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.

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.

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.

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.

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.

Establishment and characterization of a new human hepatocellular carcinoma cell line, HAK-3, and its response to growth factors.

A new human hepatocellular (HCC) cell line, HAK-3, was established from a resected HCC of a Japanese, female patient. HAK-3 retains morphologic features of the original HCC, and proliferates in a monolayered sheet (doubling time: 26 h). HAK-3 is a single aneuploid cell population with a DNA index of 2.42, the karyotype is human, chromosomes are 80-85 (mode: 83), and secretes fibronectin and tissue polypeptide antigen. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) dose-dependently accelerated the cell proliferation, while deletion-type hepatocyte growth factor (dHGF) tended to suppress the proliferation, and transforming growth factor (TGF)-alpha showed almost no influence. dHGF induced the decrease of cell adhesiveness, changed the cell morphology to spindle-shaped cells, increased cell movement, and showed chemotactic effects with the increase of its concentration gradient in cultures. HAK-3 would be useful in studies on the acceleration mechanisms of cancer cell proliferation by growth factors and of chemotaxis by dHGF.

Vitreous photography with a +90-diopter double aspheric preset lens vs the El Bayadi-Kajiura preset lens.

Two fundus lenses, a 90-diopter preset lens and a 58.6-diopter El Bayadi-Kajiura lens, were compared. The lenses were evaluated with regard to their optics, magnification, field of view, and photographic usefulness in photographing the posterior vitreous. The 90-diopter lens provided a wider field of view but lower magnification. Both lenses permitted high-quality vitreous photographs through the slit lamp, but the 90-diopter lens allowed photographs to be taken through small pupils and some lens opacities.

Risk of developing a macular hole.

We followed up 45 eyes with early-stage idiopathic macular hole (mean period, 32 months; range, 7 to 87 months) to investigate their prognoses, especially in relation to the vitreous condition. At initial examination, 18 eyes (40%) showed vitreous separation from the fovea, and 10 (22%) developed vitreous separation during follow-up. None of these 28 eyes progressed to a fully developed macular hole during follow-up. Moreover, the macular disease was apparently improved in 23 (82%) of these eyes. However, of the remaining 27 eyes without vitreous separation from the fovea, 10 eyes (37%) progressed to a fully developed macular hole. We speculate that early-stage macular hole could be reversed by spontaneous vitreous separation from the fovea, and this chance may be greater than the risk of true macular hole formation.

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.

Phenotypic consequence of the gene abnormality in the platelet glycoprotein IX gene observed in a patient with Bernard-Soulier syndrome through mammalian cell expression system.

We previously reported a patient with Bernard-Soulier syndrome (BSS) who had a homozygous missense mutation in the GPIX gene Phe55 (TTT) to Ser (TCT) replacement in the leucine-rich motif (LRM) of the GPIX polypeptide, as a probable cause of BSS phenotype. To study the effect of this mutation on the surface expression of the GPIb/IX complex and GPIX, we introduced this mutation into the cDNA of GPIX by site-directed mutagenesis and performed in vitro transfection studies with plasmid for mutant GPIX and other plasmids for GPIb/IX complex. Mutant GPIX could not increase the surface expression of GPIb-alpha, but also surface expression of GPIX itself. Immunostaining of the CHO-K1 cells transfected with two plasmids for mutant GPIX and GPIb-beta showed that mutant GPIX was weakly but certainly detected in the cytoplasm of the transfected cells. These findings indicate that this substitution is responsible for BSS phenotype and that the LRM of GPIX is a critically important element for an efficient expression of the GPIb/IX complex. They also suggest that the failure of GPIb/IX complex expression can be caused by a mechanism other than synthetic defect of the mutant protein.

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.

Effect of the vitreous on the prognosis of full-thickness idiopathic macular hole.

We studied 112 eyes with full-thickness idiopathic macular holes (mean follow-up period, 5.7 years) to ascertain the effect of the vitreous on prognosis. Eyes were divided as follows: group 1, 58 eyes with no posterior vitreous detachment; group 2, 25 eyes with no posterior vitreous detachment initially and development of complete posterior vitreous detachment during follow-up; and group 3, 29 eyes with complete posterior vitreous detachment. In group 1, the macular hole enlarged in 37 eyes (64%), and the surrounding retinal detachment enlarged in 29 (50%); visual acuity decreased in 20 eyes (34%) by two or more Snellen lines. These percentages were significantly higher than in group 3 (14%, 10%, and 10%; P = .01, P = .01, and P = .03, respectively). Eyes with a full-thickness macular hole with no posterior vitreous detachment may have a greater risk of enlargement of the macular hole and surrounding retinal detachment, and thus a worse prognosis than eyes with posterior vitreous detachment.

Prognosis of stage 2 macular holes.

