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// Lola Koniali 1 , * , Andreas Hadjisavvas 1 , 2 , * , Anastasia Constantinidou 3 , Kyproula Christodoulou 2 , 4 , Yiolanda Christou 5 , Christiana Demetriou 6 , Andreas S. Panayides 7 , Constantinos Pitris 8 , Constantinos S. Pattichis 7 , Eleni Zamba-Papanicolaou 2 , 5 and Kyriacos Kyriacou 1 , 2 1 Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 2 The Cyprus School of Molecular Medicine, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 3 Medical School, University of Cyprus and the Bank of Cyprus Oncology Centre, Nicosia, Cyprus 4 Neurogenetics Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 5 Neurology Clinic D, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 6 Department of Primary Care and Population Health, University of Nicosia Medical School, Nicosia, Cyprus 7 Department of Electrical and Computer Engineering, School of Engineering, University of Cyprus, Nicosia, Cyprus 8 Department of Computer Science, University of Cyprus, Nicosia, Cyprus * These authors contributed equally to this work Correspondence to: Kyriacos Kyriacou, email: kyriacos@cing.ac.cy Keywords: breast cancer; brain metastases; risk factors; biomarkers Received: August 21, 2019 Accepted: January 04, 2020 Published: February 11, 2020 ABSTRACT Background: Brain metastasis (BM) is an increasingly common and devastating complication of breast cancer (BC). Methods: A systematic literature search of EMBASE and MEDLINE was conducted to elucidate the current state of knowledge on known and novel prognostic factors associated with 1) the risk for BCBM and 2) the time to brain metastases (TTBM). Results: A total of 96 studies involving institutional records from 28 countries were identified. Of these, 69 studies reported risk factors of BCBM, 46 factors associated with the TTBM and twenty studies examined variables for both outcomes. Young age, estrogen receptor negativity (ER-), overexpression of human epidermal factor (HER2+), and higher presenting stage, histological grade, tumor size, Ki67 labeling index and nodal involvement were consistently found to be independent risk factors of BCBM. Of these, triple-negative BC (TNBC) subtype, ER-, higher presenting histological grade, tumor size, and nodal involvement were also reported to associate with shorter TTBM. In contrast, young age, hormone receptor negative (HR-) status, higher presenting stage, nodal involvement and development of liver metastasis were the most important risk factors for BM in HER2-positive patients. Conclusions: The study provides a comprehensive and individual evaluation of the risk factors that could support the design of screening tools and interventional trials for early detection of BCBM.

Simple Summary Breast cancer is the most commonly diagnosed type of cancer in women worldwide. Stratification of women based on their individual breast cancer risk could guide targeted preventative strategies and population screening. Integrated models that combine the effects of a polygenic risk score (PRS) with classical breast cancer risk factors could provide an individualized breast-cancer risk estimation. Although various studies have extensively evaluated the performance of such integrated models in populations of European ancestry, no previous studies have included individuals of Greek-Cypriot origin. To this end, we have assessed the predictive performance of a 15-SNP PRS (PRS15), in combination with classical breast-cancer risk factors, in women of Greek-Cypriot origin. This proof-of-concept study suggests that models combining genetic data with classical risk factors may be used in the future for the prediction of breast-cancer risk and, therefore, supports their potential clinical utility for targeted preventative strategies in Cypriot women. Abstract The PRS combines multiplicatively the effects of common low-risk single nucleotide polymorphisms (SNPs) and has the potential to be used for the estimation of an individual’s risk for a trait or disease. PRS has been successfully implemented for the prediction of breast cancer risk. The combination of PRS with classical breast cancer risk factors provides a more comprehensive risk estimation and could, thus, improve risk stratification and personalized preventative strategies. In this study, we assessed the predictive performance of the combined effect of PRS15 with classical breast-cancer risk factors in Cypriot women using 1109 cases and 1177 controls from the MASTOS study. The PRS15 was significantly associated with an increased breast cancer risk in Cypriot women OR (95% CI) 1.66 (1.25–2.19). The integrated risk model obtained an AUC (95% CI) 0.70 (0.67–0.72) and had the ability to stratify women according to their disease status at the extreme deciles. These results provide evidence that the combination of PRS with classical risk factors may be used in the future for the stratification of Cypriot women based on their disease risk, and support its potential clinical utility for targeted preventative actions and population screening.

We aimed to determine genetic diagnosis in the national primary ciliary dyskinesia (PCD) cohort of Cyprus, an island with a high disease prevalence. We used targeted next-generation sequencing (NGS) of 39 PCD genes in 48 patients of Greek-Cypriot and other ancestries. We achieved molecular diagnosis in 74% of the unrelated families tested. We identified 24 different mutations in 11 genes, 12 of which are novel. Homozygosity was more common in Greek-Cypriot than non-Greek-Cypriot patients (88% vs 46.2%, p =0.016). Four mutations (DNAH11:c.5095-2A>G, CFAP300:c.95_103delGCCGGCTCC, TTC25:c.716G>A, RSPH9:c.670+2T>C,) were found in 74% of the diagnosed Greek-Cypriot families. Patients with RSPH9 mutations demonstrated higher nasal nitric oxide (57nL/min Vs 15 nL/min, p

Simple Summary In Cyprus, approximately 9% of triple-negative (negative in common breast cancer receptors—estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2) receptors) breast cancer (TNBC) patients carry inherited mutations in the BRCA1/2 breast cancer (BC) susceptibility genes. These mutations increase the risk of BC. However, the contribution of other BC susceptibility genes has not yet been determined. To this end, we aimed to investigate the prevalence of mutations in BRCA1/2-negative TNBC patients in Cyprus. Ninety-five cancer susceptibility genes were sequenced for 163 TNBC patients. The frequency of non-BRCA mutations and especially PALB2 in TNBC patients in Cyprus appears to be higher compared to other populations, and half of the mutation-positive patients were diagnosed over the age of 60 years. Based on these results, we believe that PALB2 and TP53 along with BRCA1/2 genetic testing could be beneficial for a large proportion of TNBC patients in Cyprus, irrespective of their age of diagnosis. Abstract In Cyprus, approximately 9% of triple-negative (estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative) breast cancer (TNBC) patients are positive for germline pathogenic variants (PVs) in BRCA1/2. However, the contribution of other genes has not yet been determined. To this end, we aimed to investigate the prevalence of germline PVs in BRCA1/2-negative TNBC patients in Cyprus, unselected for family history of cancer or age of diagnosis. A comprehensive 94-cancer-gene panel was implemented for 163 germline DNA samples, extracted from the peripheral blood of TNBC patients. Identified variants of uncertain clinical significance were evaluated, using extensive in silico investigation. Eight PVs (4.9%) were identified in two high-penetrance TNBC susceptibility genes. Of these, seven occurred in PALB2 (87.5%) and one occurred in TP53 (12.5%). Interestingly, 50% of the patients carrying PVs were diagnosed over the age of 60 years. The frequency of non-BRCA PVs (4.9%) and especially PALB2 PVs (4.3%) in TNBC patients in Cyprus appears to be higher compared to other populations. Based on these results, we believe that PALB2 and TP53 along with BRCA1/2 genetic testing could be beneficial for a large proportion of TNBC patients in Cyprus, irrespective of their age of diagnosis.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited, multisystemic disorder characterized clinically by delayed muscle relaxation and weakness. The disease is caused by a CTG repeat expansion in the 3' untranslated region (3' UTR) of the DMPK gene, which leads to the expression of a toxic gain-of-function mRNA. The expanded CUG repeat mRNA sequesters the MBNL1 splicing regulator in nuclear-retained foci structures, resulting in loss of protein function and disruption of alternative splicing homeostasis. In this study, we used CAG repeat antisense oligonucleotides (ASOs), composed of locked nucleic acid (LNA)- and 2'-O-methyl (2'OMe)-modified bases in a chimeric design, to alleviate CUGexpanded-mediated toxicity. Chimeric 14-18mer LNA/2'OMe oligonucleotides, exhibiting an LNA incorporation of ∼33%, significantly ameliorated the misregulated alternative splicing of Mbnl1-dependent exons in primary DM1 mouse myoblasts and tibialis anterior muscles of DM1 mice. Subcutaneous delivery of 14mer and 18mer LNA/2'OMe chimeras in DM1 mice resulted in high levels of accumulation in all tested skeletal muscles, as well as in the diaphragm and heart tissue. Despite the efficient delivery, chimeric LNA/2'OMe oligonucleotides were not able, even at a high-dosage regimen (400 mg/kg/week), to correct the misregulated splicing of Serca1 exon 22 in skeletal muscles. Nevertheless, oligonucleotide doses were well-tolerated as determined by histological and plasma biochemistry analyses. Our results provide proof of concept that inhibition of MBNL1 sequestration by systemic delivery of a steric-blocking ASO is extremely challenging, considering the large number of target sites that need to be occupied per RNA molecule. Although not suitable for DM1 therapy, chimeric LNA/2'OMe oligonucleotides could prove to be highly beneficial for other diseases, such as Duchenne muscular dystrophy, that require inhibition of a single target site per RNA molecule.

