Oligoclonal bands (OCB) are the most widely used CSF test to support the diagnosis of MS and to predict conversion of clinically isolated syndrome (CIS) to multiple sclerosis (MS). Since OCB tests are based on non-quantitative and difficult to standardise techniques, measurement of immunoglobulin kappa free light chains (KFLC) may represent an easier to use quantitative test.KFLC were measured in CSF and serum of 211 patients using ELISA. These include patients without any inflammatory central nervous system reaction , MS (n = 20), viral CNS infections , neuroborreliosis  and other bacterial CNS infections . Furthermore a cohort of 77 patients with CIS, including 39 patients that remained CIS over follow-up of two years (CIS-CIS) and 38 patients that developed MS over the same follow-up time (CIS-MS).CSF-serum ratio of KFLC (Q KFLC) was elevated in all patients with MS, 86.8% of patients with CIS-MS and 61.5% of patients with CIS-CIS. It was significantly elevated in CIS with presence of OCB (p<0.001). Q KFLC significantly correlated with other CSF variables such as CSF leukocyte count , CSF CXCL13 levels  and also intrathecal IgG synthesis  as determined by nephelometry and quotient diagram. OCB were detected in 66.7% of CIS-CIS and in 92.1% of CIS-MS.Although the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS. In most patients who develop multiple sclerosis (MS), the disease initially presents with a first relapse-like episode known as clinically isolated syndrome (CIS) Several studies indicated that elevated immunoglobulin kappa free light chains (KFLC) and lambda free light chains (LFLC) in the CSF may offer a quantitative tool to support the diagnosis of MS Here, we provide 1) CSF reference values for KFLC based on a highly sensitive ELISA to evaluate the relevance in MS, CIS and pathogen-related CNS diseases and 2) a systematical analysis of the prognostic relevance of KFLC regarding the occurrence of further relapses in a large and clinically well-defined cohort of patients with CIS. We compare the prognostic relevance of KFLC in CIS to MRI Barkhof criteria 211-paired CSF and serum samples from the Department of Neurology, University of Ulm (Germany) were analysed. These included 77 patients with CIS collected in a prospective study with a follow-up time of two years, as previously described The inflammatory control groups consisted of 17 patients with neuroborreliosis (NB) .MRZR  was determined as previously described (23) using an enzyme-linked immunosorbent assay (ELISA) according to the instructions as supplied by the manufacturer . Quantitative expression of the intrathecal immune response was based on calculation of the CSF/serum quotients (Q) of specific antiviral IgG antibodies, and the intrathecal synthesis of antibodies was detected by calculation of the corresponding antibody indices (AI). AI values ≥1.5 were considered to be indicative of intrathecal IgG synthesis against the respective antigen, and MRZR was considered positive if two or more AI values were ≥1.5.CXCL13 was measured using ELISA  according to the instructions as supplied by the manufacturer. Samples of 50 ml CSF and 50 ml serum were used for the ELISA. Details on the application of the assay for CSF have been published previously by our group CSF leukocyte count (cells/cu.mm), total protein (g/L), lactate (mmol/L), the albumin CSF/serum concentration ratio , immunoglobulins G, A and M were obtained as previously described OCB were detected by isoelectric focusing (IEF) on agarose gels followed by immunoblotting using an IgG-specific antibody staining. Paired CSF and serum adjusted for protein concentrations were applied in the same assay as described earlier Immunoglobulin Kappa and Lambda Free Light Chains (KFLC and LFLC) were measured by quantitative enzyme-linked immunosorbent assay (ELISA) (BioVendor) according to the instructions supplied by the manufacturer. All samples were stored at −80°C until analysis according to the sampling and storage protocol of BioMS MRI scans of the brain and spinal cord were performed on a 1.5 tesla whole-body MRI  according to a previously fixed protocol including T1-weighted spin-echo (SE) axial slices with and without application of gadolinium-DTPA as well as T2-weighted SE axial slices. Barkhof criteria Statistical analysis was performed using SPSS . Differences of FLC between groups were analyzed using Kruskal-Wallis test followed by single pairwise comparisons (Mann-Whitney U-test). The Correlation was analysed using Spearman rank order correlation. To test for normality, the Kolmogorov-Smirnov-test was applied. Sensitivity was calculated as , specificity was calculated as . The positive predictive value (PPV) was calculated as , and the negative predictive value (NPV) as . For all diagnostic values the exact 95% confidence intervals were given Patients with MS and CIS showed significantly elevated CSF KFLC levels as compared with NIND (p<0.001) . CSF KFLIn NIND, CSF KFLC correlated significantly with serum KFLC  and also significantly with CSF-serum ratio of albumin   . TherefoQ KFLC was significantly elevated in cases with presence of CSF oligoclonal IgG bands (OCB)  . Q KFLC In cases of CIS, Q KFLC was positive , CSF CXCL13 levels  and also intrathecal IgG synthesis . Q KFLC was significantly higher in CIS with positive OCB as compared to CIS without OCB p<0.001, . SimilarAll patients with MS showed elevated Q KFLC, 90% of them showed positive OCBs.Of all markers investigated, OCB showed the highest sensitivity for conversion of CIS to MS (92.1%), which could not be improved by adding any of the other parameters . KFLC weIncreasing recognition of the pathogenetic relevance of B lineage cells in MS encouraged the evaluation of B cell-associated biomarker candidates Ig free light chains (FLCs) are “sideline products” of the Ig synthesis by B lymphocytes Our observation of elevated KFLC in the CSF of patients with MS and pathogen related CNS diseases support previous studies alb, which is a widely accepted quantitative measure of blood-CSF barrier function Due to a significant correlation of CSF KFLC with serum KFLC and CSF-serum ratio of albumin  we introduce reference values of Q KFLC in relation to QAs a new aspect, we analyse the prognostic relevance of KFLC and other parameters regarding the occurrence of further relapses in a large and clinically well-defined cohort of patients with CIS. Here, we showed OCB to have the highest sensitivity (92.1%) and highest negative predictive value (81.3%) for conversion of CIS to MS as compared to Barkhof criteria (highest specificity (88.2%)). We observed Q KFLC to be nearly as sensitive as CSF OCB in predicting conversion of CIS to MS .The correlation of Q KFLC with CSF leukocyte count, intrathecal IgG synthesis and the B cell attracting chemokine CXCL13 possibly is in line with the polyspecific intrathecal B cell response in MS So far, OCB are the most widely used CSF test to support the diagnosis of MS and to predict conversion of CIS to MS. OCB s in CSF that are absent in serum indicate intrathecal IgG synthesis. In the classification by Andersson et al 1994 The significant correlation of Q KFLC with positive OCB  supportsIn conclusion, our data indicates the relevance of KFLC as a marker of polyspecific IgG response in the CSF of MS and CIS patients. Similar to oligoclonal bands KFLC add information in predicting conversion of patients with CIS to MS. Although the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS. Additionally, these findings underline the relevance of CSF parameters in MS and CIS. Although CSF analysis is no longer a mandatory part of diagnostic criteria in MS


Immunotherapy for the treatment of breast cancer can be categorized as either (a) specific stimulation of the immune system by active immunization, with cancer vaccines, or (b) passive immunization, such as tumor-specific antibodies (including immune modulators) or adoptive cell therapy that inhibit the function of, or directly kill, tumor cells. We will present the current information and the future perspectives of immunotherapy in patients with breast cancer, including the prognostic role of tumor infiltrating lymphocytes, immune signatures, targeted therapies modulating the immune system, and tumor antigen cancer vaccines. Active immunotherapy in breast cancer and its implementation into clinical trials have been largely a frustrating experience in the last decades. The concept that the immune system regulates cancer development is experiencing a new era of interest. It is clear that the cancer immunosurveillance process indeed exists and potentially acts as an extrinsic tumor suppressor. Also, the immune system can facilitate tumor progression by sculpting the immunogenic phenotype of tumors as they develop. Cancer immunoediting represents a refinement of the cancer immunosurveillance hypothesis and resumes the complex interaction between tumor and immune system into three phases: elimination, equilibrium, and escape. Major topics in the field of immunology deserve a response: what do we know about tumor immunogenicity, and how might we therapeutically improve tumor immunogenicity? How can we modulate response of the immune system? Is there any gene signature predictive of response to immune modulators? The success of future immunotherapy strategies will depend on the identification of additional immunogenic antigens that can serve as the best tumor-rejection targets. Therapeutic success will depend on developing the best antigen delivery systems and on the elucidation of the entire network of immune signaling pathways that regulate immune responses in the tumor microenvironment. Evading immune destruction is an emerging hallmark of cancer. The immune system plays a dual role in cancer: it not only can suppress tumor growth by destroying cancer cells or inhibiting their outgrowth but also promotes tumor progression either by selecting for tumor cells that are more fit to survive in an immunocompetent host or by establishing conditions within the tumor microenvironment that facilitate tumor outgrowth. The conceptual framework called ‘cancer immunoediting’ integrates the immune system’s dual host-protective and tumor-promoting roles. Nonetheless, numerous studies have shown that tumors can be recognized and contained for extended periods of time by the immune response through the concerted action of the innate and adaptive immune responses . DespiteIn BC, recent evidence has demonstrated that immune-related factors play an important role in defining patient prognosis and their response to treatment. These include the extent of lymphocyte infiltration in tumor tissue and a class of gene expression signatures, both of which have the potential to more precisely define patients’ clinical evolution and identify patient subgroups with different sensitivities to standard treatments. Despite these new insights, clinicians still rely primarily on classic clinical-pathological features such as tumor size and lymph node involvement for daily patient management, and it is difficult to see how these parameters may be implemented in the clinic in the future. This review will highlight the importance of exploring the immune system in both research and clinical settings, since its role in defining BC behavior is proving to be a significant factor.P = 0.007). In