Narasimhan Kothandaraman

Proteomic analysis of human placental syncytiotrophoblast microvesicles in preeclampsia

BackgroundPlacental syncytiotrophoblast microvesicles (STBM) are shed into the maternal circulation during normal pregnancy. STBM circulate in significantly increased amounts in preeclampsia (PE) and are considered to be among contributors to the exaggerated proinflammatory, procoagulant state of PE. However, protein composition of STBM in normal pregnancy and PE remains unknown. We therefore sought to determine the protein components of STBM and whether STBM protein expressions differ in preeclamptic and normal pregnancies.Patients with PE (n = 3) and normal pregnant controls (n = 6) were recruited. STBM were prepared from placental explant culture supernatant. STBM proteins were analyzed by a combination of 1D Gel-LC-MS/MS. Protein expressions levels were quantified using spectral counts and validated by immunohistochemistry.ResultsOver 400 proteins were identified in the STBM samples. Among these, 25 proteins were found to be differentially expressed in preeclampsia compared to healthy pregnant controls, including integrins, annexins and histones.ConclusionSTBM proteins include those that are implicated in immune response, coagulation, oxidative stress, apoptosis as well as lipid metabolism pathways. Differential protein expressions of STBM suggest their pathophysiological relevance in PE.

E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

BackgroundOvarian epithelial cancer (OEC) usually presents in the later stages of the disease. Factors, especially those associated with cell-cycle genes, affecting the genesis and tumour progression for ovarian cancer are largely unknown. We hypothesized that over-expressed transcription factors (TFs), as well as those that are driving the expression of the OEC over-expressed genes, could be the key for OEC genesis and potentially useful tissue and serum markers for malignancy associated with OEC.MethodsUsing a combination of computational (selection of candidate TF markers and malignancy prediction) and experimental approaches (tissue microarray and western blotting on patient samples) we identified and evaluated E2F5 transcription factor involved in cell proliferation, as a promising candidate regulatory target in early stage disease. Our hypothesis was supported by our tissue array experiments that showed E2F5 expression only in OEC samples but not in normal and benign tissues, and by significantly positively biased expression in serum samples done using western blotting studies.ResultsAnalysis of clinical cases shows that of the E2F5 status is characteristic for a different population group than one covered by CA125, a conventional OEC biomarker. E2F5 used in different combinations with CA125 for distinguishing malignant cyst from benign cyst shows that the presence of CA125 or E2F5 increases sensitivity of OEC detection to 97.9% (an increase from 87.5% if only CA125 is used) and, more importantly, the presence of both CA125 and E2F5 increases specificity of OEC to 72.5% (an increase from 55% if only CA125 is used). This significantly improved accuracy suggests possibility of an improved diagnostics of OEC. Furthermore, detection of malignancy status in 86 cases (38 benign, 48 early and late OEC) shows that the use of E2F5 status in combination with other clinical characteristics allows for an improved detection of malignant cases with sensitivity, specificity, F-measure and accuracy of 97.92%, 97.37%, 97.92% and 97.67%, respectively.ConclusionsOverall, our findings, in addition to opening a realistic possibility for improved OEC diagnosis, provide an indirect evidence that a cell-cycle regulatory protein E2F5 might play a significant role in OEC pathogenesis.

Membrane proteins of human fetal primitive nucleated red blood cells.

In humans, primitive fetal nucleated red blood cells (FNRBCs) are thought to be as vital for embryonic life as their counterpart, adult red blood cells (adult RBCs) are in later-gestation fetuses and adults. Unlike adult RBCs, the identity and functions of FNRBC proteins are poorly understood owing to a scarcity of FNRBCs for proteomic investigations. The study aimed to investigate membrane proteins of this unique cell type. We present here, the first report on the membrane proteome of human primitive FNRBCs investigated by two-dimensional liquid chromatography coupled with mass-spectrometry (2D-LCMS/MS) and bioinformatics analysis. A total of 273 proteins were identified, of which 133 (48.7%) were membrane proteins. We compared our data with membrane proteins of adult RBCs to identify common, and unique, surface membrane proteins. Twelve plasma membrane proteins with transmembrane domains and eight proteins with transmembrane domains but without known sub-cellular location were identified as unique-to-FNRBCs. Except for the transferrin receptor, all other 19 unique-to-FNRBC membrane proteins have never been described in RBCs. Reverse-transcriptase PCR (RT-PCR) and immunocytochemistry validated the 2D-LCMS/MS data. Our findings provide potential surface antigens for separation of primitive FNRBCs from maternal blood for noninvasive prenatal diagnosis, and to understand the biology of these rare cells.