The evolution of Next Generation Sequencing (NGS) technologies represents a significant advancement in the field of molecular genetics and has set the ground, for the discovery of novel variants which cannot be easily classified as deleterious or neutral. In-vitro and in-vivo characterization of these variants of uncertain clinical significance (VUS) should be followed; however, it is often not feasible to carry out the experimental interpretation for every single VUS. In silico tools have been crucial for the prediction of the impact of VUS on protein structure, stability and function. Our aim was to combine computational approaches to investigate the impact of VUS identified in a cohort of Cypriot Triple-Negative Breast Cancer (TNBC) patients by NGS. Using a combination of structural, functional and network-based bioinformatics approaches for the classification of a nonsense PRF1 mutation in association with BC susceptibility, we propose a possible triggered interaction of the mutant PRF1 protein with the CDKN2A protein, a product of a BC susceptibility gene. Additionally, our results support that the increased probability of interaction of the mutant counterpart of perforin with its top 10 predicted interactors, could play an important role in the obstruction of cellular processes related to carcinogenesis such as cell death, necrosis, DNA damage, immortality, UV stress, DNA repair and cell cycle control. We conclude that probably the nonsense PRF1 mutation could be associated with BC predisposition. However, although in silico tools provide an important tool for the interpretation of VUS, functional studies, co-segregation analyses and/or case-control association studies are needed to draw conclusions on variant classification.

ObjectiveTo evaluate the therapeutic effects of omega-3 (ω3) fatty acids in the retina of aged mice when the blood arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is maintained between 1.0 and 1.5.Methods and analysisAged (24-month-old) wild-type C57BL/6J mice were allocated to two groups: ω3 treated and untreated. Treatment with ω3 was by daily gavage administration of EPA and docosahexaenoic acid for 60 days. Gas chromatography was used to identify and quantify fatty acids in the blood and retina. To count lipofuscin granules and measure the photoreceptor layer, eyecups were examined histologically using transmission electron microscopy and light microscopy. We also analysed eyecups using mass spectrometry-based proteomics.ResultsAA levels were lower, and EPA levels were higher, in the blood and retinas of the ω3-treated group than in the untreated group, resulting in a lower AA/EPA ratio. The ω3-treated group also showed significantly fewer lipofuscin granules and a thicker outer nuclear layer than the untreated group. Proteomic analysis revealed significantly greater expression of myelin basic protein, myelin regulatory factor-like protein, myelin proteolipid protein and glial fibrillar acidic protein in the ω3-treated group than in the untreated group. Three different pathways were significantly affected by ω3 treatment: fatty acid elongation, biosynthesis of unsaturated fatty acids and metabolic pathways.ConclusionTwo months of ω3 supplementation (when the blood AA/EPA~1.0–1.5) in aged mice reduced lipofuscin granule formation in the retina and protected the photoreceptor layer, suggesting that ω3 supplementation slows normal age-related retinal degeneration.

This paper presents the largest study in Cyprus evaluating the frequency and distribution of BRCA1/2 mutations in a high risk patient cohort. Deleterious mutations in the BRCA1/2 genes were identified in 68 of the 527 patients tested (13%). It is of interest that a quarter of those tested positive, did not have an extensive family history of breast/ovarian cancer but were diagnosed with early onset breast cancer, ovarian cancer under the age of 60 or triple negative breast cancer. The spectrum of mutations identified in our patient cohort is different compared to other Mediterranean countries. Furthermore, several of the mutations detected are novel and have not been identified in other ethnic populations. This highlights the importance of operating a national reference center for cancer genetic diagnosis which offers services tailored to the needs of the Cypriot population.

There has been tremendous progress in detection of breast cancer in postmenopausal women, resulting in two-thirds of women surviving more than 20 years after treatment. However, breast cancer remains the leading cause of cancer-related deaths in premenopausal women. Breast cancer is increasing in younger women due to changes in life-style as well as those at high risk as carriers of mutations in high-penetrance genes. Premenopausal women with breast cancer are more likely to be diagnosed with aggressive tumours and therefore have a lower survival rate. Mammography plays an important role in detecting breast cancer in postmenopausal women, but is considerably less sensitive in younger women. Imaging techniques, such as contrast-enhanced MRI improve sensitivity, but as with all imaging approaches, cannot differentiate between benign and malignant growths. Hence, current well-established detection methods are falling short of providing adequate safety, convenience, sensitivity and specificity for premenopausal women on a global level, necessitating the exploration of new methods. In order to detect and prevent the disease in high risk women as early as possible, methods that require more frequent monitoring need to be developed. The emergence of “omics” strategies over the last 20 years, enabling the characterisation and understanding of breast cancer at the molecular level, are providing the potential for long term, longitudinal monitoring of the disease. Tissue and serum biomarkers for breast cancer stratification, diagnosis and predictive outcome have emerged, but have not successfully translated into clinical screening for early detection of the disease. The use of breast-specific liquid biopsies, such as nipple aspirate fluid (NAF), a natural secretion produced by breast epithelial cells, can be collected non-invasively for biomarker profiling. As we move towards an age of active surveillance, home-based liquid biopsy collection kits are increasingly being applied and these could provide a paradigm shift where NAF biomarker profiling is used for routine breast health monitoring. The current status of established and newly emerging imaging techniques for early detection of breast cancer and the potential for alternative biomarker screening of liquid biopsies, particularly those applied to high-risk, premenopausal women, will be reviewed.

