metastasis and overall survival, whereas inflammatory markers, including the neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio, were not correlated with distant metastasis or overall survival. CONCLUSIONS: Higher pre-treatment CD8+ tumour-infiltrating lymphocyte density is a useful predictive biomarker for reduced distant metastasis and better prognosis.

SurePath® LBC improves the diagnostic accuracy of intrahepatic and hilar cholangiocarcinoma.

INTRODUCTION: The current study aimed to compare cytology using SurePath® (SP)-LBC and biliary tissue histology (BTH) for the diagnosis of biliary disease. METHODS: Between January 2014 and December 2016, 57 patients underwent endoscopic retrograde cholangiopancreatography for the diagnosis of biliary disease. Biliary cytological samples were processed using SP-LBC and subsequently BTH was performed. A final diagnosis was confirmed by surgery (23 malignant cases) and clinical follow-up (34 benign and malignant cases): 18 extrahepatic cholangiocarcinoma; 17 intrahepatic/hilar cholangiocarcinoma (intra/H-CC); eight other malignant disease; and 14 benign biliary disease. The diagnoses made using SP-LBC and BTH were classified into four categories: (1) benign; (2) indeterminate; (3) suspicious for malignancy/malignant; and (4) inadequate. In addition, diagnostic accuracy was compared between SP-LBC and BTH. RESULTS: Although 23% (13/57) of BTH samples were classified as inadequate, all SP-LBC cases were classified as adequate. Among 43 malignant cases, 11 normal, four indeterminate and 28 suspicious for malignancy/malignant were found using SP-LBC (26%, 9% and 65%, respectively), in contrast to 10 inadequate, nine normal, 10 indeterminate and 14 suspicious for malignancy/malignant observed using BTH (23%, 21%, 23%, and 33%, respectively). The identification of malignant cells was strikingly different between SP-LBC and BTH. Furthermore, limited to intra/H-CC, accuracy was significantly higher using SP-LBC than using BTH (P < .001). CONCLUSIONS: SP-LBC of the biliary tract is a useful and reliable method for diagnosing biliary malignant disease and has an advantage over BTH for detecting malignant cells and accurately diagnosing intra/H-CC.

A Combined test using both cell sediment and supernatant cell-free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients.

INTRODUCTION: The aim of this study was to examine whether a combined test using both cell sediment and supernatant cytology cell-free DNA (ccfDNA) is more useful in detecting EGFR mutation than using cell sediment DNA or supernatant ccfDNA alone in pleural effusion of lung cancer patients. METHODS: A total of 74 lung adenocarcinoma patients with paired samples between primary tumour and corresponding metastatic tumour with both cell sediment and supernatant ccfDNA of pleural effusion cytology were enrolled in this study. Cell sediment and supernatant ccfDNA were analysed separately for EGFR mutations by polymerase chain reaction. RESULTS: Out of 45 patients with mutant EGFR in primary tumours, EGFR mutations were detected in 23 cell sediments of corresponding metastases (sensitivity; 51.1%) and 20 supernatant ccfDNA corresponding metastases (sensitivity; 44.4%). By contrast, the combined test detected EGFR mutations in 27 corresponding metastases (sensitivity; 60.0%), and had a higher sensitivity than the cell sediment or the supernatant ccfDNA alone (P < .05). Out of 45 patients with mutant EGFR, 24, three and 18 were cytologically diagnosed as positive, atypical or negative, respectively. The detection rate in the combined test was highest (95.8%) in the positive group, and mutant EGFR was also detected in four of 18 samples (22.2%) in the negative group. CONCLUSIONS: A combined test using both cell sediment DNA and supernatant ccfDNA samples increases the concordance rate of EGFR mutations between primary tumour and corresponding metastases. Our findings indicate that supernatant ccfDNA is useful even in cases where the cytological diagnosis is negative.

Cytological nuclear atypia classification can predict prognosis in patients with endometrial cancer.

