Application of The Paris System to atypical urine cytology samples: correlation with histology and UroVysion® FISH.

OBJECTIVES: To evaluate whether atypical urine cytology cases may be stratified more objectively using The Paris System (TPS) and whether reclassified cases correlate with histology and UroVysion® results. METHODS: Atypical urine cytology cases subjected to UroVysion® testing over a period of 6 years were reviewed. Each case was reclassified according to TPS and correlated with histology and UroVysion® results. RESULTS: A total of 91 cases were identified; 70.3% were reclassified as 'negative for high-grade urothelial carcinoma (HGUC)' and 14.3% as 'atypical urothelial cells (AUC)'. The histological correlation was available in 45 cases. In the 'negative for HGUC' category, 67.9% had no histological evidence of malignancy, but 17.9% were diagnosed as HGUC. In the 'AUC' category, histology revealed urothelial carcinoma in 70% of the cases (of these, 71.4% were high grade). There was no histological evidence of malignancy in 30% of cases; notably, all of which were from patients under surveillance. The sensitivity and specificity of UroVysion® were 85.7% and 33.3% in the 'AUC' group and 62.5% and 100% in the 'negative for HGUC' group. CONCLUSIONS: The Paris System is an objective template for reporting urine cytology specimens, and is particularly useful in identifying HGUC cases and refining the category of 'AUC'.

Simultaneous immunostaining with anti-S100P and anti-SV40 antibodies revealed the origin of BK virus-infected decoy cells in voided urine samples.

BACKGROUND: Methods for determining the origin of BK virus (BKV)-infected cells (decoy cells) in clinical urine samples have not been established although they could enhance the diagnosis of BKV infection in immunocompromised patients. METHODS: We performed simultaneous immunostaining with anti-S100P (a urothelial marker) and anti-SV40 antibodies in 66 clinical urine samples exhibiting SV40 positivity and a decoy-cell appearance on Papanicolaou staining. The clinical voided urine samples included seven cases of renal transplantation, 47 cases of cancer therapy and 12 cases of non-neoplastic disease. SurePath(™) liquid-based cytology was used for the urine samples. RESULTS: BKV-infected cells were categorized as SV40(+)/S100P(+) and SV40 (+)/S100p(-). SV40(+)/S100P(-) cells were found in 55 cases (83.4%); nine cases (13.6%) carried both SV40(+)/S100P(-) and SV40(+)/S100P(+) cells. The former were identified as BKV infection in renal tubules and the latter in both the renal tubules and urothelial epithelia. The remaining two cases (3.0%) had only SV40(+)/S100P(+) cells of urothelial origin. CONCLUSION: Simultaneous immunostaining with anti-S100P and anti-SV40 is a useful method for determining the origin of BKV-infected cells in clinical urine samples from immunocompromised patients such as renal transplantation recipients.

Squamous-lined cyst of the pancreas: Radiological-pathological correlation.

Pancreatic cystic lesions are increasingly being detected incidentally because of the increased use of cross-sectional imaging. Squamous-lined cysts of the pancreas (lymphoepithelial cyst, epidermoid cyst, and dermoid cyst) are rare cystic lesions lined with squamous epithelium. Distinguishing squamous-lined cysts from other cystic lesions of the pancreas is important to avoid unnecessary surgery, because squamous-lined cysts of the pancreas have no malignant potential. The purpose of this review is to describe findings on computed tomography and magnetic resonance imaging and the histopathological characteristics of squamous-lined cysts, and to summarize the key points of differential diagnosis for pancreatic cystic lesions.

Steroid sulphatase and oestrogen sulphotransferase in human non-small-cell lung carcinoma.