Purpose To evaluate the therapeutic effects of omega-3 (ω-3) fatty acids in the ABCA4-/- model of Stargardt's disease. Monitoring the blood level of eicosapentaenoic acid (EPA) and arachidonic acid (AA), served to adjust the treatment dosage (AA/EPA = 1-1.5). Methods Eight-month-old mice were allocated to different groups: A) wild type (129S1), B) ABCA4-/- untreated, C) ABCA4-/- treated with ω-3. Treatment was daily administered by gavage for 3 months. Eye cups from each group were histologically examined using confocal and transmission electron microscopy. Proteomic analysis and N-retinylidene-N-retinylethanolamine (A2E) quantification was carried our using liquid chromatography mass spectrometry (LC-MS/MS). Blood and retinal fatty acids analysis was performed using gas chromatography (GC). Results A significant decrease in melanolipofuscin granules in the retinal pigment epithelium (RPE) was observed in the treatment group, in parallel with a reduction in A2E level, a major component of RPE lipofuscin. Proteomic analysis indicated decreased levels of complement 3 (C3) in the treated group. The outer nuclear layer (ONL) thickness was significantly greater in group C (75.66 ± 4.8 μm), compared to group A (61.40 ± 1.84 μm) and B (56.50 ± 3.24 μm). Increased EPA and decreased AA levels were observed in both blood and retinas in the treatment group. Conclusions Supplementation with ω-3 fatty acids (when AA/EPA = 1-1.5) suggests a protective mechanism in the ABCA4-/- animal model of Stargadt's disease.

Oculo-dento-digital dysplasia (ODDD) is a congenital disorder manifesting with multiple phenotypic abnormalities involving the face, eyes, teeth, and limbs in addition to neurologic symptomatology. This report aims to present a female patient with ODDD who was referred due to extensive oral restorative needs. The presence of hypoplastic enamel triggered further evaluation. Characteristic facies with hypoplastic alae nasi and syndactyly offered greater insight into the phenotype of the syndrome. Clinical suspicion was confirmed by genetic sequencing revealing heterozygous mutation in GJA1. It is important to be aware of genetic disorders associated with characteristic dental malformations to offer appropriate counseling and treatment.

Purpose Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopatholigical features. Our aim, in this pilot study, was to investigate the intra- and inter-patient protein composition of nipple secretions and the implications for their use as liquid biopsies. Experimental design Matched pairs of NAF (n = 15) were characterised for physicochemical properties and SDS PAGE. Four pairs were selected for semi-quantitative proteomic profiling and trypsin-digested peptides analysed using 2D LC Orbitrap Fusion mass spectrometry. The resulting data was subject to bioinformatics analysis and statistical evaluation for functional significance. Results A total of 1990 unique proteins were identified many of which are established cancer associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals was observed. Conclusions This was the most complete proteomic study of NAF to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort. This article is protected by copyright. All rights reserved

Background: Nitric oxide (NO) is low in Primary Ciliary Dyskinesia (PCD) and has been found to be correlated with Ciliary Beat Frequency (CBF). Although low nasal NO is used in PCD diagnosis, few studies have examined the potentially therapeutic effect of increasing endogenous NO. We aimed to examine the effect of increasing NO production substrate L-Arginine, on CBF in human respiratory ciliated epithelium. Methods: 27 suspect cases referred for PCD testing and 7 healthy controls underwent nasal brushing. The cells obtained were divided between solutions containing medium 199 with different L-Arginine levels (baseline: 0.33mM, 1mM and 10mM). CBF measurements were obtained at 37°C and at 1, 3 and 24 hours after brushing. Results: PCD was confirmed in 8 subjects and excluded in 19. Among the PCD subjects, ciliary motility was characterized by rotational (n=5) or dyskinetic (n=3) beating. Compared to baseline, higher levels of L-Arginine resulted in up to 8.3% CBF increase at 1 hour (p=0.009), up to 9.6% CBF increase at 3 hours (p Conclusions: L-Arginine increases CBF and merits to be evaluated as a potential therapeutic agent for mucociliary clearance disorders with residual motility. Larger animal and human studies are needed.

Renal podocytes and their slit diaphragms ensure the integrity of the renal basement membrane that forms the barrier to urinary protein loss. A putative disruption of the slit diaphragm and its main protein components, nephrin and podocin, may be implicated in the pathogenesis of lupus nephritis (LN). We studied the glomerular protein expression of nephrin and podocin in NZB/W LN mice by Western blot and immunofluorescence; mRNA levels were measured by real-time PCR. Human kidney biopsies of class II ( n = 5), IV ( n = 4), V ( n = 7) LN were evaluated for nephrin expression by immunohistochemistry. Glomerular protein expression of nephrin and podocin were significantly reduced in NZB/W LN, starting from the earlier stages (mild mesangial LN) and becoming pronounced at advanced histological forms (focal and diffuse proliferative LN). Nephrin and podocin mRNA levels were substantially decreased in diffuse proliferative disease. Decreased expression of both proteins correlated with electron microscopy findings of distorted slit diaphragms. In patients with LN, nephrin was decreased particularly in diffuse proliferative LN. The main slit diaphragm proteins, nephrin and podocin, are affected from the earlier stages of LN and their expression correlates with disease histology. Our findings suggest a novel role of podocytes and their structures in immune-mediated nephritis.

What's known on the subject? and What does the study add? Scientists have found a number of genetic factors that increase prostate cancer risk, including heritable mutations in the genes BRCA1 and BRCA2. These mutations are not common but can have major impact, as a BRCA2 mutation increases risk by up to seven-fold while a BRCA1 mutation is thought to double risk in men under 65. The IMPACT study aims to determine whether targeted screening in men with a known BRCA1 or BRCA2 mutation would lead to earlier diagnosis of prostate cancers. This data from the IMPACT study adds to the increasing evidence that BRCA mutation carriers develop more aggressive disease. Although these are early results, it appears that PSA screening is more accurate at predicting potentially aggressive prostate cancer among men at higher risk of the disease due to a genetic predisposition than general population screening. This study provides support for continued screening in men with genetic mutations. OBJECTIVE To evaluate the role of targeted prostate cancer screening in men with BRCA1 or BRCA2 mutations, an international study, IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls), was established. This is the first multicentre screening study targeted at men with a known genetic predisposition to prostate cancer. A preliminary analysis of the data is reported. PATIENTS AND METHODS Men aged 40-69 years from families with BRCA1 or BRCA2 mutations were offered annual prostate specific antigen (PSA) testing, and those with PSA > 3 ng/mL, were offered a prostate biopsy. Controls were men age-matched (+/- 5 years) who were negative for the familial mutation. RESULTS In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116 BRCA2 and 95 controls) over 33 months. At the baseline screen (year 1), 7.0% (21/300) underwent a prostate biopsy. Prostate cancer was diagnosed in ten individuals, a prevalence of 3.3%. The positive predictive value of PSA screening in this cohort was 47 center dot 6% (10/21). One prostate cancer was diagnosed at year 2. Of the 11 prostate cancers diagnosed, nine were in mutation carriers, two in controls, and eight were clinically significant. CONCLUSIONS The present study shows that the positive predictive value of PSA screening in BRCA mutation carriers is high and that screening detects clinically significant prostate cancer. These results support the rationale for continued screening in such men.