PURPOSE: We ascertained the natural course of stage 2 idiopathic macular holes to determine better treatment possibilities. METHODS: We reviewed 48 eyes with stage 2 idiopathic macular holes and followed them up for more than two years. At each examination, best-corrected Snellen visual acuity was measured by a physician masked to the hypothesis of the study. RESULTS: Stage 2 lesions progressed to stage 3 or 4 during the follow-up period in 32 (67%) and 14 (29%) of 48 eyes, respectively; two eyes (4%) remained in stage 2. In 41 (85%) of 48 eyes, the hole size enlarged during the follow-up: 32 (94%) of 34 eyes had vitreomacular attachment and nine (64%) of 14 eyes had vitreomacular separation at the final examination, for a statistically significant difference in prevalence (P = .03). Visual acuity decreased two or more Snellen lines during the follow-up period in 34 (71%) of 48 eyes, the prevalence of which was significantly higher in eyes with vitreomacular attachment at the final examination (28 of 34, 82%) than in eyes with vitreomacular separation at the final examination (six of 14, 43%) (P = .01). CONCLUSION: Even though vitreomacular separation may improve the prognosis of a macular hole, stage 2 lesions usually will develop an enlarged hole and decreased visual acuity.

Vitreous findings in cases of optic nerve pits and serous macular detachment.

We examined biomicroscopically the relationship between the optic nerve and the vitreous in 17 eyes of 16 consecutive patients with pits of the optic nerve that demonstrated an associated serous detachment of the macula. The posterior vitreous was attached in 15 eyes (88%); the posterior vitreous was partially detached with a vitreous strand terminating at the optic pit in two eyes (12%). Of the 15 eyes without posterior vitreous detachment, 11 (73%) had an anomalous Cloquet's canal that was markedly condensed and terminated at the margin of the pit. During ocular movement, we observed a back-and-forth movement of the anomalous Cloquet's canal and a pulsating translucent membrane that covered the pit. Our findings suggest that the intravitreal traction on the optic pit by the anomalous Cloquet's canal may have a role in the development of macular detachment.

Imaging of epiretinal membranes in macular holes by scanning laser ophthalmoscopy.

PURPOSE: Because recognition and removal of an epiretinal membrane are important in macular hole surgery, we used the scanning laser ophthalmoscope preoperatively to study epiretinal membranes in patients with idiopathic macular holes. METHODS: We studied 67 eyes (60 consecutive patients) with idiopathic macular holes. We evaluated the thickness and the extent of the epiretinal membrane by using a scanning laser ophthalmoscope. We then compared the fundus images obtained with the scanning laser ophthalmoscope with red-free monochromatic fundus photographs. RESULTS: Confocal imaging of the fundus with either argon blue (488 nm) or argon green (514 nm) laser illumination clearly showed the epiretinal membranes. In all eyes, we observed lesions ranging from a patchy glinting light reflex to a dense epiretinal membrane. A well-demarcated dense epiretinal membrane around the macular hole was observed in seven (44%) of 16 eyes with stage 2 macular holes and in 12 (40%) of 30 eyes with stage 3 holes, but in only two (10%) of 21 eyes with stage 4 holes. The prevalence of the dense epiretinal membrane in stage 2 or 3 holes was significantly higher than in stage 4 holes (P = .025 and .024, respectively). CONCLUSIONS: Fundus imaging using the scanning laser ophthalmoscope with argon laser illumination is useful preoperatively to evaluate epiretinal membranes in eyes with idiopathic macular holes.

Spontaneous resolution of foveal detachments and macular breaks.

PURPOSE: To evaluate the mechanism of spontaneous resolution of foveal detachments and idiopathic macular breaks. METHODS: We reviewed the records of 139 consecutive eyes (94 patients) with either a foveal detachment or a macular break in patients who were examined between 1989 and 1992. There were 26 men and 68 women (mean age, 66.9 +/- 6.9 years). They were either unoperated on or observed during the period that preceded surgery. Each patient underwent complete ophthalmic examination in addition to slit-lamp photography of the vitreomacular interface and microperimetry with the scanning laser ophthalmoscope. RESULTS: Eight eyes demonstrated spontaneous resolution. A foveal detachment was noted in five eyes (five patients) and a stage 2 macular break in three eyes (three patients). The mean duration of observation was 33 months (range, one to 144 months). Resolution of the foveal detachments occurred without the development of posterior vitreous detachment. In each eye, the presence of a pseudo-operculum, indicating vitreofoveal separation, was accompanied by flattening of the foveal detachment without detectable posterior vitreous detachment. The three eyes with stage 2 macular break resolved after premature development of a posterior vitreous detachment. CONCLUSIONS: Foveal detachment and macular break resolution seem to result from the release or weakening of vitreous traction on the fovea. Reattachment of the foveal retina preserves fair to good visual acuity. Surgical intervention is contraindicated (1) in eyes in which foveal detachment flattens and develops a pseudo-operculum and (2) when a posterior vitreous detachment develops in an eye with a stage 2 macular break. Careful biomicroscopic vitreous examination and microperimetry with the scanning laser ophthalmoscope are extremely useful methods for adequate examination of these patients.

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.