BACKGROUND: Steroid sulphatase (STS) is one of the steroid-metabolising enzymes involved in desulphating inactive steroid sulphates and oestrogen sulphotransferase (EST) sulphates active oestrogen. The roles of both STS and EST have not been examined in oestrogen-dependent non-small-cell lung cancer (NSCLC). METHODS: We evaluated the immunoreactivity of STS and EST in NSCLC cases using immunohistochemistry. The function of STS and EST was further demonstrated using NSCLC cell lines. RESULTS: The immunoreactivity of STS and EST was detected in 49.5% and 27.8% of NSCLC cases, respectively. The immunoreactivity of STS was significantly higher in female adenocarcinoma cases. The STS-positive NSCLCs were also significantly correlated in an inversed manner with tumour size and cell proliferation and tended to be associated with better clinical outcome. However, the immunoreactivity of EST was significantly correlated with intracellular oestradiol concentration. Results of in vitro analysis demonstrated that oestrone sulphate (E1-S) induced and pregnenolone sulphate (Preg-S) inhibited the proliferation in STS-expressing cell lines. The inhibition by Preg-S was reversed by a specific progesterone receptor blocker. Simultaneous addition of E1-S and Preg-S significantly suppressed the proliferation. CONCLUSION: In NSCLC patients, STS is considered a good prognostic factor. Results of our present study also indicated the benefits of potential progesterone therapy for NSCLC patients.

Intratumoral concentration of estrogens and clinicopathological changes in ductal carcinoma in situ following aromatase inhibitor letrozole treatment.

BACKGROUND: Estrogens have important roles in ductal carcinoma in situ (DCIS) of the breast. However, the significance of presurgical aromatase inhibitor treatment remains unclear. Therefore, we examined intratumoral concentration of estrogens and changes of clinicopathological factors in DCIS after letrozole treatment. METHODS: Ten cases of postmenopausal oestrogen receptor (ER)-positive DCIS were examined. They received oral letrozole before the surgery, and the tumour size was evaluated by ultrasonography. Surgical specimens and corresponding biopsy samples were used for immunohistochemistry. Snap-frozen specimens were also available in a subset of cases, and used for hormone assays and microarray analysis. RESULTS: Intratumoral oestrogen levels were significantly lower in DCIS treated with letrozole compared with that in those without the therapy. A great majority of oestrogen-induced genes showed low expression levels in DCIS treated with letrozole by microarray analysis. Moreover, letrozole treatment reduced the greatest dimension of DCIS, and significantly decreased Ki-67 and progesterone receptor immunoreactivity in DCIS tissues. CONCLUSION: These results suggest that estrogens are mainly produced by aromatase in DCIS tissues, and aromatase inhibitors potently inhibit oestrogen actions in postmenopausal ER-positive DCIS through rapid deprivation of intratumoral estrogens.

Mutation analysis in the BRCA2 gene in primary breast cancers.

Breast cancer, one of the most common and deleterious of all diseases affecting women, occurs in hereditary and sporadic forms. Hereditary breast cancers are genetically heterogeneous; susceptibility is variously attributable to germline mutations in the BRCA1 (ref. 1), BRCA2 (ref. 2), TP53 (ref. 3) or ataxia telangiectasia (ATM) genes, each of which is considered to be a tumour suppressor. Recently a number of germline mutations in the BRCA2 gene have been identified in families prone to breast cancer. We screened 100 primary breast cancers from Japanese patients for BRCA2 mutations, using PCR-SSCP. We found two germline mutations and one somatic mutation in our patient group. One of the germline mutations was an insertion of an Alu element into exon 22, which resulted in alternative splicing that skipped exon 22. The presence of a 64-bp polyadenylate tract and evidence for an 8-bp target-site duplication of the inserted DNA implied that the retrotransposal insertion of a transcriptionally active Alu element caused this event. Our results indicate that somatic BRCA2 mutations, like somatic mutations in the BRCA1 gene, are very rare in primary breast cancers.

Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus.

A locus for familial melanoma, MLM, has been mapped within the same interval on chromosome 9p21 as the gene for a putative cell cycle regulator, p16INK4 (CDKN2) MTS1. This gene is homozygously deleted from many tumour cell lines including melanomas, suggesting that CDKN2 is a good candidate for MLM. We have analysed CDKN2 coding sequences in pedigrees segregating 9p melanoma susceptibility and 38 other melanoma-prone families. In only two families were potential predisposing mutations identified. No evidence was found for heterozygous deletions of CDKN2 in the germline of melanoma-prone individuals. The low frequency of potential predisposing mutations detected suggests that either the majority of mutations fall outside the CDKN2 coding sequence or that CDKN2 is not MLM.