locally advanced BC treated with neoadjuvant chemotherapy, the presence of TILs in the primary biopsy predicts pathologic complete response [+ and CD20+ cells. The same group has recently evaluated the prognostic and predictive value of TILs in a large cohort of lymph node-positive early-BC patients prospectively randomly assigned to receive either high-dose anthracycline-based chemotherapy or a combinatorial regimen involving anthracyclines and docetaxel within the BIG02-98 trial [+ were strongly associated with good prognosis among patients with TN BC, whereas TILs had no prognostic value among patients with HER2+ BC. However, TILs+ were associated with improved therapeutic responses to high-dose anthracyclines in patients with HER2+ BC [− to TIL+ and that the post-chemotherapy TIL+ status was associated with an improved clinical response. Furthermore, Ladoire and colleagues [+ infiltration in the tumor. This post-treatment infiltration is associated with an improved outcome. It is worthy to highlight that one of the proposed mechanisms of action of chemotherapy is the induction of an anti-tumoral immune response. The group led by Zitvogel [+ BCs present specific features that could have specific clinical implications in terms of prognosis. These data from population-based studies or randomized trials will need additional validation using a prospective trial before implementation. A potential implication could be to use TILs as inclusion criteria in large adjuvant trials that include TN BC or HER2-positive BC. Another potential prognostic implication relates to the potentially good prognosis of patients who were TIL− at baseline but became TIL+ after neoadjuvant therapy [+ is associated with good outcome is an argument to develop immune strategies in patients with BC. TIL assessment could identify a subset of TIL− TN and HER2+ BC patients who deserve additional therapy. Genomic predictor of chemo-immunization could represent a new prognostic parameter that will allow clinicians to select which TIL− BCs are candidates for new therapeutics in the (neo)adjuvant setting, thus decreasing toxicity, decreasing cost, and making feasible (neo)adjuvant trials. TILs are represented mainly by non-activated T cells which often become activated after exposure to chemotherapy. More recently, the nature of TILs has been better characterized. Ruffell and colleagues [+/CD56− T cells but that a minority consisted of natural killer (NK) cells or CD20+ cells. The majority of CD3+ cells were either CD4+ or CD8+ T cells. Interestingly, CD8+ cells did not express Granzyme B at baseline, which means that they did present inactivation status, but they did express Granzyme B after neoadjuvant chemotherapy in one third of the patients. Lastly, a minority of TILs presented T and NK cell features [+ tumors are important to understand which molecular mechanisms lead to lymphocyte infiltration. Genomic instability may promote anti-tumor immune response through tumor-associated antigens. Some mechanisms of chemokine release by the tumor have been described and correlated with lymphocyte attraction. TILs have been associated with CXCL9 and CXCL13 expression by the tumor [+ tumors present a specific methylation pattern on immune-related genes, including CCL5 [Over the past few decades, a growing body of evidence has emerged demonstrating that the immune system participates both in tumor development (via chronic inflammation orchestrated by the innate immune system) and in tumor elimination and control (through the actions of the adaptive immune system) . The preresponse . In this98 trial . TILs+ wHER2+ BC . InteresHER2+ BC  have replleagues  have repZitvogel  has repoZitvogel  that TIL therapy . Neverthlleagues  have repfeatures . The gening CCL5 . A clusting CCL5 .+ subtypes since these tumors are by ‘nature’ highly proliferative. Several investigators have tried to overcome the limitations of these ‘first generation’ signatures by focusing on the BC microenvironment or immune response (or both) to define promising ‘second generation’ prognostic signatures  study) in which patients with ERcyclines . The immsurvival ,38. Antisurvival ,40. Corrapproach .ex vivo-activated T cells and NK cells, or blockade of Treg cells could be useful to amplify the anti-tumor response. Forkhead box P3 (FOXP3)+ Treg cells are crucial for the induction and maintenance of peripheral tolerance to self-antigens. While exerting their function, Treg cells can also suppress immune responses to tumor antigens, alloantigens, and allergens [In recent years, the better knowledge of BC biology has provided an opportunity to develop some types of immunotherapy to overcome the relative non-immunogenic property of BC and improve immune response. Some molecules such as PD-1 and its ligand PDL-1, CTLA-4, and immune cells such as regulatory T (Treg) cells are involved in the induction of tolerance to antigens, and their upregulation is associated with increased risk of developing BC . The mAbllergens ,43. The llergens . FOXP3 wllergens . PD-1 isllergens  showed tAnother interesting immune molecule is CTLA-4 (CD152), which is similar to PD-1, but its immune-inhibitory signals are different. CTLA-4 knockout mice display early lethality, unlike PD-1 knockouts, which demonstrate late-onset and organ-specific autoimmunity. Anti-CTLA4 mAb treatment has shown robust tumor responses in phase III trials, but with considerable adverse events . Still, + BC may improve outcome by involving and enhancing critical host immunity [Hence, immunotherapeutics that augment CD8 T-cell anti-tumor activity - such as anti-PD1 and anti-CTLA4 mAbs - given in combination with trastuzumab in patients with HER2immunity ,48,49.Given this evidence, the evaluation of baseline immune response and the identification of easy-to-define surrogate markers of immune system activation could be helpful in the management of BC to identify patients who may benefit from these combination therapies, even eliminating the need for combination cytotoxic chemotherapy.de novo aberrant expression. Many TAAs  have been identified and been shown to be specifically recognized by T cells [Vaccines constitute an active and specific immunotherapy designed to stimulate the intrinsic anti-tumor immune response by presenting tumor-associated antigens (TAAs) expressed on normal tissues that are overexpressed on tumor cells. Malignant cells can express both normal self-antigens and specific TAAs that arise from genetic mutations or epigenetic changes or both, recognized by the immune response through either their loss or  T cells . InductiThe identification of immunological and genetic features affecting immune response in patients with minimal tumor burden represents the optimal background for development of clinical studies in the adjuvant setting as it should allow the immune system to mount a response before it is overtaken. An active immunization may provide a non-toxic therapeutic modality able to induce anti-tumor immune responses in patients with cancer . HoweverWe have provided an overview of the recent evidence demonstrating that the immune system plays a critical role in defining BC prognosis and response to different treatments. However, more studies are necessary to precisely define the nature and role of TILs, establish the clinical value of immune-based prognostic and predictive gene signatures, and optimize standard treatments to effectively work in tandem with the anti-tumor immune response. Several questions are raised by all remarkable data presented in this review; answers to these questions should be considered possible areas of research in the following years. Should all patients with cancer be treated with an active immunotherapy approach or only individuals potentially more ‘responding’? How can we predict that the individual will develop an immune response against a particular antigen used in the vaccine formulation? Is there any genetic signature predicting response to immunotherapy? What are the risks associated with such a vaccination ? What is the durability of immune protection? Can we combine vaccine therapy with therapeutic mAbs or small target oriented molecules? What is the potential effect of antibodies modulating the immune system in patients with metastatic BC? Continued basic research into the molecular mechanisms regulating carcinogenesis and immunosurveillance/tolerance will identify new potential targets, introducing vaccine therapy in prevention trials for patients at high risk for developing cancer. A better understanding of the dialogue between cancer and immune cells could help to improve immunotherapeutic approaches and foster the development of new drugs that increase the ability of the immune response to provide effective and sustained anti-tumor immunity.Recent advances in breast cancer treatment and the translational research behind them", edited by Jenny Chang. Other articles in this series can be found at http://breast-cancer-research.com/series/treatment.This article is part of a series on "ADCC: Antibody-dependent cellular cytotoxicity; BC: Breast cancer; CTLA-4: Cytotoxic T lymphocyte-associated antigen 4; ER: Estrogen receptor; FEC: Fluorouracil/epirubicin/cyclophosphamide; FOXP3: Forkhead box P3; mAb: monoclonal antibody; NK: Natural killer; PD-1: Programmed death-1; TAA: Tumor-associated antigen; TIL: Tumor-infiltrating lymphocyte; TN: Triple-negative; Treg: Regulatory T.The authors declare that they have no competing interests.


To the Editor: We applaud Mann et al. on their use of a school-based absenteeism surveillance system to compare daily all-causes absenteeism data against a historic baseline to detect outbreaks of influenza-like illness (ILI) as an adjunct to traditional disease reporting  2009, workplace absenteeism data from the Transport for London attendance/absence reporting system were compared with the historical baseline 3-year mean for comparative weeks of the year. The proportion of Transport for London employees absent because of self-reported or medically certified ILI, during June 28–October 17, 2010, generated surveillance alerts when compared with historical baseline data above the 95th and 99th percentile thresholds (SDs 1.96 and 2.58). For the same period, cause-specific workplace influenza absenteeism data were highly correlated with routinely published ILI surveillance, including the National Pandemic Flu Surveillance and sentinel General Practitioner systems  (2).In Australia, workplace all-causes absenteeism for a major Australia-wide employer has been included as a nonspecific indicator of influenza surveillance by the Australian government for >15 years. A recent study during a severe influenza season in Australia confirmed that employee absenteeism was highly correlated with laboratory-confirmed influenza, and such information could be used to provide surveillance alerts up to 2 weeks before other traditional influenza surveillance data sources (The use of workplace absenteeism data, particularly from large employers, has the potential for overcoming the major limitation of school-based absenteeism data in detecting outbreaks of ILI: the effects of school holidays and local planned school closures. Near real-time workplace absenteeism is an effective surveillance tool and should be more widely incorporated in influenza surveillance systems.