Abstract B5: Haptoglobin proved to be a novel biomarker for intraoperative triage of epithelial ovarian cancer at early stage

Introduction: Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer-related deaths worldwide, and the prognosis can be greatly improved if cancer is detected at early stage. Intra-operative suspicion of malignancy for women going for ovarian cystectomy is critical for triage of patients for the most suitable surgical procedures. In this study, we aimed to evaluate the diagnostic accuracy of haptoglobin level in ovarian cyst fluid as a potential biomarker for intra-operative triage of EOC in both local (Singapore) and regional (Indonesia and Vietnam) centers and to explore its possible clinical applications. Methods: We measured haptoglobin concentration in ovarian cyst fluid samples, that were carefully collected during surgery without spillage, from 113 benign tumors, 24 early stage and 31 late stage cancers in Department of Obstetrics and Gynaecology, National University Hospital in Singapore and 6 regional centers in Southeast Asia from 2004–2009, using an in-house sandwich enzyme-linked immunosorbent assay (ELISA). We also tested the feasibility of using a rapid colorimetric assay (Phase™ range haptoglobin assay, Tridelta Development Limited, Wicklow, Ireland) which measures haptoglobin in 5 minutes and could be potentially used in the operation theater for intra-operative cancer triage and compared it with ELISA and frozen section results. Results: Our data indicated that cyst fluid haptoglobin level was significantly elevated in both early and late stage EOCs (6.51±2.2 and 6.00±1.9 mg/ml, respectively) as compared to benign tumors (0.83±0.9 mg/ml, p A rapid way of measuring haptoglobin level was needed in order to be used for intra-operative diagnosis. Thus a rapid colorimetric assay was employed and the result was almost as good as ELISA. Haptoglobin level measured by the rapid assay was able to separate malignant from benign tumors with sensitivity of 98.1% (95% CI 88.6–99.9%), specificity of 92.1% (95% CI 84.5–96.3%), positive predictive value (PPV) of 86.7% (95% CI 74.9–93.7%) and negative predictive value (NPV) of 93.4% (95% CI 93.4–99.9%). Hence haptoglobin measured by both ELISA and rapid colorimetric assay can used as an alternative to frozen section diagnosis (especially in developing countries where frozen section is not available) or as low-cost triage methods. A point-of-care diagnostic device that employs the principle of the colorimetric assay has been developed and is currently being tested for rapid intra-operative cancer triage. Conclusion: Cyst fluid haptoglobin is a reliable maker for intra-operative diagnosis of EOC at early stage not only in local but also in regional sample sets, with high sensitivity for malignant tumors and a low false-positive rate allowing for optimal surgical procedure to be carried out. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B5

Diagnostic accuracy of haptoglobin within ovarian cyst fluid as a potential point-of-care test for epithelial ovarian cancer: an observational study

To investigate haptoglobin within ovarian cyst fluid (OCF) as a diagnostic biomarker for epithelial ovarian cancer (EOC) and develop an in vitro diagnostic point‐of‐care device test (IVDPCT) for use in the operating theatre.

Abstract POSTER-CTRL-1211: Haptoglobin identified within ovarian cyst fluid as an accurate intraoperative diagnostic biomarker for epithelial ovarian cancer

Introduction: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy, with a poor prognosis and low survival rate; most cases are diagnosed at a late stage due to the fact that symptoms at early stages are usually non-specific in nature. There is currently no screening method proven to be effective in improving the outcome of EOC patients; existing biomarkers for EOC, such as CA-125, generally suffer from a lack of specificity in early stage disease, which is the ideal time for therapeutic intervention. Aim: We hypothesized that a single protein biomarker within the ovarian cyst fluid (OCF) could be identified, isolated, characterized and validated for application in a point-of-care device (POC), which could subsequently be used in operating theaters for triage for frozen section (FS). Methods: We screened the OCF proteome by mass-spectrometry (MALTI-TOF/MS), confirmed the identity of the protein by western blot and SELDI immunocapture analysis. Next, demonstrated using tissue microarray (TMA) that cellular expression of haptoglobin varied in normal, benign and malignant ovarian tissue. We developed a simple ELISA and a rapid colorimetric assay that allowed semi-quantification of OCF haptoglobin intraoperatively. Finally, we validated a point-of-care test kit to accurately identify EOC using OCF with a higher predictive value than can be achieved using RMIs, and an equivalent accuracy to intraoperative FS. Results: The OCF haptoglobin concentration in benign tumors was 0.70±0.09 mg/ml compared to 6.22±0.53 mg/ml and 6.57±0.65 mg/ml in early- and late-stage EOCs, respectively (P Conclusion: This is the first study whereby an intraoperative tumor marker has been utilised in the differentiation between benign and malignant ovarian lesions. Its accuracy suggests that it could be utilised as a replacement or an adjunct to frozen section, particularly in situations where histopathological expertise is scarce. Citation Format: Mahesh Choolani, Loganath Annamalai, Lin Liu, Khalil Razvi, Changqing Zhao, Gregory Rice, Aniza P Mahyuddin, Arijit Biswas, Jerry Chan, Narasimhan Kothandaraman. Haptoglobin identified within ovarian cyst fluid as an accurate intraoperative diagnostic biomarker for epithelial ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1211.