Carcinoembryonic antigen-related cell adhesion molecules as surrogate markers for EGFR inhibitor sensitivity in human lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LADCA) patients with epidermal growth factor receptor (EGFR) mutations are in general associated with relatively high clinical response rate to EGFR-tyrosine kinase inhibitors (TKIs) but not all responded to TKI. It has therefore become important to identify the additional surrogate markers regarding EGFR-TKI sensitivity. METHODS: We first examined the effects of EGFR-TKIs, gefitinib and erlotinib, upon cell proliferation of lung adenocarcinoma cell lines. We then evaluated the gene profiles related to EGFR-TKI sensitivity using a microarray analysis. Results of microarray analysis led us to focus on carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, CEACAM 3, 5, 6, 7, and 19, as potential further surrogate markers of EGFR-TKI sensitivity. We then examined the correlation between the status of CEACAM 3, 5, 6, 7, and 19 immunoreactivity in LADCA and clinicopathological parameters of individual cases. RESULTS: In the cases with EGFR mutations, the status of all CEACAMs examined was significantly higher than that in EGFR wild-type patients, but there were no significant differences in the status of CEACAMs between TKI responder and nonresponder among 22 patients who received gefitinib therapy. However, among 115 EGFR mutation-negative LADCA patients, both CEACAM6 and CEACAM3 were significantly associated with adverse clinical outcome (CEACAM6) and better clinical outcome (CEACAM3). CONCLUSION: CEACAMs examined in this study could be related to the presence of EGFR mutation in adenocarcinoma cells but not represent the effective surrogate marker of EGFR-TKI in LADCA patients. However, immunohistochemical evaluation of CEACAM3/6 in LADCA patients could provide important information on their clinical outcome.

Macrophage migration inhibitory factor and DJ-1 in gastric cancer: differences between high-incidence and low-incidence areas.

BACKGROUND: There is a need for sensitive and specific blood-borne markers for the detection of gastric cancer. Raised serum macrophage inhibitory factor (MIF) levels have been proposed as a marker for gastric cancer diagnosis but, to date, studies have only encompassed patients from high-incidence areas. METHODS: We have compared the serum concentration of MIF in a large cohort of UK and Japanese gastric cancer patients, together with appropriate control subjects (age and gender matched). Carcinoembryonic antigen and H. pylori IgG were also measured, as was DJ-1, a novel candidate protein biomarker identified by analysis of gastric cancer cell line secretomes. RESULTS: Marked elevations of the serum concentration of MIF and DJ-1 were seen in Japanese patients with gastric cancer compared with Japanese controls, a trend not seen in the UK cohort. These results could not be accounted for by differences in age, disease stage or H. pylori status. CONCLUSION: In regions of high, but not low incidence of gastric cancer, both MIF and DJ-1 have elevated serum concentrations in gastric cancer patients, compared with controls. This suggests that differing mechanisms of disease pathogenesis may be at play in high- and low-incidence regions.

Immunohistochemical analysis of gastrin-releasing peptide receptor (GRPR) and possible regulation by estrogen receptor ßcx in human prostate carcinoma.

Gastrin-releasing peptide (GRP) belongs to the family of bombesin-like peptides. GRP was demonstrated to stimulate the proliferation and invasiveness of androgen-independent prostate carcinoma. GRP mediates its action through the membrane-bound receptor, GRP receptor (GRPR), which is characterized by a high-affinity binding for both GRP and bombesin. In human prostate cancer tissue, GRPR mRNA was reported to be detectable in more than 90% but its immunolocalizaition has not been reported. Therefore, in this study we immunolocalized GRPR in 51 human prostate cancer cases and correlated the findings with several clinicopathological parameters in order to better understand the function and regulation of GRPR in human prostate cancer. GRPR was immnolocalized in carcinoma cells and their values were significantly associated with Gleason score and immunoreactivity of estrogen receptor ßcx (ERßcx) that is one of splicing variants of ligand dependent transcription factor, ERß, and considered to be prognostic factor of prostate cancer patients. The amounts of GRPR and ERßcx mRNA in three prostate cancer cell lines PC-3, DU-145 and LNCaP evaluated by quantitative RT-PCR (qPCR) analysis were also significantly correlated. In addition, we established stable transformants of prostate carcinoma cell line PC-3 introduced with ERßcx, and confirmed that GRPR mRNA was induced in ERßcx over-expressing PC-3 cells by qPCR analysis. These results also suggest that ERßcx contributes to prostate cancer development possibly through mediating GRPR expression in carcinoma cells.