OBJECTIVE: Endometrial cancer is one of the leading causes of malignancy in females. Nuclear findings are important for patients with cancer, and can provide valuable information to treating oncologists. We investigated whether nuclear findings were a useful prognostic factor in patients with endometrial cancer. METHOD: We investigated 71 cases of endometrial carcinoma with paired histology and cytology at Kurume University Hospital. We classified endometrial endometrioid adenocarcinoma (EEC) G1 and G2 as type I carcinomas, and uterine papillary serous carcinoma (UPSC), clear cell carcinoma (CC) and EEC G3 as type II carcinomas. For the establishment of the cytological nuclear atypia classification, we examined the following nuclear factors on the cytological smears: mitotic figures, prominent nucleoli, nuclear area and anisonucleosis. RESULTS: There was a significant difference in mitotic figures (P < 0.001) and anisonucleosis (P = 0.026) in cytological smears between type I and type II carcinomas. Based on these findings, we categorized cytological nuclear atypia into three groups, nuclear atypia-1 (57.7%), nuclear atypia-2 (19.7%) and nuclear atypia-3 (22.5%), and this classification system correlated well with prognosis in patients with endometrial cancer (P < 0.001). Furthermore, this classification system was able to extract patients with a good prognosis from those with high-grade carcinomas, such as UPSC+CC+EEC G3, and patients with a poor prognosis from those with EEC G1. CONCLUSIONS: Our system of cytological nuclear atypia classification based on endometrial cytology can predict patient prognosis. Cytological nuclear atypia classification and histological typing may be useful for the treatment and follow-up of patients with endometrial cancer, and should be routinely incorporated into cytological reports.

Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors.

Aberrant activation of the Ras/Raf-1/extracellular-regulated kinase (ERK) pathway has been shown to be involved in the progression of human hepatocellular carcinoma (HCC). However, the mechanism of dysregulation of ERK activation is poorly understood. Recently, we identified Sprouty-related protein with Ena/vasodilator-stimulated phosphoprotein homology-1 domain (Spred) as a physiological inhibitor of the Ras/Raf-1/ERK pathway. In this study, we found that the expression levels of Spred-1 and -2 in human HCC tissue were frequently decreased, comparing with those in adjacent non-tumorous tissue. Moreover, Spred expression levels in HCC tissue were inversely correlated with the incidence of tumor invasion and metastasis. Forced expression of Spred-1 inhibited HCC cell proliferation in vitro and in vivo, which was associated with reduced ERK activation. Spred-1 overexpression also reduced the secretion of matrix metalloproteinase-9 (MMP-9) and MMP-2, which play important roles in tumor invasion and metastasis. In addition, Spred-1 inhibited growth factor-mediated HCC cell motility. These data indicate that the reduction of Spred expression in HCC is one of the causes of the acquisition of malignant features. Thus, Spred could be not only a novel prognostic factor but also a new therapeutic target for human HCC.

Variations of Weiss's ring.

BACKGROUND: Weiss's ring is an important index for diagnosing a posterior vitreous detachment (PVD). The authors studied the configurations of Weiss's ring because the ring has several variations. METHODS: Weiss's ring was examined biomicroscopically in 223 eyes with PVD and documented videographically using a scanning laser ophthalmoscope. The configurations of Weiss's ring were classified into four groups: a complete ring, a partial ring, a ball-like opacity, and a hole without a ring. RESULTS: In the 223 eyes with PVD, Weiss's ring was categorized as a complete ring in 63 eyes (28.3%), a partial ring in 128 eyes (57.4%), a ball-like opacity in 17 eyes (7.6%), and a hole without a ring in 15 eyes (6.7%). A complete ring was observed in 8 (57.1%) of 14 eyes with hyperopia (+3 diopters or more), in 25 (28.7%) of 87 eyes with emmetropia (between -1 and +1 diopter), and in 10 (16.1%) of 62 eyes with myopia (-3 diopters or more). Six months after the initial examinations, the contour of Weiss's ring remained unchanged in 86 (96.6%) of 89 eyes with fresh PVD. CONCLUSIONS: Scanning laser ophthalmoscope is a useful tool for observing Weiss's ring. A "classic" annular opacity around a hole in the prepapillary posterior vitreous cortex represents less than one third of the configurations of Weiss's ring, indicating that the term "ring" is erroneous.

Expression of angiogenic factors, basic fibroblast growth factor and vascular endothelial growth factor, in human biliary tract carcinoma cell lines.