Hepatitis C is a fatal liver disease caused by the hepatitis C virus. In this study, blood donors, from various districts of the KPK province and the federally administered tribal area (FATA) of Pakistan were tested for anti-HCV antibodies and HCV RNA by ICT (Immuno-chromatographic test), ELISA and RT-PCR. Out of the 7148 blood donors, 224 (3.13%) were positive for anti-HCV antibodies by ICT, 135 (1.89%) by ELISA while 118 (1.65%) blood donors had active HCV infection as detected by RT-PCR. We suggest that ELISA should be used for anti-HCV screening in public sector hospitals and health care units. Hepatitis C is caused by the hepatitis C virus (HCV), which was identified in 1989 . ContamiEarlier studies have reported the prevalence of anti-HCV antibodies among the blood donors or general population from KPK province  using Immunochromatographic tests -8 while The aim of this study was to analyze the prevalence of the anti-HCV antibodies or HCV RNA among the blood donors of KPK and the federally administered areas (FATA) of Pakistan from January to September 2009. Three different methods were used to find out the prevalence of anti-HCV antibodies or HCV RNA. The scope of ICT and ELISA techniques for screening blood was also evaluated.Blood samples was taken from the voluntary blood donors and examined either at Hayat Abad Medical Complex (HMC) or at the Institute of Biotechnology and Genetic Engineering, KPK Agricultural University Peshawar.Initially all the blood donors were tested for anti-HCV antibodies by immuno-chromatographic tests. Each positive sample was tested twice. The immune-chromatographic strips used in this study were from two different sources. Samples positive by ICT technique were further evaluated using ELISA.Sera positive by ICT were tested for anti-HCV antibodies by ELISA  according to the manufacturer's instructions. All the ELISA positive samples were processed for RNA extraction.RNA isolation from the HCV positive ELISA samples and subsequent RT-PCR was carried out with the help of RNA extraction and RT-PCR kit from Sacace  according to the manufacturer's instructions, inside the Cepheid smart cycler .A total of 7148 voluntary blood donors were initially screened for anti HCV antibody by ICT. 3.13% of the volunteers were detected positive for anti-HCV antibodies  donors had HCV RNA in their blood (Table rd generation ELISA which indicated that 1.89% of the blood donors had antibodies against HCV. Seventeen (12.6%) blood donors who were positive for anti-HCV by ICT turned out to be negative by ELISA. These results reveal that screening of blood and blood products by ICT devices may not predict the true picture of anti-HCV prevalence. In KPK, screening of the blood and blood products at maximum health care units is carried out with ICT devices only. We suggest that ELISA should replace ICT procedures for screening in all health care units, especially those which are concerned with immigration of the workers. Companies are developing ELISAs for the detection of HCV core antigen. These assays can be used in addition to anti-HCV assays and may provide a valuable tool in the identification of individuals undergoing HCV seroconversion. These assays may be more appropriate as a supplement to anti-HCV antibody tests than using NAT for screening blood donations in countries with limited resources [Viral hepatitis is rapidly spreading among the general population of Pakistan. The lack of proper screening facilities or expertise in screening blood and blood products for possible HCV infection at our public sector hospitals is partly contributing towards the spread of HCV. Previous studies on HCV prevalence based on anti-HCV antibodies among the blood donors have revealed high prevalence rate in KPK province ,8-10. Hiesources .For confirmation of the active infection, we investigated all the ELISA positive samples by Real-time PCR which revealed that 1.65% of the blood donors had HCV RNA in their blood. Presence of anti-HCV and the absence of HCV RNA in the blood may be attributed to the self limiting nature of the disease in some people or it may be due to the presence of antibodies against HCV in treated subjects.Prevalence of active HCV infection among the blood donors of KPK province of Pakistan and the federally administered tribal region of Pakistan is 1.65% which is lower in comparison to the previous estimates. The scope of ICT devices for screening against anti-HCV seems to be limited. Proper and reliable HCV screening should include latest ELISA procedures.The authors declare that they have no competing interests.IA designed the study, advised about the protocols and prepared the manuscript. NK, LR and LS carried out ICT, ELISA and RT-PCR. MR, SK and SA helped in sampling. NA, AI, IM and ZAS critically reviewed the manuscript. All authors read and approved the final manuscript.


We introduced non-surgical therapy with a novel enzyme-targeting radiosensitization treatment, Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas, Type II (KORTUC II) into early stages breast cancer treatment. The purpose of this study was to examine changes in tumor shadows and microcalcifications on mammography (MMG) following KORTUC II for elderly patients with breast cancer. We also sought to determine whether MMG was useful in evaluating the therapeutic effect of KORTUC II. In addition to MMG, positron emission tomography-computed tomography (PET-CT) was performed to detect both metastasis and local recurrence. In all 10 patients, tumor shadows on MMG completely disappeared in several months following the KORTUC II treatment. The concomitant microcalcifications also disappeared or markedly decreased in number. Disappearance of the tumors was also confirmed by the profile curve of tumor density on MMG following KORTUC II treatment; density fell and eventually approached that of the peripheral mammary tissue. These 10 patients have so far have also shown neither local recurrence nor distant metastasis on PET-CT with a mean follow-up period of approximately 27 months at the end of September, 2010. We conclude that breast-conservation treatment using KORTUC II, followed by aromatase inhibitor, is a promising therapeutic method for elderly patients with breast cancer, in terms of avoiding any surgical procedure. Moreover, MMG is considered to be useful for evaluating the efficacy of KORTUC II. Therefore, we introduced non-surgical therapy with a novel enzyme-targeting radiosensitization treatment, Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas, Type II (KORTUC II)  into earThe purpose of this study was to examine changes in tumor shadows and microcalcifications on mammography (MMG) following KORTUC II for elderly patients with breast cancer. We also sought to determine whether MMG was useful in evaluating the therapeutic effect of KORTUC II. In addition to MMG, positron emission tomography-computed tomography (PET-CT) was performed to detect both metastasis and local recurrence.2.Patient data are summarized in Disappearance of the tumors was also confirmed by the profile curve of tumor density on MMG following the KORTUC II treatment; density fell and eventually approached that of the peripheral mammary tissue. The examples of changes in size of tumor shadows and microcalcifications are shown in These 10 patients have so far shown neither local recurrence nor distant metastasis also on PET-CT with a mean follow-up period of approximately 27 months at the end of September, 2010.3.Recently, breast-conserving surgery has become the most common surgical procedure for breast cancer treatment. However, even this type of surgery often has an unacceptable cosmetic outcome. Therefore, various types of minimally invasive options have been used as alternatives to surgical therapy, such as radiofrequency ablation (RFA), focused ultrasound ablation (FUS), and cryotherapy. These minimally invasive approaches are currently being investigated. Although excellent locoregional control can be obtained with these procedures, long-term control rates remain unknown. Moreover, RFA and cryotherapy demand insertion of a relatively large needle into the breast. General anesthesia is essential to perform RFA, and FUS is very time-consuming and requires MRI to monitor the thermal distribution. It is also important to note that these non-surgical approaches require adjuvant radiation therapy to non-ablated tissue in order to eliminate residual cancerous tissue.KORTUC II, a form of radiation therapy intensified with a newly-developed radiosensitizer for intratumoral injection, is a logical technique for the ablation of cancerous nests throughout the breast. KORTUC II has an advantage over other non-surgical ablation therapies, as it can treat breast cancer with one treatment. General anesthesia, insertion of a large needle, and expensive equipment to monitor thermal distribution are unnecessary with this method.Precise assessment of therapeutic efficacy is important when evaluating breast cancer treatments. MMG has been reported to be reliable for the detection of breast tumors and microcalcifications. Therefore, this study used MMG and PET-CT as diagnostic tools for the assessment of the therapeutic effects of KORTUC II on primary breast tumors. Tumors disappeared on MMG in several months following KORTUC II treatment. Moreover, PET-CT confirmed that the absence of recurrence and distant metastasis. Therefore, KORTUC II is considered to be an ideal non-surgical treatment for elderly patients with early stages breast cancer.4.Ten elderly female patients with breast cancer  but no clinical evidence of distant metastasis were enrolled in the KORTUC II trial. Each patient signed an informed consent form before participation in the study. Patient data are summarized in For each patient, radiation therapy (RT) with 4 MV X-ray was delivered with an EXL-20TP linear accelerator equipped with a multileaf collimator  ,11. Hypoth fraction onwards. At the injection of the agent, small amount of 1% lidocaine hydrochloride was used for pain relief at the injection site. The agent is composed of 0.5% hydrogen peroxide and 0.83% sodium hyaluronate, which is safe for injection and effectively preserves oxygen concentration in the tumor tissue for more than 24 hours following intratumoral injection [The new radiosensitizer was injected into the breast tumor tissue twice a week under ultrasonographic guidance, just prior to each administration of RT from the 6njection . For sodHormonal status was examined on a specimen obtained by needle biopsy at pre-treatment. Patients with estrogen receptor-positive tumors also underwent hormonal therapy using aromatase inhibitor for five years following KORTUC II. MMG was performed approximately twice a year during follow-up according to the Japanese Standard Methods established by the Japanese Central Committee on Quality Control of Mammographic Screening.The therapeutic effects of KORTUC II were evaluated in terms of the changes in size of tumor shadows and microcalcifications shown on the monitor  before and following KORTUC II. Changes of tumor density on MMG were also analyzed using image analysis software image-J . PET-CT was also performed annually during follow-up to detect both distant metastasis and local recurrence.5.We conclude that breast-conservation treatment using KORTUC II followed by aromatase inhibitor is a promising therapeutic method for elderly patients with breast cancer, in terms of avoiding any surgical procedure. Moreover, MMG is considered to be useful for evaluating the efficacy of KORTUC II.