Relationship between Bunina bodies and TDP-43 inclusions in spinal anterior horn in amyotrophic lateral sclerosis.

AIMS: Amyotrophic lateral sclerosis (ALS) is characterized by upper and lower motor neurone involvement with Bunina bodies (BBs) and TDP-43 inclusions. To elucidate the relationship between BBs and TDP-43 inclusions, we examined the spinal cord from 18 patients with ALS. METHODS: Five serial sections from lumbar cord were first stained with haematoxylin and eosin to detect BBs and subsequently immunostained with anti-TDP-43 antibody. Immunoelectron microscopy was performed on vibratome sections from two cases of ALS. RESULTS: BBs were found in 15 out of 18 cases. TDP-43 inclusions were found in all the cases. The average incidence of anterior horn cells with BBs and TDP-43 inclusions relative to the total number of neurones was 17.1% and 46.4%, respectively. The concurrence of both inclusions in the same neurones was found in 15 cases. The incidence of co-localization of BBs and TDP-43 inclusions was 15.7% of total neurones. The frequency of TDP-43 inclusions was significantly higher in neurones with BBs than in those without. Ultrastructurally, TDP-43-immunoreactive filamentous structures were intermingled with early-stage BBs, but not associated with advanced-stage BBs. CONCLUSION: These findings suggest that there is a close relationship in the occurrence between BBs and TDP-43 inclusions.

Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients.

Previous studies suggested that one or more genes on chromosome 5q21 are responsible for the inheritance of familial adenomatous polyposis (FAP) and Gardner's syndrome (GS), and contribute to tumor development in patients with noninherited forms of colorectal cancer. Two genes on 5q21 that are tightly linked to FAP (MCC and APC) were found to be somatically altered in tumors from sporadic colorectal cancer patients. One of the genes (APC) was also found to be altered by point mutation in the germ line of FAP and GS patients. These data suggest that more than one gene on chromosome 5q21 may contribute to colorectal neoplasia, and that mutations of the APC gene can cause both FAP and GS. The identification of these genes should aid in understanding the pathogenesis of colorectal neoplasia and in the diagnosis and counseling of patients with inherited predispositions to colorectal cancer.

A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1.

A strong candidate for the 17q-linked BRCA1 gene, which influences susceptibility to breast and ovarian cancer, has been identified by positional cloning methods. Probable predisposing mutations have been detected in five of eight kindreds presumed to segregate BRCA1 susceptibility alleles. The mutations include an 11-base pair deletion, a 1-base pair insertion, a stop codon, a missense substitution, and an inferred regulatory mutation. The BRCA1 gene is expressed in numerous tissues, including breast and ovary, and encodes a predicted protein of 1863 amino acids. This protein contains a zinc finger domain in its amino-terminal region, but is otherwise unrelated to previously described proteins. Identification of BRCA1 should facilitate early diagnosis of breast and ovarian cancer susceptibility in some individuals as well as a better understanding of breast cancer biology.

BRCA1 mutations in primary breast and ovarian carcinomas.

Loss of heterozygosity data from familial tumors suggest that BRCA1, a gene that confers susceptibility to ovarian and early-onset breast cancer, encodes a tumor suppressor. The BRCA1 region is also subject to allelic loss in sporadic breast and ovarian cancers, an indication that BRCA1 mutations may occur somatically in these tumors. The BRCA1 coding region was examined for mutations in primary breast and ovarian tumors that show allele loss at the BRCA1 locus. Mutations were detected in 3 of 32 breast and 1 of 12 ovarian carcinomas; all four mutations were germline alterations and occurred in early-onset cancers. These results suggest that mutation of BRCA1 may not be critical in the development of the majority of breast and ovarian cancers that arise in the absence of a mutant germline allele.