X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited peripheral neuropathy caused by mutations in GJB1, the gene that encodes the gap junction protein connexin32 (Cx32). Cx32 is expressed by myelinating Schwann cells and forms gap junctions in non-compact myelin areas but axonal involvement is more prominent in X-linked compared to other forms of demyelinating Charcot-Marie-Tooth disease. To clarify the cellular and molecular mechanisms of axonal pathology in CMT1X, we studied Gjb1-null mice at early stages (i.e. 2- to 4-month-old) of the neuropathy, when there is minimal or no demyelination. The diameters of large myelinated axons were progressively reduced in Gjb1-null mice compared to those in wild type littermates. Furthermore, neurofilaments were relatively more dephosphorylated and more densely packed starting at 2 months of age. Increased expression of β-amyloid precursor protein, a marker of axonal damage, was also detected in Gjb1-null nerves. Finally, fast axonal transport, assayed by sciatic nerve ligation experiments, was slower in distal axons of Gjb1-null vs. wild type animals with reduced accumulation of synaptic vesicle-associated proteins. These findings demonstrate that axonal abnormalities including impaired cytoskeletal organization and defects in axonal transport precede demyelination in this mouse model of CMT1-X.

The DNA repair pathway is known to play a role in the etiology of breast cancer. A number of studies have demonstrated that common germline variants in genes involved in the DNA repair pathway influence breast cancer risk. To assess whether alterations in DNA repair genes contribute to breast cancer, we genotyped 12 single nucleotide polymorphisms (SNPs) in 1,109 Cypriot women with breast cancer and 1,177 age-matched healthy controls. We found significant associations with breast cancer for SNPs in the BRCA2 and MRE11A genes. Carriers of the BRCA2 rs1799944 variant (991 Asp) were found to have an increased risk of breast cancer (OR = 1.41, 95% CI 1.08–1.83, P = 0.01) with P trend = 0.0076. Homozygous carriers of the MRE11A rs601341 A allele had an increased risk of breast cancer (OR = 1.36, 95% CI 1.08–1.71, P = 0.009) with P trend = 0.0087. This study suggests that genetic variants in BRCA2 and MRE11A are associated with breast cancer risk.

Population-based studies have reported significant associations between specific genetic polymorphisms and breast cancer susceptibility. A number of studies have demonstrated that common variants of genes involved in the DNA repair pathway act as low penetrance breast cancer susceptibility alleles. We aimed to investigate the association of single nucleotide polymorphisms (SNPs) in the DNA repair genes XRCC1, XRCC2 and XRCC3 and breast cancer in MASTOS, a population-based case-control study of 1,109 Cypriot women with breast cancer diagnosed between 40 and 70 years and 1,177 age-matched healthy controls. Five coding SNPs were genotyped including rs1799782, rs25489 and rs25487 in XRCC1, rs3218536 in XRCC2 and rs861539 in XRCC3. Homozygous XRCC1 280His carriers had an increased risk of breast cancer (odds ratio 4.68; 95% CI 1.01-21.7; P = 0.03). The XRCC2 188His allele was associated with a marginal protective effect for breast cancer (odds ratio 0.79; 95% CI 0.62-1.00; P = 0.05). No significant associations were observed between the other three SNPs and breast cancer. This study suggests that genetic variation in SNPs in XRCC1 and XRCC2 genes may influence breast cancer susceptibility.

Background Perivascular epithelioid cell neoplasm (PEComa) is a rare mesenchymal tumor. Gynecological PEComas account for just over one-fourth of the overall PEComa cases reported in the literature. Surgery is the most recommended primary treatment while adjuvant therapy is generally reserved for high-risk cases. However, the best management of this neoplasia has not been well established, primarily because of the paucity of cases described to date. Objectives The aim of this systematic review is to summarize what is known thus far regarding the etiopathogenesis, clinical and pathologic features of PEComas, focusing also on the most valid treatment options for uterine cases. Data sources Pubmed articles on PEComas published in various journals over the past 70 years were analyzed. Conclusions and key findings Although the optimal treatment of gynecological PEComas is controversial, surgical resection remains the cornerstone. The use of adjuvant treatment is warranted in high risk patients to increase disease control. A multidisciplinary approach should be key in treatment decision-making regarding gynecological PEComas.

Breast cancer still represents a serious health problem and is currently the most frequent malignancy in the female population in developed countries. In Cyprus, there are 300 new cases annually. In the present study, histology, electron microscopy, immunohistochemistry, and Western blot analysis were used to investigate 100 cases of invasive breast carcinoma. In addition, mutation analysis for the BRCA1 gene was carried out in patient DNA from 26 families with multiple cases of breast/ovarian cancers. Of note are the results of molecular biology which show that there are no germline truncating mutations in the BRCA1 gene in these 26 Cypriot breast cancer families. Furthermore, Western blot analysis revealed the presence of multiple BRCA1 bands in homogenates of tumor and normal tissues, and immunoelectron microscopy showed the presence of nuclear staining for BRCA1 antibodies.

cope : A high adherence to the Mediterranean diet (MD) was previously associated with a decreased risk of breast cancer (BC) among Greek-Cypriot women. Additionally, particular polymorphisms were shown to modulate this MD-BC association. Herein, we aimed to investigate the effect of polymorphisms-MD interactions on the levels of specific metabolites that could be related to dietary adherence or enzymatic activity, which is itself modulated by polymorphisms. Methods and results : Greek-Cypriot women who were BC controls and had the lowest or the highest MD adherence (vegetables, fruit, legumes, fish) as assessed by principal component analysis (n = 564) were included. Participants were previously genotyped for 9 polymorphisms of the one-carbon metabolism, oxidative stress and xenobiotic metabolism. The serum levels of 14 metabolites that are key players in the aforementioned pathways were measured by UPLC-MS/MS. ANCOVA was used to assess polymorphism-MD interactions on metabolites’ levels within a multivariate linear regression model. Statistically significant interactions between GSTM1 deletion polymorphism and MD on flavin mononucleotide (FMN) and on 5-methyltetrahydrofolate (5-MTHF) concentrations were observed. The MTHFR rs1801133 interacted significantly with MD on 5-MTHF concentration. Conclusion : Serum levels of FMN and 5-MTHF were shown to be influenced by interactions between GSTM1 deletion or MTHFR (rs1801133) polymorphisms and a dietary pattern, characteristic of MD. This article is protected by copyright. All rights reserved

In Cyprus all couples carrying α0-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for α-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)n repeat polymorphic site located between the ψα1- and α2-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two α-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)n repeat polymorphisms analysis. No misdiagnosed cases for α-thalassaemia have been reported to date. Copyright © 2001 John Wiley & Sons, Ltd.

The Biopsy Analysis Support System (BASS), previously used for image analysis of immunohistochemically stained sections of breast carcinoma, has been extended to include indexing and content-based retrieval of biopsy slide images from a database of 57 captured cases. Images from histopathological biopsy slides are described and these are accessed in terms of the properties of either individual nuclei or groups of cell nuclei present in the slide. Visual similarity of cases is specified in terms of a diagnostic index, commonly known as the H-score, which incorporates the heterogeneity of nuclear staining intensity, as well as the percentage of nuclei staining at specific intensities. The system provides a platform that can be exploited in telepathology and teleconsultation, but further research is needed to explore its full potential and accuracy in a diagnostic clinical environment.