Background: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. Results: We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. Conclusions:cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process. Developmental Dynamics 241:169–189, 2012. © 2011 Wiley Periodicals, Inc.A genome-wide catalog of Drosophila conserved DNA sequence clusters.cis-Decoder discovers functionally related enhancers.Functionally related enhancers share balanced sequence element copy numbers.Many enhancers function during multiple phases of development. We have also developed a set of integrated alignment algorithms, collectively known as cis-Decoder, that identify multi-copy and unique elements within CSCs that are shared with other CSCs , a late temporal neuroblast (NB) determinant  identifies multiple peaks of conserved DNA that are flanked by less conserved DNA sequences   elements, of ≥ 6 bp in length, and reveals that these account for more than half of the CSC's conserved sequences (vvl-41 and vvl-43) were tested for their regulatory behavior in this study (see below). Our previous analysis of enhancer sequence conservation has shown that individual enhancers can be identified by the maintenance of their CSB cluster integrity across Drosophila species, while ICR regions show greater sequence length variability  B. Most requences B. The 6.Drosophila CSCs was created by EvoPrinting most of the euchromatic genome of Drosophila melanogaster and nearly all of the previously in vivo characterized enhancers that are included in the REDfly database , one of the obscura group (D. pseudoobscura or D. persimilis), and two to four orthologous regions selected from the more evolutionary distant species: D. willistoni, D. virilis, D. mojavensis, and/or D.grimshawi species. Most of the EvoPrints represented a combined evolutionary divergence of >150 My .As a first step in the identification of structurally related CSCs, a genome-wide database of EvoPrint readouts to identify CSCs. A relaxed EvoPrint highlights sequences that are present in all or all but one of the orthologous DNAs used to generate the print  were avoided in generating EvoPrints, and second and third scoring pair-wise alignments were included in the analysis when rearrangements were detected , due to their having non-conserved intervals >150 or >200 but <250 bases, are present two or three times in the database. The database contains >100,000 non-redundant clusters. To expedite database searches, in addition to cataloging individual CSCs and their CSBs, RPS elements of 6 bp or longer were pre-identified by intra-CSC CSB alignments and stored in the database. Most CSCs that contain more than 150 bp of conserved DNA have RPS elements that account for >50 % of their sequences . The UCSC browser also provides information concerning chromatin accessibility and transcription factor–binding data.The CSC database contains two types of file entries: (1) ∼2,000 files originated from genomic regions spanning previously characterized genes and (2) ∼1,000 entries consisted of genomic regions that cover more than one known or predicted gene or large regions of CSCs not associated with flanking genes. Genomic regions that contain highly repetitive DNA sequences that lack identifiable sequence conservation, such as most of chromosome 4 and specific regions of the X, were not included in the database. Care was taken when annotating the clusters to identify CSCs within non-coding, coding, and 3′UTR regions. Database searches can be modified to include all CSCs or focus on just coding or non-coding regions. It is important to note that CSCs were named according to their proximity to genes: whether the CSCs are indeed enhancers for nearby genes requires functional tests and knowledge of endogenous gene expression patterns. To allow the user to find the location of the CSC relative to flanking genes, a link is provided to the UCSC BLAT server  and/or their similar identifying names. It should be noted that many REDfly entries were made from data that often did not delimit the exact boundaries of the enhancer. In addition many REDfly entries included multiple CSCs or truncated CSCs whose ends were restriction enzyme sites used for cloning purposes and were not within less-conserved ICRs. To reduce the number of truncated entries, EvoPrinted regions were expanded to include flanking ICRs. Also, since many REDfly entries are redundant, care was taken to eliminate this redundancy by eliminating repeated and overlapping entries.We have also included CSCs from all previously in vivo characterized enhancers by cis-Decoder search algorithms in identifying related tissue- and/or temporal-specific enhancers, we show how a single enhancer can be used to identify other functionally related enhancers. A detailed step-by-step tutorial describing the use of the search protocol is given at the cis-Decoder website .In addition to the comparative analysis of enhancer sub-structure, our goal in establishing the CSC database and accompanying search algorithms was to identify functionally related enhancers. The assumption that initiated this study is that many functionally related enhancers share overlapping sets of conserved sequence elements. To demonstrate the utility of cis-Decoder input window an EvoPrinted enhancer that spans a single CSC. cis-Decoder then parses and annotates constituent CSBs in forward and reverse/complement directions. By alignment of the CSBs to one another, the program next identifies multi-copy and palindromic elements that are ≥6 bp. A table is generated that shows the copy-number of each repeat, the element frequency in the database, and the number of database CSCs that contain two or more of each element. Based on our earlier analysis of known enhancers, matches of less than 6 bp in length were not considered, because searches with 5 bases or less yielded results that were not informative . Another set of algorithms then rates the individual database CSCs using the following similarity indices when compared to the input CSC: (1) A repeat balance profile, that assesses relative shared repeat copy numbers and weighs them according to the RPS length  The number of shared repeats (full-length RPS elements and shorter elements contained within longer input repeats); (4) Total number of shared elements including RPS and uniquely shared sequences; (5) Percent coverage of aligning input sequences, which reflects the number of conserved bases in the database CSC that align with the input enhancer CSBs, normalized to the total number of conserved sequences in the database cluster; (6) The number of user-specified required elements present in the database CSC; (7) The longest shared sequence between the input and database CSCs (viewed at the cis-Decoder scorecard by placing the cursor on the sequence length number); and (8) The total number of conserved bases within the database CSC  that controls the embryonic expression of the gene encoding Cas, a zinc-finger transcription factor expressed during late embryonic CNS NB lineage development . The cas-6 CSC is located 392 bp 5&prime to the cas gene predicted transcriptional start site. As described above, one of the first steps in the cis-Decoder analysis is parsing CSBs from the input EvoPrinted enhancer in both forward and reverse directions, and then aligning the CSBs with one another  to discover RPS elements . Thus, we focused the search to this limited set of CSCs. Once these CSCs were identified, one-on-one alignments between the input and database CSBs were automatically performed to discover additional shared sequence elements. As expected, the highest scoring database CSC for most of the indices was cas-6 itself , truncated portions of the cas-6 repeat sequences that we term sub-repeats (orange), and ≥6-bp sequences that are unique matches between cas-6 and cg7229-5 (blue). In many cases, different multi-copy repeats are nested within larger unique matches. For example, within the largest unique aligning sequence shown in Although the search required the hepamer sequence ATGCAAA to be present at least three times in the database CSC, most of the highest-scoring CSCs (both for correlation coefficients and shared RPS elements) contained at least three RPS elements with the full octamer motif [ATG CAAAT], including cis-Decoder also generates lists sequence elements that are shared between the input and database CSC. For example, cas-6 and cg7229-5 CSCs. Fifty-seven percent of the cg7229-5 conserved sequences aligned with cas-6 conserved sequences .equences C. In addcg7229-5 that correspond to cas-6 RPS elements is shown by color-coded highlights  exhibit early expression in a subset of ventral cord midline cells, while sqz-11 and vvl-41 (identified using cas-8 as the input CSC) exhibit onset in a larger number of midline cells while other enhancers do not activate reporter expression in the midline precursor cells  and vvl-41 (identified cas-8 as input). cas-6 and cas-8 enhancers both drive reporter expression in overlapping subsets of cells that represent sub-patterns of endogenous cas expression , was found to share 92 RPS and unique sequence elements with cas-6 . Although these two CSCs have extensive overlap of shared elements, the repeat balance index and correlation coefficient reveal that their shared elements are not balanced in copy number  enhancer  intronic gap enhancer  include multiple polyA/polyT motifs, presumably targets of Hunchback, that are found in even balance (five copies) between the two enhancers  complex genes. Multiple alternative binding sites within these enhancers have been identified for Suppressor of Hairless [Su(H)], the transcription factor utilized by the Notch pathway  to discover other similarly structured CSCs, using as required sequences a single Su(H)-binding site (TGGGAA) and a single bHLH-binding site (CAGCTG). This search resulted in 101 database hits, including CSCs from known Su(H) targets m2, m6, and mγ . In addition, searching with the Notch-targeted deadpan NB enhancer , that contains two alternative Su(H)-binding sites , and the m4 PNS enhancer . Additionally, nab-1 expression was similar to that of the dnabe310 enhancer-trap expression in third-instar larvae CNS . The c492a P-element insertion site was localized to the 4th intron of an uncharacterized gene, cg32264  are more accurate indices when searching for functionally related enhancers, since they take into account not only the number of shared elements but also the RPS copy number balance between the input enhancer and database CSC. The percent alignment coverage is likewise an important indicator of the relationship between the input and database CSCs. Thus, sorting the scorecard by the repeat balance index or by the correlation coefficient increases the likelihood that functionally related enhancers rank at the top of the list. For example, all of the late temporal NB enhancers identified in this study had repeat balance index scores of greater than 1.0, correlation coefficient rankings of above 0.4, and percent coverage of ≥40%.Although each of the cis-Decoder searches, we used the cas-6 as the input enhancer (for search conditions see above). The search returned 111 database hits, of which 27 that shared many repeat elements with cas-6 were tested for enhancer activity in flies. Of these, 12 proved to be late temporal network enhancers, with each being expressed in a different subset of midline, brain, and/or ventral cord neuroblasts. Eleven were expressed exclusively either in adult brain, larval precursors, or in embryonic neurons, and four were considered negative, since their reporter expression was undetectable or found in other tissues other than the nervous system. As for enhancers that were missed in the search, we have identified late temporal network enhancers that do not contain