Three-dimensional double-echo steady-state (3D-DESS) magnetic resonance imaging of the knee: contrast optimization by adjusting flip angle.

BACKGROUND: The three-dimensional double-echo steady-state (3D-DESS) sequence is being used to investigate sequence parameters for the assessment of knee cartilage by magnetic resonance imaging (MRI). With the flip angle (FA) values that are commonly used, contrast between cartilage and synovial fluid may be too low to delineate minor lesions of the cartilage surface. PURPOSE: To investigate the value of FA maximizing contrast between cartilage and synovial fluid using the 3D-DESS sequence. MATERIAL AND METHODS: In 10 healthy volunteers (five men, five women; age range 23-48 years), 3D-DESS imaging was performed to capture median sagittal sections of the knee by adjusting FA from 10 degrees to 90 degrees. Cartilage-synovial fluid contrast-to-noise ratio (CNR) was measured to determine the value of FA maximizing CNR. Water-excitation pulse was used to suppress fat signals. MRI was performed at 1.0 T using a circular polarization flex coil. RESULTS: Cartilage-synovial fluid CNR was highest with 90 degrees FA, yielding images with good contrast between cartilage and synovial fluid. CONCLUSION: Simply by increasing FA to 90 degrees in 3D-DESS imaging, the contrast between cartilage and synovial fluid increased substantially. Subtle cartilage lesions may thus be detectable using 3D-DESS sequences.

Molecular epidemiological and mutational analysis of DNA mismatch repair (MMR) genes in endometrial cancer patients with HNPCC-associated familial predisposition to cancer.

Recently, a high rate of endometrial cancer has been reported in women with hereditary non-polyposis colorectal cancer (HNPCC), suggesting a relationship between familial endometrial cancers and HNPCC. Familial endometrial cancers constitute only about 0.5% of all endometrial carcinomas and it is essential to examine family histories in detail. A mutational analysis of three DNA mismatch repair (MMR) genes (hMLH1, hMSH2 and hMSH6) in patients with endometrial cancer who meet our criteria for familial predisposition to HNPCC-associated endometrial cancers was performed. Mutations were detected in 18 of the 120 patients (15.0%). Most HNPCC-related endometrial cancers do not meet the New Amsterdam Criteria for HNPCC. These clinical criteria may identify only some HNPCC-associated endometrial cancers. Establishing the correct family history for endometrial cancer patients is important for diagnosing familial endometrial carcinomas. An analysis of MMR genes may be useful for patients with endometrial cancer showing familial aggregation. In addition, gynecologists must be accurately informed, and it is important to perform large-scale, multicenter studies both nationwide and internationally.

Aurora kinase B is a predictive factor for the aggressive recurrence of hepatocellular carcinoma after curative hepatectomy.

BACKGROUND: Patterns of cancer recurrence hold the key to prognosis after curative resection. This retrospective study aimed to identify a predictor and therapeutic candidate for aggressive recurrence of hepatocellular carcinoma (HCC). METHODS: Primary HCC tissues from 107 patients who had curative resection were analysed. Genome-wide gene expression profiles were investigated using a microarray technique, and clustering analysis was carried out based on the first diagnosis of recurrence according to the Milan criteria. Immunohistochemical expression and array-based comparative genomic hybridization (array-CGH) were also assessed. RESULTS: Microarray analysis revealed overexpression of Aurora kinase B, a chromosome passenger protein kinase, as the most significant predictor of the aggressive recurrence of HCC. Aurora kinase B protein expression was significantly associated with aggressive recurrence (P < 0.001) and prognosis (P < 0.001). Multivariable analysis identified Aurora kinase B as the only independent predictor of aggressive recurrence of HCC (P = 0.031). Array-CGH analysis showed that genomic instability was closely related to Aurora kinase B expression (P = 0.011). CONCLUSION: Aurora kinase B is an effective predictor of aggressive HCC recurrence, in relation to the genomic instability. It might be worth considering as a molecular target for the adjuvant therapy of HCC.