RAK antigens p120, p42, and p25 exhibit molecular and immunological similarity to the proteins encoded by HIV-1 and are expressed by 95% of breast and gynecological cancer cases in women and prostate cancer cases in men. Binding of the monoclonal antibody (MAb) RAK-BrI to cancer RAK antigens has been found to be inhibited by a peptide derived from the variable loop V3 of HIV-1. Since MAb RAK-BrI has been developed against denatured froms of breast cancer proteins, and it binds to a short epitope, GRAF, this MAb does not recognize the native, three-dimensional structure of proteins. Subsequently Western blot, after electrophoretic separation in gels with SDS, has been used to detect these unique cancer markers. The current studies were focused on the immunohistochemical evaluation of the novel marker RAK. Serial sections, 5 microm thick, were cut from frozen or Formalin-fixed, paraffin-embedded tissue blocks and immunostained with MAb RAK-BrI. All of the 53 cases of breast cancer tested RAK positive and no differences were observed in the immunohistochemical staining of lobular and ductal carcinoma cases. In contrast, MAb RAK-BrI antigens were detected in only 3 of 15 cases of macroscopically normal breast removed during mastectomy for breast cancer. It is noteworthy that Western blots of breast samples from the same series demonstrated a high expression of three RAK antigens in 20/20 of invasive breast carcinomas, while there was only a very weak expression of RAK antigens in 2/7 of the macroscopically "normal" breast samples. Due to the suspected viral origin of RAK markers, immunohistochemical staining with MAb RAK-BrI might be a useful tool in the early detection of malignant changes occurring in breast tissues.

Patients with beta-thalassaemia major frequently suffer from hypersiderosis which leads to hemochromatosis of major organs such as the heart and liver. Little information exists about the ultrastructural pathology of the human heart in beta-thalassaemia patients. Five Cypriot patients with elevated blood ferritin and intractable heart failure were investigated. Cardiac biopsies from these patients were studied by light and electron microscopy, as well as by X-ray microanalysis. Ultrastructural examination revealed the presence of disrupted myocytes showing loss of myofibers, dense nuclei, and a variable number of pleomorphic electron dense granules. These cytoplasmic granules or siderosomes consisted of iron-containing particles as confirmed by X-ray microanalysis. It is likely that the ultrastructural changes observed in myocytes of patients with beta-thalassaemia are largely due to iron deposition.

A detailed morphometric analysis of glomerular basement membrane (GBM) thickness was carried out on biopsies from 16 patients exhibiting normal histology and unremarkable immunofluorescence. Eleven of these patients presented with proteinuria, 8 in the nephrotic syndrome range, while 5 had hematuria as well. The remaining 5 patients presented with hematuria only. Eight patients had an initial diagnosis of minimal change disease, 4 were diagnosed as thin-membrane nephropathy, 2 had Alport syndrome, and the remaining 2 had hypertensive nephropathy. Quantitative morphometric analysis of GBM identified 3 subsets of patients. The first subset consisted of 6 patients: 5 adults, with an average GBM width of 361 +/- 34 nm, and 1 child. The second subset included 8 patients with thin GBMs and a mean thickness of 253 +/- 15 nm. The last subset comprised 2 patients with Alport syndrome showing marked variability in GBM thickness. This study has confirmed the presence of thin GBMs in hematurics, but has also revealed GBM thinning in 50% of patients with a diagnosis of minimal change disease.

acute renal failure; pregnancy; partial lipo- towards the end of the gestation period, between 34dystrophy; low C3; membranoproliferative nephritis and 38 weeks, and it is invariably the result of pre-eclampsia and its complications or the result of severeblood loss [5–7]. Occasionally post-infectious glom-erulonephritis may occur during pregnancy and can be

We describe a large Cypriot family with an interstitial type of nephropathy, inherited as an autosomal dominant trait that led to end stage renal failure between 51 to 78 years of age (mean 62.2 years). Twenty-three people are known to be affected, but several younger relatives with normal renal function may remain undiagnosed because of the absence of precise clinical and laboratory diagnostic criteria. This nephropathy is associated with medullary renal cysts, hypertension, hyperuricemia, and gout. Several relatives have typical medullary cystic disease (MCD), while in the others the findings are compatible with this diagnosis. Due to the similarity of clinical and pathologic findings, earlier reports had suggested that MCD may be allelic to autosomal recessive familial juvenile nephronophthisis, which was mapped recently to chromosome band 2q13. Linkage analysis of the present family with a closely linked marker excluded linkage to the above locus. Linkage was also excluded to the PKD1 locus of adult polycystic kidney disease type 1, and up to 5 cM on either side, on chromosome 16. We suggest that because of the element of hyperuricemia and gout found in this family, although with reduced penetrance, it may represent a variant of autosomal dominant MCD of the adult type. This variability may be the result of allelic or locus heterogeneity. Molecular genetic approaches including linkage analysis on appropriate families will certainly assist in classifying such related genetically heterogeneous disorders.

A computer-aided detection system for tissue cell nuclei in histological sections is introduced and validated as part of the Biopsy Analysis Support System (BASS). Cell nuclei are selectively stained with monoclonal antibodies, such as the anti-estrogen receptor antibodies, which are widely applied as part of assessing patient prognosis in breast cancer. The detection system uses a receptive field filter to enhance negatively and positively stained cell nuclei and a squashing function to label each pixel value as belonging to the background or a nucleus. In this study, the detection system assessed all biopsies in an automated fashion. Detection and classification of individual nuclei as well as biopsy grading performance was shown to be promising as compared to that of two experts. Sensitivity and positive predictive value were measured to be 83% and 67.4%, respectively. One major advantage of BASS stems from the fact that the system simulates the assessment procedures routinely employed by human experts; thus it can be used as an additional independent expert. Moreover, the system allows the efficient accumulation of data from large numbers of nuclei in a short time span. Therefore, the potential for accurate quantitative assessments is increased and a platform for more standardized evaluations is provided.

Thin basement membrane nephropathy (TBMN) or benign familial hematuria, is one of the commonest kidney disorders, characterized by recurrent benign hematuria, which is often associated with a family history (Gregory, 2005). It is generally considered to be a nonprogressive, life long disorder with a rather benign course (Tryggvason & Patrakka, 2006), although data from some studies show that a proportion of patients manifest more severe symptoms, than originally described and eventually develop endstage renal disease (ESRD) (Dische, Weston, & Parsons, 1985; Frasca et al., 2005; Pierides et al., 2009; Tiebosch et al., 1989). TBMN was first described in 1926 (Baer, 1926) and at presentation is usually characterized by microscopic painless hematuria, by little or negligible proteinuria and normal renal function. TBMN is an autosomal dominant disorder and more than 50% of the cases have a family history of hematuria. In about 40% of families with a confirmed diagnosis of TBMN the condition co-segregates with heterozygous COL4A3/COL4A4 mutations (Lemmink et al., 1996). Mutations in the type IV collagen gene family which also includes the COL4A5 gene, also cause Alport syndrome (Hostikka et al., 1990). It is believed that these disorders are the result of defective, synthesis and/or assembly, of the critical glycoprotein components, that form the glomerular basement membranes (GBMs), among which type IV collagen, is the major constituent. Clinically TBMN must be differentially diagnosed, between IgA nephropathy and Alport syndrome which are the other two main causes of hematuria, particularly in children. The need for differential diagnosis, often necessitates the examination of a kidney biopsy at the electron microscopical level, which also enables measurement of the thickness of the GBMs. Indeed Rogers et al (Rogers, Kurtzman, Bunn, & White, 1973) were the first to associate recurrent benign hematuria with the presence of thin GBMs. Currently the most widely

Renal podocytes and their slit diaphragms ensure the integrity of renal basement membrane and prevent urinary protein loss. We have previously reported that decreases of the podocyte slit diaphragm proteins nephrin and podocin represent early events in the podocytopathy of lupus nephritis (LN). We asked whether immunosuppressive agents such as glucocorticoids and cyclophosphamide may have direct effects on podocytes. We assessed in New Zealand Black/New Zealand White (NZB/W) F1 LN mice glomerular nephrin and podocin expression and localization by the use of Western blot and immunofluorescence; mRNA levels were measured by real-time polymerase chain reaction (PCR) and renal histology by light and electron microscopy. Early treatment with glucocorticoids and cyclophosphamide halted the histologic alterations associated with LN, preserving podocyte foot processes. Nephrin and podocin protein expression significantly increased in both glucocorticoid and cyclophosphamide groups as early as after three months of therapy. Real-time PCR revealed similar enhancement in nephrin and podocin mRNA levels after three to six months of treatment. This study documents that early treatment in experimental LN with glucocorticoids or cyclophosphamide preserves slit diaphragm proteins in podocytes and halts histological changes of the glomeruli, thus raising the possibility of a direct protective effect of these drugs on podocytes.