three or more complete or partial octamer sequences, or do not score highly using cas-6 as input. The low-scoring enhancers included sqz-11 and vvl-41, which were discovered using cas-8 as the input CSC (mentioned above). Likewise, ct-3 and ct-14 did not contain three octamer sequences, and they also proved to be late temporal network NB enhancers. Finally, we have identified five other late temporal network enhancers that do not contain octamer motifs but do contain other repeated elements found in late temporal network enhancers (data not shown). It is clear from these results that a search for enhancers using a mandatory sequence, such as the octamer motif, is insufficient to detect the full genomic repertoire of late temporal network enhancers. To identify as many functionally related enhancers as possible, multiple database searches using different search criteria, are recommended. Our current understanding of the role of octamer motifs in conferring temporal gene expression is incomplete, in that we are unable to fully distinguish between embryonic late temporal network enhancers, and octamer-site rich larval or adult brain enhancers. Nevertheless, the fact that only four of the 27 clusters tested were not expressed in the CNS, speaks to the efficacy of cis-Decoder search algorithms in detecting neural enhancers.To estimate the number of false-positive predictions and functionally related enhancers that were missed in cis-Decoder algorithm with other web-based tools for discovery and analysis of cis-regulatory elements. However, not all search programs use evolutionary comparisons, and those that do use different levels of evolutionary divergence to identify conserved sequences in enhancers. The comparative analysis of enhancer discovery programs nevertheless points to factors present in various computational formats that appear to be important for successful cis-regulatory element prediction  and kni(+1) gap enhancers  Functionally related enhancers can be identified based on their balanced copy numbers of shared conserved repeat elements. (2) Enhancers that have extensive shared conserved sequence elements (often >60%), but do not have balanced shared repeat copy numbers, may display significantly different regulatory behaviors. (3) Shared repeat and unique elements between functionally related enhancers are not found in any fixed order or orientation. (4) Similarly regulating families of enhancers need not share specific sets of conserved sequence elements, since different enhancers can accomplish the same regulatory behavior with different but overlapping sets of conserved elements. (5) Enhancers that share conserved repeat elements and perform related Drosophila CNS developmental enhancers are highly complex, based on their conserved sequence composition, and many have proven to be multifunctional. The observed complexity of enhancers, specifically with regard to multi-copy repeat motifs, also suggests that enhancer function is realized through a complex process involving combinatorial interactions among many factors and cannot be easily explained by single activator/repressor transcription factor switches. In addition, the fact that functionally diverse enhancers can display such extensive overlap in their conserved sequences underscores the combinatorial complexity of cis-regulation  programs consist of an integrated set of search and alignment algorithms that help discover conserved sequence elements that are shared between similarly regulated enhancers. The following is a description of the sequential steps and accompanying algorithms used by cis-Decoder protocol to identify repeat and palindrome elements within the input cluster and to scan a genomic CSC database for other CSCs that contain the conserved sequence elements present in the input CSC.Drosophila CSC database is based on relaxed EvoPrints  of >90% of the eukaryotic genome. Clusters were identified and named using EvoPrint cutter, an Image J macro written by Wayne Rasband of NIMH. CSBs were extracted in forward and reverse directions, a database repository was populated with CSC details including a list of CSBs, and records of repeat and palindromic elements and their number within each cluster as well as across all the clusters in the database.The cis-Decoder extracts and annotates the CSBs in both forward and reverse directions and discovers repeat and palindromic sequences within the input CSC. The system also invokes a database search to find accruals for each repeat that is found in database repository along with a display of the number of database clusters containing more than one copy of each repeat. The user sets search constraints to limit search to specific list of repeat motifs as well as sequence type .Upon input of a user-provided CSC, cis-Decoder then searches the database repository for clusters that conform to the search constraints. An alignment between the input and database cluster is generated that shows alignments to input enhancer multi-copy repeat motifs along with input enhancer repeats that were excluded from the database search, unique alignments, and database cluster repeat sequences found as a subset of the unique alignments.cis-Decoder then generates lists of shared sequences of input and database CSCs, and generates a “repeat balance map” visual representation of the relative frequency of appearance of the RPSs in the input versus database CSC , the Pearson Correlation Coefficient (a measure of the strength of a linear relationship between the relative occurrence of repeat and unique matches in the input vs. the database cluster), Number of Shared Repeats, Total Shared Elements , Percent Coverage , Number of Required Repeats (set as a search criterion), Longest Shared Sequence, and the number of Conserved Bases in the database CSC. S. cerevisiae), and SV40 3'UTR , and photographed using a Nikon  microscope equipped with Nomarski (DIC) optics. Embryo developmental stages were determined by morphological criteria .Embryo collection and fixation were performed according to the procedures described by Patel . For in GAL4 expression in the larval brain and CNS of wandering larvae was analyzed using mCD8::GFP ] were performed as described previously (Gao et al.,


One of the main issues of molecular evolution is to divulge the principles in dictating the evolutionary rate differences among various gene classes. Immunological genes have received considerable attention in evolutionary biology as candidates for local adaptation and for studying functionally important polymorphisms. The normal structure and function of immunological genes will be distorted when they experience mutations leading to immunological dysfunctions.Here, we examined the fundamental differences between the genes which on mutation give rise to autoimmune or other immune system related diseases and the immunological genes that do not cause any disease phenotypes. Although the disease genes examined are analogous to non-disease genes in product, expression, function, and pathway affiliation, a statistically significant decrease in evolutionary rate has been found in autoimmune disease genes relative to all other immune related diseases and non-disease genes. Possible ways of accumulation of mutation in the three steps of the central dogma (DNA-mRNA-Protein) have been studied to trace the mutational effects predisposed to disease consequence and acquiring higher selection pressure. Principal Component Analysis and Multivariate Regression Analysis have established the predominant role of single nucleotide polymorphisms in guiding the evolutionary rate of immunological disease and non-disease genes followed by m-RNA abundance, paralogs number, fraction of phosphorylation residue, alternatively spliced exon, protein residue burial and protein disorder.Our study provides an empirical insight into the etiology of autoimmune disease genes and other immunological diseases. The immediate utility of our study is to help in disease gene identification and may also help in medicinal improvement of immune related disease. In particular, studies on the functional and evolutionary attributes of human immune system have attained a major focus since it is an orchestra of various defense mechanisms whereby human body maintains functional and organizational integrity against foreign encroachment. The evolutionary history of insects, chicken and mammals indicates that the majority of immune response genes are subjected to positive selection than remainder of the genes  gradual increase in the ratio of non-synonymous to synonymous substitutions (ω) . Such evolutionary dynamics of disease and non-disease genes linked to immunological genes is a bit surprising because disease genes intuitively experience more mutational changes than non-disease genes, yet they are unable to escape the evolutionary pressure. It is obvious that mutation or variation will occur either in gene or m-RNA or protein level or in all the three levels to confer disease phenotypes. We intend to investigate in which level the mutation persuade the persistence of selection pressure.The available resources of immunological disease genes facilitate us to investigate the evolutionary pressure acting on the autoimmune disease genes (AD) and other classes of disease genes resulting from malfunctioning immunological genes (ID) with respect to the immunological genes (IG) without known association to disease. Our result depicts a significant [The first wave of information from the human genome analysis has revealed that single nucleotide polymorphisms (SNPs) is the major resource of genetic and phenotypic variations in human. Scanning for the signatures of positive selection in human population suggests that SNPs in protein coding regions show regional evidence of less intense purifying selection ,15. InveP<0.05 level in Mann-Whitney test). Though the mutagenic nature of recombination rate may reflect the possibility of higher non-synonymous substitutions, the prevalence of Hill-Robertson interference in the genomic regions with higher RR have been proposed to increase the efficacy of purifying selection [P<0.05 level in Mann-Whitney test). In addition to that, RR has long been thought to be one of the principal forces behind the gene duplication frequency [P<0.05 level in Mann-Whitney test). Higher duplicability may enforce the slower evolutionary rate on AD genes in contrast to other two classes since duplicated genes encounter more purifying selection than singletons even though shortly after the duplication, they experience a considerable relaxation of selection pressure [SNP, RR = 0.120, P = 1.0 × 10-3; Spearman's ρ RR, paralogs number = 0.060, P = 1.0 × 10-3) and elicit the selection pressure on disease genes.Secondly, the shuffling of genes brought about by genetic recombination is a major engine of genetic variation. Recombination rate (RR) has been found to have a positive correlation with DNA diversity in many organisms, both in animals -18 and ielection ,21. Moreelection . Analyzirequency ,24. Calcpressure . From thWith the advent of genome scanning technology it has uncovered that the human genome becomes structurally dynamic due to the presence of thousands of heritable copy of mutation and are equally important as SNPs . It was P<0.05 level in Mann-Whitney test). Such nature of IG is also biologically relevant since it was proposed that AS is crucial for a functional immune system as it offers the potency of high degree of diversity and the competence of individual cells to rapidly adapt and respond towards the changing environmental conditions [Over the past decade, it has been postulated that alternative splicing (AS) is a critical post transcriptional event directing an enhancement of transcriptome and proteome diversity, particularly in higher organisms . The frenditions ,33.Since, alternative splicing can bolster organism complexity by effectively increasing the proteome size, the m-RNA abundance would be higher for the immunological genes. However, we already noticed IG genes are lowly expressed. Accounting EST data, the trend remain exactly same i.e. the EST count/m-RNA abundance is lower for the IG (27.02) compared to ID 35.11) and AD (48.72) genes. Hence, we ask what drives the lower m-RNA abundance of AS rich immunological genes. In the recent year it has been clarified that up to one-third of human AS events create a premature termination codon (PTC) that would cause the resulting mRNA to be degraded by nonsense-mediated mRNA decay (NMD) ,35 and i.11 and AAnother implication of alternative splicing is to promote intrinsically disordered protein, thus enabling functional and regulatory diversity in human proteome ,38. Calcto combat against an almost infinite diversity of physiological or synthetic antigens is predominantly rendered by intrinsically disordered regions of proteins . AssociaIn recent years there has been growing evidence for extensive natural variations like SNPs to be the major contributor of alternative splicing variation in humans . NumerouAll proteins are potentially subjected to Post-translational Modifications (PMs) to accomplish many important roles in regulating the biological processes such as regulation of gene expression, activation/deactivation of enzymatic activity, protein stability or destruction, mediation of protein-protein interactions etc . HoweverFurthermore, it is well established that buried residues in a protein are important determinants of protein stability while surface residues are involved in protein function . Here weSystematic approach to the analysis of SNPs indicated that SNPs resulting in deleterious amino acid changes predominantly affect the stability of the protein . We thenHere, we noticed different probable factors in the three levels (DNA-m-RNA-Protein) that can explain evolutionary rate differences among AD, ID and IG genes. To assess the contribution of each variable, we compute Principal Component Analysis (PCA). The dominant eigen vectors  that appear from this analysis can be interpreted as the most important contributors directing protein evolution . PCA wit-3 in each case and ds for autoimmune disease genes = 0.0232; immunological disease genes = 0.0254; immunological genes = 0.0291; Mann-Whitney's P < 0.05 in each case). Significant differences in synonymous substitutions rates among the gene groups indicate the role of neutral substitutions in driving the evolutionary rate discrepancies among them. Now, the slower evolving disease linked immune genes raise a fundamental question why non disease immune genes evolve at a higher rate compared to disease related immune genes since it was previously documented by several studies that non-disease genes evolve at a slower rate than disease genes [Recent years have witnessed rapid progress in elucidating the molecular causes of various diseases. Here we analyzed the evolutionary disparity between the functional and non-functional immune systems. We noticed that autoimmune disease genes are more conserved than other immunological disease genes and both sets of genes evolved significantly at a slower rate than immunological genes. Though the evolutionary rates differences among the gene groups are statistically significant, the difference of mean values between autoimmune and immunological disease genes is small. However, the differences of mean values among the groups turned out to be prominent when we analyzed non-synonymous and synonymous substitution rates separately . Thus, we deciphered that the basic difference in the involvement of proteome expansion machinery put differential selective pressures on malfunctioning immune genes and the functional immune genes. Moreover, it is also observed in our search that autoimmune disease and other immunological disease genes are more prone to post-translational phosphorylation which may regarded as a possible reason for slower evolutionary rate. In protein structure level, the higher residue burial is observed in two types of disease genes compared to non-disease genes and the propensity of single nucleotide polymorphisms to substitute hydrophilic, amphipathic amino acid by hydrophobic amino acid in disease groups could be prompted as a reason of lower sequence divergence in autoimmune disease and other immunological disease genes than immunological genes. Conferring structural stability to the autoimmune disease genes also has a biological significance since incidence of autoimmunity sharply increases in the stable protein forms in the cell [The evolutionary conservation of disease related immunological genes in spite of their higher association with non-synonymous single nucleotide polymorphisms is an artifact of its beneficial impact on disease related genes Figure . Single the cell .Assessing the results from multivariate regression analysis we conclude that the relative dominance of individual factors modulating the differential substitution rate experienced by autoimmune disease, other immunological disease and immunological disease genes is in the order of single nucleotide polymorphisms > m-RNA abundance > paralogs number > phosphorylation residue > alternatively spliced exon > protein residue burial> protein disorder. To the best of our knowledge, this is the first extensive comparison of disease and non-disease related immunological genes from evolutionary perspective. This finding also shades light into the mutational spectrum acting on DNA-mRNA-protein level of the three classes of genes. Our study will surely enrich the knowledge of disease gene identification and may also help in medicinal improvement of autoimmune disease.Immune related disease genes mainly consist with Autoimmune disease, Immunoproliferative disease, Immunologic deficiency syndromes, hypersensitivity, Graft rejection, Purpura, thrombocytopenia, and Glomerulonephritis. There exists a clear demarcation between the basic disease mechanism of autoimmune disease and rest of the immune related disease genes. Thus immune related disease genes are broadly categorized into the two groups - autoimmune disease genes and other immunological disease genes. These two types of genes inherited by Mendelian and non-Mendelian fashion were downloaded from Biobase and Genetic Association Database  respectiThe gene sequences, paralogs information, pair-wise non-synonymous substitution rates (dn) and synonymous substitution rates (ds) with Chimp (1:1) orthologs corresponding to both types of immunological disease genes as well as non-disease genes were retrieved from Ensembl .The gene expression profile data was extracted from BioGPS dataset . The sigi /l, where ρi stands for recombination rate at a base position and l for the genic length corresponding to that gene [Non-synonymous SNPs and CNVs information were downloaded from Polydoms  and Datahat gene .Data for alternatively spliced isoforms and exons for the genes in the dataset were downloaded from the Alternative Splicing Annotation Project . Splice Disorder predictions were carried out using the program FoldIndex  implemenResidue-wise burial in proteins was computed by a standalone version of sequence-based prediction program RVP-Net . This prWe performed Mann-Whitney U test for pair-wise comparisons since the values are not normally distributed in our dataset. Multiple regression analysis, Principal component analysis were performed for relative contribution analysis of each factors to evolutionary rate. All the statistical tests were carried out by the SPSS (13.0) package.AD: Autoimmune Disease; ID: Immunological Disease; IG: Immunological Genes; RR: Recombination Rate; AS: Alternative Splicing; NMD: Non-sense Mediated mRNA Decay; PMs: Posttranslational Modifications; ssSNP: splice site SNPs; PCA: Principal Component Analysis; PTC: Premature Termination Codon.The authors declare that they have no competing interests.SP made the analyses and drafted the manuscript. TCG guided the work and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary Table S1. Genomic and proteomic features of Autoimmune disease, Immunological disease gens and Immunological genes.Click here for file


Prion diseases or transmissible spongiform encephalopathies are a unique category of infectious protein-misfolding neurodegenerative disorders. Hypothesized to be caused by misfolding of the cellular prion protein these disorders possess an infectious quality that thrives in immune-competent hosts. While much has been discovered about the routing and critical components involved in the peripheral pathogenesis of these agents there are still many aspects to be discovered. Research into this area has been extensive as it represents a major target for therapeutic intervention within this group of diseases. The main focus of pathological damage in these diseases occurs within the central nervous system. Cells of the innate immune system have been proven to be critical players in the initial pathogenesis of prion disease, and may have a role in the pathological progression of disease. Understanding how prions interact with the host innate immune system may provide us with natural pathways and mechanisms to combat these diseases prior to their neuroinvasive stage. We present here a review of the current knowledge regarding the role of the innate immune system in prion pathogenesis. Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious neurodegenerative conditions characterized by vacuolar degeneration of the central nervous system (CNS) and deposition of an abnormal isoform of the host-encoded prion protein (PrP). These diseases affect a wide variety of animal species and display limited zoonotic potential. Usually displaying prolonged incubation periods, they are clinically recognized via progressive neurological deterioration resulting from synaptic and neuronal loss and associated activated glial responses to CNS damage. Identification of genetically inherited forms of these diseases implicate further the critical role of the prion protein in disease pathogenesis and their classification as protein-misfolding disorders, with similarities to other progressive dementias such as Alzheimer’s and Parkinson’s diseases and amyotrophic lateral sclerosis. Sporadic prion diseases, with no known genetic risk factor or exposure to infection, have also been identified. The disease-associated isoform of the prion protein gains several properties including ability to transmit infection, limited protease resistance, and increased ability to fibrillize and form amyloid, these observations on both etiology and biochemical nature of the agent resulted in the prion hypothesis . i.e., the disease-associated isoform of the prion protein (PrPSc), with the means to self-propagate via an auto-catalytic process of template-mediated refolding of the nascent cellular prion protein (PrPC). The pathway and mechanisms from refolding of the prion protein to neurodegeneration are still unknown. The peripheral pathogenesis of these diseases have been extensively studied using animal models, as mice are naturally susceptible to both sheep scrapie and bovine spongiform encephalopathy (BSE). Following natural peripheral exposure to prion agents, infection is usually sequestered to lymphoid organs prior to invasion of the nervous system (termed neuroinvasion) [The prion hypothesis proposed that the infectious agent may be solely composed of a proteinaceous particle, nvasion) ,3. In thnvasion) ,5. Usingnvasion) ,7. The lnvasion) ,9,10,11.nvasion) ,13. Direnvasion) ,15. Concnvasion) .i.e., gene-knockout) that effect both peripheral and CNS innate or adaptive immune responses. Despite these difficulties several clear