Hemispheric asymmetry of the arcuate fasciculus: a preliminary diffusion tensor tractography study in patients with unilateral language dominance defined by Wada test.

OBJECTIVE: Lateralization of language function is a prominent feature of human brain function, and its underlying structural asymmetry has been recently reported in normal right-handed subjects. By means of diffusion tensor tractography (DTT), we investigated the asymmetry of the language network, namely, the arcuate fasciculus in patients in whom the unilateral language dominance was defined by Wada test. METHODS: DTT was performed in 24 patients with a focal lesion or an epileptic focus outside the C-shaped segment of the arcuate fasciculus. The arcuate fasciculus was reconstructed by placing two regions-of-interest in the deep white matter lateral to the corona radiata. The pathway was then divided into one terminating in the temporal lobe (FT tract) and the other in the parietal lobe (FP tract). The relative number and volume of the FT and FP tracts in each hemisphere were submitted to repeated measure ANOVA separately, with the hemisphere as a within-subject factor and with the side of pathology as a between subject factor. RESULTS: The FT tract showed a significantly larger number and volume in the language dominant hemisphere than in the non-dominant hemisphere, while, for the FP tract, no significant hemispheric difference was observed in the relative number or volume. There was a tendency that the FT tract was less lateralized when the pathology was located in the dominant hemisphere than in the non-dominant hemisphere. CONCLUSION: Dominance of the FT tract in the language dominant hemisphere was demonstrated for the first time in a patient population and implicated a clinical utility of DTT for non-invasive evaluation of language lateralization. Our preliminary study might indicate reorganization of the language network in conjunction with pathology.

Imaging Differences between Neuromyelitis Optica Spectrum Disorders and Multiple Sclerosis: A Multi-Institutional Study in Japan.

BACKGROUND AND PURPOSE: Both clinical and imaging criteria must be met to diagnose neuromyelitis optica spectrum disorders and multiple sclerosis. However, neuromyelitis optica spectrum disorders are often misdiagnosed as MS because of an overlap in MR imaging features. The purpose of this study was to confirm imaging differences between neuromyelitis optica spectrum disorders and MS with visually detailed quantitative analyses of large-sample data. MATERIALS AND METHODS: We retrospectively examined 89 consecutive patients with neuromyelitis optica spectrum disorders (median age, 51 years; range, 16-85 years; females, 77; aquaporin 4 immunoglobulin G-positive, 93%) and 89 with MS (median age, 36 years; range, 18-67 years; females, 68; relapsing-remitting MS, 89%; primary-progressive MS, 7%; secondary-progressive MS, 2%) from 9 institutions across Japan (April 2008 to December 2012). Two neuroradiologists visually evaluated the number, location, and size of all lesions using the Mann-Whitney U test or the Fisher exact test. RESULTS: We enrolled 79 patients with neuromyelitis optica spectrum disorders and 87 with MS for brain analysis, 57 with neuromyelitis optica spectrum disorders and 55 with MS for spinal cord analysis, and 42 with neuromyelitis optica spectrum disorders and 14 with MS for optic nerve analysis. We identified 911 brain lesions in neuromyelitis optica spectrum disorders, 1659 brain lesions in MS, 86 spinal cord lesions in neuromyelitis optica spectrum disorders, and 102 spinal cord lesions in MS. The frequencies of periventricular white matter and deep white matter lesions were 17% and 68% in neuromyelitis optica spectrum disorders versus 41% and 42% in MS, respectively (location of brain lesions, P < .001). We found a significant difference in the distribution of spinal cord lesions between these 2 diseases (P = .024): More thoracic lesions than cervical lesions were present in neuromyelitis optica spectrum disorders (cervical versus thoracic, 29% versus 71%), whereas they were equally distributed in MS (46% versus 54%). Furthermore, thoracic lesions were significantly longer than cervical lesions in neuromyelitis optica spectrum disorders (P = .001), but not in MS (P = .80). CONCLUSIONS: Visually detailed quantitative analyses confirmed imaging differences, especially in brain and spinal cord lesions, between neuromyelitis optica spectrum disorders and MS. These observations may have clinical implications.