Cilia and flagella are hair-like structures which extend from the cell membrane and are involved in a variety of biological functions in eukaryotes. Cilia are found in a wide range of human tissues and cell types including: the epithelium of the upper and lower respiratory tracts, the epithelium of the kidney, bile and pancreatic ducts, the embryonic node, and the ependymal cells in the brain and in the oviduct epithelium.

Background Diet has long been suspected to impact on breast cancer risk. In this study we evaluated whether the degree of adherence to a Mediterranean diet pattern modifies breast cancer risk amongst Greek-Cypriot women. Methods Subjects included 935 cases and 817 controls, all participating in the MASTOS case-control study in Cyprus. The study was approved by the Cyprus National Bioethics Committee. Information on dietary intakes was collected using an interviewer administered 32-item Food Frequency Questionnaire. Information on demographic, anthropometric, lifestyle, and other confounding factors was also collected. Adherence to the Mediterranean Diet pattern was assessed using two a-priory defined diet scores. In addition, dietary patterns specific to our population were derived using Principal Component Analysis (PCA). Logistic regression models were used to assess the association between the dietary patters and breast cancer risk. Results There was no association with breast cancer risk for either score, however, higher consumptions of vegetables, fish and olive oil, were independently associated with decreased risk. In addition, the PCA derived component which included vegetables, fruit, fish and legumes was shown to significantly reduce risk of breast cancer (ORs across quartiles of increasing levels of consumption: 0.89 95%CI: 0.65-1.22, 0.64 95%CI: 0.47-0.88, 0.67 95%CI: 0.49-0.92, P trend < 0.0001), even after adjustment for relevant confounders. Conclusions Our results suggest that adherence to a diet pattern rich in vegetables, fish, legumes and olive oil may favorably influence the risk of breast cancer. This study is the first investigation of dietary effects on breast cancer risk in Cyprus, a country whose population has traditionally adhered to the Mediterranean diet.

Proteomic analysis of breast cancer tissue has proven difficult due to its inherent histological complexity. This pilot study presents preliminary evidence for the ability to differentiate adenoma and invasive carcinoma by measuring changes in proteomic profile of matched normal and disease tissues. A dual lysis buffer method was used to maximize protein extraction from each biopsy, proteins digested with trypsin, and the resulting peptides iTRAQ labeled. After combining, the peptide mixtures they were separated using preparative IEF followed by RP nanoHPLC. Following MALDI MS/MS and database searching, identified proteins were combined into a nonredundant list of 481 proteins with associated normal/tumor iTRAQ ratios for each patient. Proteins were categorized by location as blood, extracellular, and cellular, and the iTRAQ ratios were normalized to enable comparison between patients. Of those proteins significantly changed (upper or lower quartile) between matched normal and disease tissues, those from two invasive carcinoma patients had >50% in common with each other but

Inability to correctly repair DNA damage is known to play a role in the development of breast cancer. Single nucleotide polymorphisms (SNPs) of DNA repair genes have been identified, which modify the DNA repair capacity, which in turn may affect the risk of developing breast cancer. To assess whether alterations in DNA repair genes contribute to breast cancer, we genotyped 62 SNPs in 29 genes in 1,109 Cypriot women with breast cancer and 1,177 age-matched healthy controls. Five SNPs were associated with breast cancer. SNPs rs13312840 and rs769416 in the NBS1 gene were associated with a decrease in breast cancer risk (OR TT vs. TC/CC = 0.58; 95% CI, 0.37–0.92; P = 0.019 and OR GG vs. GT/TT = 0.23, 95% CI 0.06–0.85, P = 0.017, respectively). The variant allele of MRE11A rs556477 was also associated with a reduced risk of developing the disease (OR AA vs. AG/GG = 0.76; 95% CI, 0.64–0.91; P = 0.0022). MUS81 rs545500 and PBOV1 rs6927706 SNPs were associated with an increased risk of developing breast cancer (OR GG vs. GC/CC = 1.21, 95% CI, 1.02–1.45; P = 0.031; OR AA vs. AG/GG = 1.53, 95% CI, 1.07–2.18; P = 0.019, respectively). Finally, haplotype-based tests identified significant associations between specific haplotypes in MRE11A and NBS1 genes and breast cancer risk. Further large-scale studies are needed to confirm these results.

Thin basement membrane nephropathy is one of the main causes of hematuria in both children and adults. It is often associated with a family history and its true incidence is unknown. Accurate diagnosis of thin basement membrane nephropathy relies on the presence of attenuated glomerular basement membranes, a finding that can be appreciated only by examination in the electron microscope. In Cyprus the department of electron microscopy has received 990 consecutive renal biopsies for diagnosis. The aim of this study is to define the incidence of thin basement membrane nephropathy in this population sample based on the results of electron microscopy.

Cardiac damage caused by iron overload toxicity is the main cause of death in thalassemia patients. Biopsy samples of poorly chelated thalassemia patients who suffered congestive cardiac failure (CCF) show extensive iron deposition in the myocardium. In one patient who survived CCF, a cardiac biopsy was performed during the removal of a thrombus caused by a port-a-cath, which was used for the administration of intravenous (iv) deferoxamine (DFO). Ultrastructural pathology studies of the cardiac biopsy indicated extensive iron deposition in myocytes with accumulation of iron mainly in lysosomes, leading in some cases to their disruption. Damage to other intracellular components of the myocytes and loss of myofibers was also observed. The patient became intolerant to iv and subcutaneous (sc) DFO 2 years after the CCF, and was then treated with deferiprone (L1) for 7 years. Within 1 year of L1 treatment at 75-80 mg/kg/day, serum ferritin levels were reduced to0.45 mg/L and she became asymptomatic, needing no further drugs for her cardiomyopathy. Lowering the L1 dose to 50-70 mg/kg/day caused an increase in serum ferritin levels. Maintenance of normal iron stores during the last 3 years as detected by cardiac and liver magnetic resonance imaging (MRI) T2 and T2* and normalization of serum ferritin levels (0.15 mg/L) was observed following L1 therapy at 80-85 mg/kg/day. Deferiprone (80 mg/kg/day) appears to be effective in the rapid clearance of cardiac iron, in the reversal of iron overload related cardiomyopathy, in the maintenance of normal iron stores and the overall long-term survival of thalassemia patients.