messages have been received regarding the role of specific components of the innate immune system, some of which are less inter-dependent than the systems regulating the adaptive immune response, which we review and summarize below.Originating from the stromal cell compartment FDC are specialized cells that capture and retain antigen for presentation to cognate B-cells and subsequent generation of specific antibody responses and thus reside within primary B-cell follicles and germinal centers within lymphoid tissues ,18. Due The innate immune system is considered to be a protective system that is older in evolutionary terms than the adaptive immune system. This system was originally proposed to respond in a non-specific way to invading pathogens in an attempt to eliminate them or their activity, whilst alerting or informing the adaptive immune response. As such the innate immune system confers no immunological memory or lasting protective immunity. The identification and characterization of specific pattern recognition receptors, such as Toll-like (TLR), C-type lectin (CLR), NOD-like (NLR) and RIG-I-like (RLR) receptors, has revealed the diversity of the innate immune system to detect and tailor specific responses to pathogen-associated molecular patterns (PAMPs) ,22,23,24C expressed by M-cells has been hypothesized to function as an uptake receptor for pathogenic bacteria [Sc has also been observed on enterocyte derived extracellular vesicles suggesting an M-cell independent pathway for prions to cross epithelial barriers [The epithelial cell layers constitute the first physical barrier to infection. Prion infection via the skin is experimentally facilitated by scarification or breaking of the keratinized epidermal barrier layers to allow access to the epithelial layer beneath . Prion ibacteria . Translobarriers . Lumenalbarriers . Althougbarriers , indicatbarriers . Thus thFollowing uptake and translocation across the epithelial barrier layer, prions are then free to encounter other elements of the innate immune system. Directly beneath the FAE and within basolateral pockets of the M-cells themselves are areas enriched in classical DC, macrophages (Mϕ) and lymphocytes termed the subepithelial dome (SED), precisely located so as to process incoming antigen. The final physical barrier to prion pathogenesis appears to be the distance between site of entry and FDC and subsequently between FDC and peripheral nerves. Innervation of the lymphoid organs has been shown to be critical for prion neuroinvasion . ManipulThe complement system is composed of numerous blood borne proteins that are generally synthesized in the liver and circulate as inactive precursors or pro-proteins. Significant amounts of complement components are also synthesized locally by tissue-resident mononuclear phagocytes (MNPs) ,39, epitComplement has been shown to be activated early during prion pathogenesis by as yet undetermined mechanisms and may constitute the first active response to infection. Prion protein has been shown to be directly bound by C1q and Factor H  and thisSc may alter the downstream processing by classical DC, indeed Flores-Langarica and colleagues imply the receptor calreticulin in this process suggesting targeting towards a protein degradative pathway. This is counter-intuitive to the concept that classical DC may retain intact antigen via a FcγRIIB and complement-mediated process for subsequent presentation [Sc has been shown to modulate prion propagation in a similar fashion [The local production of complement components by MNP has been shown to alter their function . C1q enhentation  which seentation , whilst  fashion .Complement component C1q also regulates dendritic cell development from monocytes via DC-SIGN signaling ,52. ThisSc appear to differ dependent upon the strain of infectious agent [The role of complement during prion pathogenesis is further complicated by the observation that the individual complement components mediating interaction with PrPus agent . Cell-auus agent .The role of the complement system within the CNS has also been extensively reviewed recently ,58. As wC) and their ability to traffic to the brain [Mast cells have been implicated in prion pathogenesis due to their high expression levels of cellular prion protein (PrP or PrPhe brain . In facthe brain . Mast cehe brain . This acC by mast cells likely occurs via proteolytic or lipolytic cleavage mechanisms removing the glycosylphosphatidylinisotol (GPI) anchor from the protein [C substrate for conversion to the disease-associated isoform late in the disease process, when significant damage has occurred to the CNS already, adding to the exponential accumulation of misfolded protein within the clinical stage of disease. Investigation of GPI-anchorless PrP transgenic mice has revealed that they are capable of forming amyloid in the brain without clinical prion disease symptoms, whilst co-expression of anchorless and native PrPC increased prion pathogenesis [The role of mast cells within the brain is thought to be neuro-modulatory and mast cell trafficking to the brain is linked to steroidal hormones and sexual activity or anxiety behaviors . The pre protein ,63. Actiogenesis . Novel sogenesis , suggestogenesis  or prionogenesis .MNP constitute a system of highly phagocytic cells that have a common origin in the bone marrow and circulate or reside in the body tissues in order to sample their environment via uptake of foreign (and self) material. The development of MNP relies upon expression of the colony stimulating factor 1 receptor (CSF-1R) during their differentiation in order to respond to CSF-1. MNP play a vital role in the bridge between the innate and adaptive immune systems by processing and presenting of antigenic compounds to stimulate specific antibody production. During the initial processing by MNP material may be directly disposed of by degradation via lysosomal or proteasomal pathways. These actions of MNP aid in the development of immunity or tolerance to the vast multitude of foreign material encountered. Numerous (particularly intracellular) pathogens have adapted to abuse the functions of MNP as a vehicle for pathogenesis, allowing for their uptake into MNP and trafficking to lymphoid organs for further spread. It is most likely that the uptake and spread of prions to lymphoid organs occurs using MNP as a ‘Trojan horse’ via a similar mechanism.The role of MNP in prion pathogenesis have been subject to recent review and are considered diverse . This reRegardless of their major functionality all MNP likely use similar surface receptor based pathways and mechanisms to sense their local micro-environment and tailor their responses to pathogens or tissue damage. The exact receptor-mediated mechanism determining the response to prion agents has not been identified or definitively characterized. MNP-mediated uptake of prion agent is enhanced by complement opsonization see  above. Ti.e., due to the factors mentioned above) the balance is switched in favor of degradation of agent, thereby preventing or dramatically slowing pathogenesis. This may occur both within any given cell (and not exclusively to degradative or phagocytic cell types), in fact it is well known that there are limited in vitro cell infection models in the prion field due to the relevant inability to infect numerous cell types with a variety of prion strains even within the same species [The expression of cellular prion protein in MNP has been associated with phagocytic ability and modulation of inflammatory responses ,71,72. E species , and witSc has been shown to be inhibited by cysteine protease inhibitors [C to PrPSc conversion [Cellular degradation of PrPhibitors . It has hibitors  one possnversion . The rolnversion . The celnversion ,81. + CD8+ (in this case CD8αα) DC subsets restricts intraperitoneal but not oral pathogenesis suggesting that alternative DC subsets may be employed following different infection routes. CD8 knockout mice revealed no alteration of prion pathogenesis, suggesting no direct role for CD8 [+ cells had the biggest impact upon pathogenesis [in vivo [in vitro [APC have long been identified as being critical to prion pathogenesis. While evidence exists for both cell-free and cell-mediated transport of prions to lymphoid tissues , removal for CD8 . This paogenesis . Extract[in vivo  and in vin vitro  respecti106-126, also reveal enhanced survival and proliferative responses to CSF-1 [per se but may require priming by other CNS cell types. Following priming the microglial cells undergo chemotaxis to the site of insult [Within the CNS the innate immune response is mediated by specialized MNP known as microglia. Microglial development is also dependent upon signaling via the CSF1-R , howeverto CSF-1 . Followif insult , impairmf insult . Once acf insult . In fact106-126) has been shown to be directly neurotoxic, activate MNPs and act as a chemotactic agonist for the FPRL1 receptor [106-126 fragment to prion pathogenesis is elusive at best. FPRL1 is strongly expressed on granulocytes and MNP cell types and promotes chemotaxis to sites in response to allergic inflammation [Gene expression data reveal that PrP expression is generally down-regulated during granulocyte differentiation  though sreceptor ; the relammation . Eosinop−/− mice are deficient in NK/T cells they are unsusceptible to prion infection due to their B-cell deficiency and failure to generate mature FDC. Selective reconstitution of the Rag2−/− model for prion pathogenesis studies has not been reported. An increase in γδ T cells in the peripheral blood mononuclear cells (PBMCs) has been reported following scrapie infection in sheep [Natural killer (NK) and γδ T cells are thought to play little role in the pathogenesis of prion diseases. Both cell types require triggering or activation by danger signals, for NK cell this is usually via cytokines and for γδ T cells little is known about their activating signals. NK cells primarily respond to virally-infected cells and function to destroy these cells via expression of perforin and granzyme. Perforin-knockout mice revealed unaltered prion pathogenesis . NK cellin sheep .C is released in exosomes [C from macrophages [i.e., from draining to contralateral non-draining lymph nodes) has been prevented by blocking the egress and thus recirculation of B-cells [The megakaryocyte lineage and platelets have been associated with the expression of prion protein ,158 and exosomes , similarrophages . While nrophages . The overophages . These drophages ,165,166. B-cells . The rou B-cells ,168. Hem B-cells . C) by the hematopoietic compartment is not required for prion pathogenesis [C dependent mechanisms. Little is known about the uptake mechanisms of prions and the factors that lead to cell clearance, infection or passivity that may facilitate transport. PrPC is not required for uptake of prions in vitro, even by non-phagocytic cell types such as neurons [C expression we present here data on the expression of the prion protein gene (Prnp) transcripts in various innate immunity relevant cellular compartments  and prion pathogenesis is via transgenic mouse models. These models have been used to determine the effect of knockout or overexpression of a particular protein or cell type on prion pathogenesis. The classic experiment revealed that knockout of nfection ,141. A pnfection   of mononuclear phagocyte cells appears critical to both peripheral and central prion pathogenesis through as yet unidentified receptors and signaling pathways. Investigation of the expression and function or cellular prion protein has clearly identified widespread expression throughout cell types of the innate immune system and has also suggested possible immune-system specific functions of PrP separate from its possible roles within the CNS.