In order to clarify angiogenic mechanism in biliary tract carcinoma, expressions and functions of basic fibroblast growth factor (bFGF) and its receptors (FGFR-1-4), and vascular endothelial growth factor (VEGF) and its receptors were investigated by using human biliary tract carcinoma cell lines (KMC-1, KMC-2, KMBC and KMG-C). Expression of bFGF was confirmed in KMC-1 and KMC-2, and that of FGFR-1-4 in all the cell lines except no FGFR-2 in KMC-2. Expression of VEGF was detected in all the cell lines, whereas the cell lines did not express VEGF receptors. Addition of anti-bFGF neutralizing antibody to the medium did not suppress cell proliferation, whereas exogenous bFGF with or without heparin accelerated cell proliferation in all cell lines. Addition of anti-bFGF neutralizing antibody or anti-VEGF neutralizing antibody to the co-culture of human umbilical vascular endothelial cells (HUVEC) and KMC-2 suppressed the proliferation of HUVEC. Surgically obtained cholangiocarcinoma tissues (n=7) were immunohistochemically negative to bFGF, while six of the seven were positive to VEGF. These findings suggested that human biliary tract carcinoma cells express both bFGF and VEGF not as autocrine growth factors but as angiogenic factors. On the other hand, expression of VEGF was found at a higher frequency than bFGF both in the cell lines and tissues.

Three-dimensional analysis of macular diseases with a scanning retinal thickness analyzer and a confocal scanning laser ophthalmoscope.

OBJECTIVE: To compare three-dimensional analysis of macular diseases obtained using the scanning retinal thickness analyzer (RTA) with that obtained using the confocal scanning laser ophthalmoscope, Heidelberg Retina Tomograph (HRT). PATIENTS AND METHODS: Both the RTA and the HRT were used to examine 50 eyes of 36 patients with diabetic macular edema, macular edema following branch retinal vein occlusion, age-related macular degeneration, and idiopathic macular holes. RESULTS: In most macular diseases, the retinal thickness map constructed using the RTA agreed with the image obtained with the HRT. The two maps were not consistent with each other, however, in patients with dense retinal hemorrhages and with extrafoveal fixation. CONCLUSIONS: Although both the RTA and the HRT give additional information to clinically evaluate macular diseases, they do have limitations. The discrepancy between these two analyses in some specific macular pathologies might be caused by the different wavelengths of the laser beam and the different methodologies used to scan the retina.

Expression and function of interleukin-8 in human hepatocellular carcinoma.

Expression and functions of interleukin (IL)-8, a pro-inflammatory cytokine with angiogenesis action, was examined in 23 surgically resected hepatocellular carcinoma (HCC) specimens and 7 HCC cell lines. In all HCC tissues, IL-8 expression was confirmed with reverse-transcription polymerase chain reaction method and enzyme-linked immunosorbent assay, and immunohistochemistry showed HCC cells were the major producer of IL-8 in the tissues. Microvessel density was measured by the double immunohistochemical staining of muscular vessels in HCC tissues, but the density was not related to the level of IL-8 in the HCC tissues. On the other hand, in the co-culture of human umbilical vein endothelial cells (HUVEC) and a HCC cell line (KIM-1), IL-8 produced by KIM-1 significantly accelerated the proliferation of HUVEC. In addition, cases with a high IL-8 level in cancerous tissue had a significantly higher frequency of portal vein invasion, venous invasion and bile duct invasion (p<0.05). In the cultures of 7 HCC cell lines IL-8 secretion into culture medium increased with the treatment of IL-1beta or tumor necrosis factor-alpha. This showed IL-8 expression is regulated by inflammatory cytokines. IL-8 produced by HCC is an angiogenesis factor of HCC, but it could have a much more important role in the invasion and metastasis of HCC.

Retinal thickness measurements with optical coherence tomography and the scanning retinal thickness analyzer.