Introduction. Detection of the very earliest stages of breast cancer remain challenging and the differentiation of those carcinomas that develop into aggressive invasive forms from those that remain benign has not been achieved. Consequently, removal of the tumor by total mastectomy, local excision (LE) plus adjuvant radiotherapy (RT), or LE alone remain the main treatments, even if they prove unnecessary. Previously, using proteomics analysis, we identified proteins that were significantly changed in expression between matched normal and tumor tissues from Cypriot patients with different stages of the disease. Heat shock protein 10 (HSP10), which is responsible for the mitochondrial protein folding, was one of the proteins identified. A range of methods were used to verify HSP10 as a possible biomarker for breast cancer. Materials and Methods. Protein extracts from breast cancer cell lines and breast tissue biopsies from Cypriot patients with different stages of the disease were analysed by (i) Western Blotting (WB) and (ii) multiple reaction monitoring mass spectrometry (MRM MS) using two proteotypic peptides unique to HSP10, in proteins extracted form fresh frozen as well as from FFPE breast tissues. Tissue microarray analysis (TMA) was carried out on core biopsies from the Leeds Breast Tissue Bank. Results. WB indicated that HSP10 was ubiquitously and evenly expressed across breast cancer cell lines representative of different phenotypes. WB and MRM MS revealed that expression was significantly increased in invasive carcinoma compared to normal tissue, but was patient specific for fibroadenoma and DCIS cases. TMA of 360 samples indicated a strong correlation between staining and type of breast cancer (invasive ductal and lobular), grade and ER positive phenotype. Discussion. Proteomics analysis using iTRAQ quantitation indicated an average of 1.6 fold increase in HSP10 in invasive carcinomas compared to matched normal tissue. By comparison, WB and MRM MS analysis indicated a significantly amplified discrimination in the expression of HSP10 as has been observed with other biomarker candidates. HSP10 protein levels were in agreement with mRNA results observed in breast cancer cell lines reported in the Cancer Cell Line Encyclopaedia. In conclusion, HSP10 represents an excellent biomarker candidate for further validation. Citation Format: Sadr-ul Shaheed, Andreas Hadjisavvas, Kleitos Sokratous, Paul Loadman, Chris Sutton, Kyriacos Kyriacou. Analysis of HSP10 as a putative biomarker of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2015-1576

Autosomal-dominant medullary cystic kidney disease type 1: Clinical and molecular findings in six large Cypriot families . Background Autosomal-dominant medullary cystic kidney disease (ADMCKD), a hereditary chronic interstitial nephropathy, recently attracted attention because of the cloning or mapping of certain gene loci, namely NPHP1, NPHP2 and NPHP3 for familial juvenile nephronophthisis (NPH) and MCKD1 and MCKD2 for the adult form of medullary cystic kidney disease. Our aim was to present and discuss the clinical, biochemical, sonographic and histopathological findings in six large Cypriot families in whom molecular analysis has confirmed linkage to the MCKD1 locus on chromosome 1q21. Methods The clinical, biochemical, sonographic and histopathological findings in 186 members of six large Cypriot families with ADMCKD-1 are presented. Creatinine clearance was calculated according to the Cockroft-Gault formula and was corrected to a body surface area (BSA) of 1.73m 2 . DNA linkage analysis was performed with previously identified flanking polymorphic markers. Results This disease is characterized by the absence of urinary findings in the vast majority of patients, leading to end-stage renal failure (ESRF) at a mean age of 53.7 years. Hypertension and hyperuricemia are common, especially in males, the former encountered more frequently in advanced chronic renal failure (CRF). Gout has been noted in a small percentage of male patients. Loss of urinary concentrating ability was not a prominent early feature of the disease, while severe natriuresis was observed in a few males toward ESRF. Renal cysts are mainly corticomedullary or medullary, and they are present in about 40.3% of patients and appear more frequently near ESRF. Conclusion ADMCKD type 1 is a common cause of ESRF among our dialysis population. The disease is difficult to diagnose clinically, particularly in the early stage when renal cysts are not usually present, making them a weak diagnostic finding. A dominant pattern of inheritance and DNA linkage analysis are helpful in the diagnosis of this disease.

A novel form of autosomal recessive distal hereditary motor neuronopathy (distal HMN) is reported. The presence of pyramidal signs within the early stages of the disease with persistence of knee hyperreflexia form distinctive clinical features. We have mapped the HMN-J gene to chromosome 9p21.1-p12, within an estimated interval of 1.2-Mb.

Accurate diagnosis of muscle disease is dependent on a careful clinical examination followed by the appropriate laboratory investigations, which in a contemporary diagnostic center should also include ultrastructural investigations. As is the case in other tissues, the interpretation of the ultrastructural abnormalities observed in muscle must take into consideration several factors, in particular the small sample size, possible artifacts, and the nonspecificity of changes. Despite the fact that the majority of ultrastructural changes seen in muscle are not specific, electron microscopic examination still provides important and unique clues regarding patterns of change that characterize certain disease entities. Since this detailed ultrastructural information cannot at present be obtained by any other means, it is anticipated that electron microscopy will still play a vital role in the diagnosis of the nonneoplastic muscle diseases, well into the twenty-first century.

Introduction. Despite oestrogen and human epidermal growth factor receptor (HER2) targeted therapies improving patient survival, breast cancer continues to be a significant cause of premature death since many tumors do not respond to treatment or acquire resistance. Hence there remains the need to identify novel targets for therapy and biomarkers for prediction of response to treatment. A quantitative proteomics study of matched normal and tumor biopsies identified 63 proteins to be significantly increased or decreased in stage-specific tumors. Some have previously been associated with breast cancer, while others such as cofilin-1 represent new targets for investigation. Cofilin-1 was subject to a range of analyses to determine its association with breast cancer. Materials and Methods. Western blotting (WB) was performed on protein extracts from breast cancer cell lines and matched normal and tumor biopsies from Cypriot patients with different stages of the disease. Immunohistochemistry (IHC) was carried out on core biopsies from the Leeds Breast Tissue Bank. Multiple reaction monitoring (MRM) mass spectrometry was performed on trypsin-digested protein extracts from biopsies. Data from protein (Human Protein Atlas) and genomics (BioGPS, TiGER, UniGene) databases were assimilated with the experimental results. Results. WB indicated that cofilin-1 was ubiquitously expressed in tumor cell lines, representative of Luminal A, Luminal B, basal-like, claudin-low and HER2 phenotypes, at higher levels than normal breast cell lines. WB also indicated increased expression of cofilin-1 in invasive carcinoma tissues, compared to matched normal. Patients with ductal carcinoma in situ or fibroadenoma exhibited less clear results, either increasing slightly or remaining unchanged. MRM analysis of three cofilin-1 peptides in tissue extracts of invasive carcinoma patients indicated expression only in tumor. IHC of core biopsies exhibited strong staining for cofilin-1 in ductal invasive carcinoma tissues with no staining in normal breast cells. Genomics and proteomics databases indicate that cofilin-1 is expressed in a diverse range of normal tissues including major organs, gastro-intestinal tract, skin and tonsils. However, mRNA and protein levels are observed to be increased in skin, lung, liver, pancreatic and breast tumors. Discussion. The original quantitative proteomics data indicated only relatively small changes in expression (less than 2-fold) due to dynamic range limitations of the technique. Additional analytical approaches substantiated that cofilin-1 is significantly up-regulated in advanced breast cancer patients. Further patient sets are required to confirm the importance of cofilin-1 levels in early stages of breast cancer compared to benign tissues. Bioinformatics provided further confidence in our findings, highlighting the value of utilising databases for evidence of disease-specific proteins. Citation Format: Chris Sutton, Sadr ul-Shaheed, Paul Loadman, Valerie Speirs, Andrew Hanby, Andreas Hadjisavvas, Kyriacos Kyriacou. Expression profiling of cofilin-1 in breast cancer cell lines and biopsies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2505. doi:10.1158/1538-7445.AM2013-2505