Less than 25% of patients with a pelvic mass who are presented to a gynecologist will eventually be diagnosed with epithelial ovarian cancer. Since there is no reliable test to differentiate between different ovarian tumors, accurate classification could facilitate adequate referral to a gynecological oncologist, improving survival. The goal of our study was to assess the potential value of a SELDI-TOF-MS based classifier for discriminating between patients with a pelvic mass.Our study design included a well-defined patient population, stringent protocols and an independent validation cohort. We compared serum samples of 53 ovarian cancer patients, 18 patients with tumors of low malignant potential, and 57 patients with a benign ovarian tumor on different ProteinChip arrays. In addition, from a subset of 84 patients, tumor tissues were collected and microdissection was used to isolate a pure and homogenous cell population.Diagonal Linear Discriminant Analysis (DLDA) and Support Vector Machine (SVM) classification on serum samples comparing cancer versus benign tumors, yielded models with a classification accuracy of 71-81% , and 73-81% on the independent validation set. Cancer and benign tissues could be classified with 95-99% accuracy using cross-validation. Tumors of low malignant potential showed protein expression patterns different from both benign and cancer tissues. Remarkably, none of the peaks differentially expressed in serum samples were found to be differentially expressed in the tissue lysates of those same groups.Although SELDI-TOF-MS can produce reliable classification results in serum samples of ovarian cancer patients, it will not be applicable in routine patient care. On the other hand, protein profiling of microdissected tumor tissue may lead to a better understanding of oncogenesis and could still be a source of new serum biomarkers leading to novel methods for differentiating between different histological subtypes. Ovarian cancer is the leading cause of gynecologic deaths in Western countries . The majWith the introduction of high-throughput mass spectrometry techniques such as SELDI-TOF, the search for biomarkers which could enhance the discriminatory accuracy of existing tumor markers was fueled ,16. The In this study, we aimed to incorporate the essential aspects of a classification study to evaluate the suitability of SELDI-TOF in generating a reliable and reproducible biomarker profile in serum and tissue to differentiate between different ovarian tumors. Instead of healthy women, patients with a benign ovarian tumor were chosen as control group. Patients with a tumor of low malignant potential were also included as a separate group because of their different biological and clinical behavior. Comparison of these patient groups enables the identification of protein profiles that differentiate between various tumors of the ovary. Prospectively collected samples, strict protocols, and extensive use of technical replicates for quality control, combined with an independent validation cohort in the serum experiments were used to obtain a robust and reliable outcome.In addition to the serum profiling we used SELDI-TOF for protein profiling directly in laser-microdissected tumor-tissue lysates from a subset of the same patients. The use of dissected homogeneous tumor cell populations leads to more reproducible protein patterns without the problems associated with serum, such as its huge dynamic range. As a result we expect that using tissue samples more accurate classification models can be generated for differentiating between tumor types. By profiling and comparing tumor tissue with serum we aim to identify peaks found in both serum and tissue resulting in the identification of reliable tumor produced serum biomarkers. A clinical serum-based assay using a tumor-specific biomarker panel could assist the clinician in differentiating between different tumor types, without the need for a tissue biopsy, subsequently facilitating adequate treatment selection or referral to a gynecological oncologist.Pairwise comparison of serum spectra from patients with ovarian cancer and benign tumors identified 145 differentially expressed peaks for the CM10, out of a total of 265 peaks. 71 peaks out of 156 peaks were differentially expressed for the Q10 data . Analysis of serum from cancer patients versus serum of patients with a tumor of low malignant potential yielded 38 differentially expressed peaks for CM10 and 13 peaks for Q10. No differentially expressed peaks were found when comparing the serum of patients with a benign tumor and the serum of patients with a tumor of low malignant potential. The ten most differentially expressed peaks between patients with ovarian cancer and a benign tumor, their adjusted p-values, and their fold changes are shown in Table The average classification accuracy for discriminating ovarian cancer from benign tumors in serum samples was 79-81% for the CM10 training cohort, with a sensitivity of 75% and a specificity of 83-86% using DLDA and SVM classifiers Table . When clClassification analysis was also performed including patients with a tumor of low malignant potential as a third class. Classification results were inferior to the results obtained when comparing cancer and benign tumors. Average accuracy reached only 50-64% across different classifiers and ionization surfaces, versus an accuracy of 71–81% when comparing the two groups. The predictive accuracy of the resulting two-class classifiers was assessed on an independent validation set measured on Q10 arrays. The spectra of the independent validation set showed good reproducibility. Analyses using DLDA and SVM classifiers resulted in a classification accuracy of 73-81% with a sensitivity of 81-88% and specificity of 62-81% for the Q10 validation data , 80% (Q10), and 84% (CM10/Q10) respectively. The confusion matrix for DLDA on the Q10 data  were found to be differentially expressed between the tissue lysates of those same groups. This might be due to the fact that the proteins differentially expressed in tissue lysates are intracellular proteins which do not enter the bloodstream, or are present at very low concentrations making them difficult to detect. SELDI-TOF may not be sensitive enough to detect tiny amounts of tumor derived peptides in serum which has a huge dynamic range. The use of pre-fractionation methods in combination with mass-spectrometry techniques could be used to see whether tumor derived proteins, present in very small concentrations could be picked up. The lack of common peaks in de protein spectra of serum and tissue of the same patients also reveals that the proteins differentially expressed in serum of the different groups are not tumor produced proteins but epiphenomena associated with the different tumor groups.Despite the initial promise of high throughput mass spectrometry techniques such as SELDI-TOF, there are a number of limitations which have only recently been addressed ,28. Our However, our data also show that SELDI-TOF-generated serum profiles are not able to outperform existing clinical methods such as the combination of CA125 and ultrasonography when it comes to differentiating between patients with an ovarian tumor. When searching for tumor-derived proteins in serum by comparing the serum and tissue profiles no tissue-derived proteins could be detected in serum via SELDI-TOF. However, the differentially expressed proteins found in tissue lysates could still lead to a better understanding of oncogenesis and be a source of new biomarkers when detected in serum by other more sensitive techniques. This could result in a clinical serum-based assay using a tumor-specific biomarker panel.Although we believe that high-throughput methods like SELDI-TOF can still be put to good use when looking for differences or similarities in large patient populations, novel methods in mass spectrometry such as qualitative and quantitative proteomic analysis by nanoscale LC-MS (Liquid Chromatography-Mass Spectrometry) are being used more frequently . These mAfter informed consent was obtained, samples were prospectively collected from newly diagnosed patients at the Academic Medical Center. The study was approved by the Ethical Committee at the Academic Medical Center. Both training and validation cohort consisted of women with non-familial epithelial ovarian cancer and patients with tumors of low malignant potential. The control group consisted of patients with a range of benign ovarian tumors such as serous and mucinous cystadenoma, mature cystic teratoma, or fibroma of the ovary. Patients were matched for age, BMI, and sample storage time Table . The valClinical and pathological characteristics of the three separate patient groups: cancer, low malignant potential (LMP) and benign tumor. The characteristics are given for the serum training, serum validation and tissue experiments. Age, body mass index (BMI) and sample age are given as a mean value with their standard deviation. In one patient differentiation grade was not available (NA).Serum samples were collected before treatment using a strict protocol. Blood was collected by the same operator at least two hours after the patient’s last meal and left to clot for 30 minutes. After centrifugation (at 1750 x g) serum was immediately frozen and stored at −80°C. Samples used were only thawed once.From 84 patients included in the serum experiments, tissue samples were available Table . HistoloProtein profiles of serum and tissue samples were generated using anionic surfaces of CM10 and cationic surfaces of Q10 ProteinChip arrays . Protocols were performed as described previously . In ordePre-processing was done with commercial ProteinChip Software  as described earlier . In summTo see whether a different pre-processing method would achieve similar results, we also pre-processed the serum samples using the mean spectrum technique from the publicly available Cromwell software developed by the bioinformatics group at the MD Anderson Cancer Center . Pre-proi) visual inspection of the spectra, (ii) assessment of intra- and inter-chip reproducibility based on technical replicates, (iii) hierarchical clustering of the pre-processed data. Based on visual inspection, seven tissue samples on one of the Q10 arrays were left out, since their spectra had very high and irregular baseline levels. Hierarchical clustering identified one serum training sample on a CM10 array as a potential outlier. This sample was discarded and pre-processing was repeated on the remaining samples.Quality control consisted of  with patient status and chip as main effects. The ANOVA analysis showed clear evidence of a chip effect for a number of peaks on the Q10 arrays for both serum and tissue samples. However, no significant interaction effect between patient status and chip was detected. None of the peaks that could differentiate between chips could also differentiate between patient groups (data not shown). A moderated t-test was then used to identify peaks that were pairwise differentially expressed between patient groups. Resulting p-values were corrected for multiple testing using the Benjamini-Hochberg False Discovery Rate (FDR) adjustment. Tests were considered significant if the adjusted p-values were <0.05.et al.[N samples were performed to generate 500 different training sets (size n) and associated test sets (size N–n). In each of the random splits, the number of samples for the patient groups was balanced in both the training and test sets. The accuracy of the resulting classifiers was assessed on the corresponding test sets. To investigate the influence of the training set size on the accuracy of the classifiers, we varied the training set size  or n = 20,35,50 (two-class) for the tissue dataset. If not mentioned otherwise, results reported are for the largest training set size. The optimal values for the cost hyperparameter of the SVMs were estimated using 5-fold cross-validation on the training set. Feature selection was used to extract the peaks most informative for predicting patient status. For each training set, an optimal classifier was identified from the 5, 10,…,50 peaks with the highest correlation between expression and patient status as determined by either t-statistics (two-class) or F-statistics (three-class). The optimal number of peaks was again selected with 5-fold cross-validation on the training set. Such a double cross-validation scheme provides an almost unbiased estimate of the true error [To test whether patient status could be predicted from the peak profiles obtained, we used two classification models often used for microarray and proteomics data, linear Support Vector Machines (SVM) and Diagonal Linear Discriminant Analysis (DLDA) . These tet al.. Random ue error .In the serum experiments an independent group of validation samples was available. These samples were classified by models trained on the serum training cohort only and with optimal values for the hyperparameters , as estimated on the training cohort by the double cross-validation scheme described above. Statistical analyses were performed using Bioconductor packages and in-house scripts in the statistical software package R .ANOVA: Analysis of variance; BMI: Body mass index; DLDA: Diagonal linear discriminant analysis; FDR: False discovery rate; FIGO: Fédération Internationale de Gynécologie Obstétrique; LCM: Laser capture microdissection; SVM: Support vector machine.The authors declared no conflict of interests.WW collected samples and clinical information, carried out the SELDI-TOF experiments, interpreted results and drafted the manuscript. PDM carried out the statistical analyses, interpreted the results and drafted the manuscript. DM interpreted the results and critically read the manuscript. SJ carried out the SELDI-TOF experiments and interpreted the results. HCJH advised on statistical analyses and interpretation of results. GGK interpreted the results. MRB was responsible for the design of the study and sample collection. JMA contributed materials and was responsible for the design of the study. All authors read and approved the final manuscript.Representative SELDI spectra. Spectra (x-axis: m/z ratio (Da); y-axis: normalized intensity) obtained for the different chip types (CM10 and Q10) and specimens (serum and tissue) from the same patient with a serous adenocarcinoma.Click here for fileRepresentative SELDI spectra. Spectra (x-axis: m/z ratio (Da); y-axis: normalized intensity) showing a differentially expressed peak at 10,884 Da (see Table Click here for file