PURPOSE: To assess the reproducibility of retinal thickness measurements in normal subjects and to compare foveal thickness using optical coherence tomography (OCT) and the scanning retinal thickness analyzer (RTA). METHODS: Two examiners performed foveal thickness measurements in 24 eyes of 12 healthy subjects using both OCT and the scanning RTA. Intraclass correlation coefficients (ICC) (intra-examiner and inter-examiner) were calculated for the paired foveal thickness measurements obtained with each instrument. RESULTS: The average foveal thicknesses measured with OCT and the scanning RTA were 155.1 +/- 14.9 microm and 107.8 +/- 18.6 microm, respectively. The intra-examiner ICCs from the two sessions using the OCT and the scanning RTA were 0.99 and 0.78 and 0.89 and 0.99, respectively. The inter-examiner ICCs of the OCT and the scanning RTA were 0.99 and 0.99, respectively. There was a significant correlation between the foveal thickness measurements with these two instruments (R2 = 0.629, P < 0.0001). CONCLUSION: The foveal thicknesses measured using OCT and the scanning RTA in healthy subjects agreed with the previously reported data on foveal thickness. Both instruments can reproducibly quantitate foveal thickness.

Effects of peroxynitrite on rabbit cornea.

BACKGROUND: Nitric oxide (NO) reacts rapidly with the superoxide anion to generate peroxynitrite, which has been found in the aqueous humor in eyes with uveitis. We evaluated the functional and anatomic effects of peroxynitrite on rabbit cornea. METHODS: One eye of each rabbit received an anterior chamber injection of 3-morpholinosydonimine N-ethylcarbamide (SIN-1), which simultaneously generates both NO and the superoxide anion. The corneal thickness was measured using an ultrasonic pachymeter before and after the injection. The eyes were fixed and the corneal specimens were prepared for electron microscopy. RESULTS: Anterior chamber injections of SIN-1 caused a significant increase in the corneal thickness (25.1+/-3.0 microm) 30 min after injection. Transmission electron microscopy showed swollen mitochondria and large vacuoles in the cytoplasm, and scanning electron microscopy revealed obscuring of the mosaic pattern by increased ruffling of endothelial cell surface and borders. CONCLUSION: The results suggest that peroxynitrite generated in the aqueous humor may cause corneal endothelial cell damage, which leads to transient corneal edema.

Expression and localization of vascular endothelial growth factor receptors in human hepatocellular carcinoma and non-HCC tissues.

Flt-1 (VEGF receptor-1) and KDR/Flk-1 (VEGF receptor-2) are the high-affinity receptors for the angiogenesis factor, vascular endothelial growth factor (VEGF). VEGF expression has been confirmed in human hepatocellular carcinoma (HCC), and VEGF is thought to be involved in the angiogenesis within HCC tissues. However, expressions of VEGF receptors in HCC have not been reported. We immunohistochemically examined expressions and localizations of Flt-1 and KDR in 28 surgically resected HCC tissues. In non-cancerous area, Flt-1 and KDR were mainly found in macrophages including Kupffer cells; both receptors were found in vascular endothelial cells in the portal veins and arteries within portal tracts; and KDR was also found in some sinusoidal endothelial cells. In cancerous area, Flt-1 and KDR were found in some macrophages, and also in the endothelial cells of intratumoral blood vessels. In 25 moderately and/or poorly differentiated HCCs, KDR expression in the blood space endothelial cells was clear and continuous in 20 cases, and focal in 5 cases. These results suggest that there would be an angiogenesis mechanism via VEGF/Flt-1 or VEGF/KDR in HCC, and the VEGF/KDR system would take a more important role.

[Burkitt's lymphoma occurring as a primary lymphomatous effusion].

A 39-year-old man was admitted with massive ascites. Specimens of ascitic fluid contained numerous cells with a FAB-L3 appearance, and small noncleaved cell lymphoma morphology. These cells expressed CD10, CD19, CD20, CD38, CD45, HLA-DR, and IgM antigens, and were positive for IgM and c-myc protein in cytoplasmic immunostaining tests. Clonal rearrangements of IgH and c-myc genes were detected by Southern blot analysis. No mass lesions were found by physical examination, and systemic computed photography did not reveal enlargement of lymph nodes, spleen, or liver. Bone marrow aspiration showed no infiltration of malignant cells. Ga scintigraphy indicated hot lesions only in the abdomen. These findings suggested that Burkitt's lymphoma had developed in the peritoneal cavity as a primary lymphomatous effusion. Chemotherapy with methotrexate, cyclophosphamide, vincristine, doxorubicin, etoposide, and dexamethasone was effective, and the patient has been free from the disease for 1 year since completion of consolidation treatment with autologous peripheral blood stem cell transplantation.