Breast cancer is the most common malignancy affecting the female population in industrialized countries. Prognostic factors, such as steroid receptors visualized in biopsy slides, provide critical information to oncologists regarding the hormonal status of the individual tumors. These factors influence the choice of treatment and help in predicting patient survival and probability of recurrence. The objective of this paper is to introduce a new computer-aided system for the classification of breast cancer nuclei based on neural networks. Currently, medical experts assess steroid receptors in breast cancer biopsy slides mostly manually using four- or five-level grading schemes. These schemes are based on the assessment of two parameters: number of nuclei positive and their staining intensity. Available computerized systems define their own grading schemes based on automated measurements of low-level features, such as optical density, texture, area, and others. However, the findings produced by these systems may not be readily comprehensible by the majority of medical experts who have been accustomed to manual assessment schemes. Moreover, findings from one system cannot be directly compared to findings obtained from other computerized systems. To date, no standardized assessment scheme exists for computerized systems, while interobserver and intraobserver variabilities limit the utility of the routinely used manual assessment schemes. In this paper a new system for computer-aided biopsy analysis is introduced. Here, we focus on the systeJ;Il’s nuclear classification module. The input to this module consists of a set of six local and global features: optical density, two chromaticity indices, a variance based texture measure, global nuclei density mean, and variance. The output of the nuclei classification module consists of a membership label in a zero to four grading scheme for each detected nucleus. The classification module is based on a feedforward neural network trained in a supervised fashion to classify the nuclear feature vectors. The sample data comprises 3015 nuclei from 28 images that were classified by a human expert. A Sammon plot visualization of the six dimensional input feature space shows that the classification problem is quite difficult. The neural network used in the classification module achieved 72% accuracy. Our results indicate that by using a nuclear classification module such as the one introduced in this paper it is possible to translate low-level system measurements into a vocabulary that is familiar to medical experts. Thus, a contribution is made to the standardization of grading schemes in addition to improving the accuracy in grading breast cancer nuclei.

We present a case of third trimester pregnancy occurring in a 29 year old woman after intracytoplasmic sperm injection (ICSI) treatment with spermatozoa from a globozoospermic man. We believe this is the first reported case. A 37 year old man was diagnosed with globozoospermia with normal sperm count and motility. In-vitro fertilization and subzonal insemination treatments failed to achieve fertilization of any eggs. The ICSI method produced 50% fertilization, 75% cleavage rate and a singleton pregnancy with a female fetus. No pregnancy or fetal abnormalities have been noted after > 7 months of gestation.

Introduction – Identification of markers of different stages of breast cancer remains a challenge. Although biofluids such as urine and plasma are preferred sources to discover potential biomarkers, tissue proteins are diluted compared to their site of expression and difficult to analyse due to degradation or due to the wide dynamic range of plasma proteins. As mastectomy is frequently used as part of treatment, it is possible to get sufficient histologically-defined tissue to perform proteomics profiling at the source of the disease and therefore more accurately identify changes in expression, for subsequent biomarker verification. Two dimensional separation of stable isotope-labelled total peptide mixtures from matched normal and disease biopsies from 10 patients with adenoma, DCIS or invasive carcinoma were compared in order to identify markers of different stages of the disease. Methods – Two lysis buffers, RIPA and urea, in combination with sonication, were used sequentially to maximise protein extraction from matched normal and diseased breast biopsies of 10 patients. The biopsies were split into three groups each comprising matched normal and diseased protein extracts from 4 patients one of which is common to all the groups to act as an internal control. Each protein extract in each group was trypsin-digested and iTRAQ (isobaric tag for relative and absolute quantitation) labelled. The digests from each group were pooled together and the resulting peptide mixture separated using isoelectric focusing followed by reverse phase nanoHPLC. Fractions from nanoHPLC were analysed by MALDI (matrix assisted laser desorption/ionisation) MS and MS/MS, and the data searched against a sequence database to identify proteins in the original sample. Results – The proteins identified in each group of 4 patients were compared and those which were observed in all three data sets, were subjected to further quantitative analysis. For each protein from each patient, a normal:tumor ratio was determined from the iTRAQ data. Those proteins for each patient that were significantly changed in expression (a ratio of 1.3 equates with a significant increase) were clustered together for further processing. Those proteins whose response was changed across a patient group (adenoma, DCIS or invasive carcinoma) were investigated for associations with cancer biology. Those which may represent novel disease stage-specific biomarkers were verified using immunohistochemistry and Western blotting. Discussion – A number of proteins significantly increased or decreased have previously been associated with breast cancer. In addition, there were a number of new proteins identified which were associated with adenoma, DCIS and invasive carcinoma stages of disease, and these may be suitable targets for drug development as well as for developing potential biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4874. doi:10.1158/1538-7445.AM2011-4874

Introduction - Quantitative proteomic profiling of breast tissue has proven difficult due to its inherent histological complexity. Substantial differences in stroma, vasculature and cellular composition of normal and tumors have further exacerbated analysis. In order to address these issues, a novel strategy for proteomics analysis was developed to determine differences between normal and disease tissue and between patients with adenoma and invasive carcinoma. Methods - A dual lysis buffer method for extracting proteins, in combination with sonication, was used to maximise recovery from normal and diseased breast biopsies. Protein extracts from matched normal breast and tumors of three patients (one with adenoma and two with invasive carcinoma) were trypsin-digested, were iTRAQ (isobaric tag for relative and absolute quantitation) labelled, pooled and the resulting peptide mixture separated using isoelectricfocusing in the first dimension and reverse phase nanoHPLC in the second dimension. The eluate from nanoHPLC was collected onto matrix assisted laser desorption/ionisation plates, analysed by MALDI MS and MS/MS, the subsequent data searched against a sequence database to identify proteins in the original sample. Results - A total of 481 proteins with associated normal: tumor iTRAQ ratios for each patient. iTRAQ ratios were indicative of increased or decreased levels of each protein in tumor compared to normal tissue. Proteins were categorised by location as blood (13% of the identified proteins), extracellular (12%) and cellular (75%). The average ratios for each group of proteins provided a gross indication of the relative levels of each tissue component in the matched normal and tumor biopsies and demonstrated marked difference in adenoma compared to invasive carcinoma. iTRAQ ratios were normalised to enable comparison of significantly changed proteins between patients. There were many cellular proteins in common between the two invasive carcinoma patients (54 increased and 51 decreased in tumors) but few between either of the invasive carcinoma patients and the adenoma patient. Discussion - A number of proteins significantly increased or decreased have previously been associated with breast cancer (Tropomyosin beta chain, Periostin, Small breast epithelial mucin and Breast cancer estrogen-inducible protein were increased and Selenium-binding protein 1, Secreted frizzled-related protein 1 precursor and Kinectin were decreased), supporting the approach we have developed for proteomics analysis of breast tissues. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5555.