OCT in successful surgery of retinal detachment associated with optic nerve head pit.

The authors describe the course of a patient with retinal detachment associated with optic nerve head pit for whom optical coherence tomography (OCT) was performed before and after vitrectomy and gas tamponade. An 18-year-old woman presented with macular elevation and a lamellar macular hole associated with optic nerve head pit. OCT showed that the retinal elevation consisted of both separation of the inner and outer retinal layers and detachment of the outer layers from the retinal pigment epithelium. After vitreous surgery and postoperative intraocular gas tamponade, the macular elevation gradually resolved, and OCT showed that improvement in central vision corresponded with flattening of the outer layer detachment in the fovea. OCT is a useful tool for monitoring the therapeutic effect of vitrectomy and gas tamponade for retinal detachment associated with optic nerve head pit.

Retinal detachment and retinoschisis detected by optical coherence tomography in a myopic eye with a macular hole.

The authors describe a myopic patient with a full-thickness macular hole that was accompanied by both retinal detachment and retinoschisis. A 51-year-old woman presented with a localized retinal elevation of three disc diameters around the macular hole in a myopic eye. Optical coherence tomography (OCT) showed features of both retinal detachment and retinoschisis. After vitreous surgery and postoperative intraocular gas tamponade, the macular elevation resolved, and separation of the neurosensory retina from the retinal pigment epithelium band disappeared on the OCT images. OCT is a useful tool for examining macular conditions associated with a macular hole in myopic patients.

Scanning laser ophthalmoscope correlations with biomicroscopic findings and foveal function after macular hole closure.

OBJECTIVE: To investigate the relation between foveal findings and visual function in eyes with a resolved idiopathic macular hole after vitreous surgery. METHODS: We divided 28 eyes with postoperative idiopathic macular hole resolution into 3 groups based on postoperative biomicroscopic foveal findings of complete closure, partial closure, or atrophic closure. To evaluate foveal retinal function, scanning laser ophthalmoscope (SLO) microperimetry was performed preoperatively and 6 months postoperatively. RESULTS: Postoperatively in 18 eyes (64%), the foveal images became normal or almost normal and were classified as having complete closure, 6 eyes (21%) were classified as having partial closure, and 4 eyes (14%) as having atrophic closure. The corresponding visual acuity levels 6 months postoperatively were, respectively, 0.10, 0.35, and 0.64 (P<.01) based on LogMAR analysis. Preoperative SLO microperimetry detected an absolute scotoma at the bottom of all macular holes; postoperatively, the absolute scotoma disappeared in the 18 eyes with complete hole closure, but a relative scotoma was detected in 6 eyes. Of 6 eyes with partial closure, 1 had an absolute scotoma and 5 had a relative scotoma. An absolute scotoma was detected in 4 eyes with atrophic closure. CONCLUSIONS: After macular hole closure, SLO findings correlate both with biomicroscopic findings and foveal function. Better anatomical foveal recovery in eyes after macular hole closure results in better improvement of vision than in eyes in which the foveal anatomical findings are not as good.

Retinal detachment associated with a macular hole in severely myopic eyes.

PURPOSE: To investigate factors associated with extensive retinal detachment in severely myopic eyes with a macular hole. METHOD: Fifty-two consecutive eyes with a macular hole and severe myopia were retrospectively studied. RESULTS: An extensive retinal detachment, defined as extending beyond the cuff of subretinal fluid, was observed in 37 eyes (71%). Extensive retinal detachment developed in 36 (95%) of 38 eyes with a posterior staphyloma and in one (7%) of 14 eyes without a posterior staphyloma (P<.0001). Extensive retinal detachment also developed in 32 (89%) of 36 eyes with complete posterior vitreous detachment and in five (31%) of 16 eyes without posterior vitreous detachment (P<.0001). CONCLUSION: Posterior staphyloma rather than anteroposterior vitreomacular traction may contribute to the development of retinal detachment associated with a macular hole in severely myopic eyes.