A Second Look or, Not to Mention the Occasional Capsizing of a Windsurfer

Of all of the epithelial ovarian cancers (EOC), clear cell adenocarcinoma (CCA) has the worst clinical prognosis. Furthermore, the conventional EOC biomarker CA125 is more often negative in CCA than in other subtypes of EOC. This study sought to discover a new diagnostic biomarker that would allow more reliable detection of CCA. Using mass spectrometry, we compared proteins in conditioned media from cell lines derived from CCA and other types of EOC. We identified 30 extracellular or released proteins specifically present in CCA-derived cell lines. Bioinformatics analyses identified a serine protease inhibitor, tissue factor pathway inhibitor 2 (TFPI2), as a potential biomarker for CCA. Real time RT-PCR and Western blot analyses revealed that TFPI2 was exclusively expressed in CCA-derived cell lines and tissues. For clinical validation, we measured levels of TFPI2 and CA125 in a set of sera from 30 healthy women, 30 patients with endometriosis, and 50 patients with CCA, using an automated enzyme-linked immunosorbent assay systems. Serum levels of TFPI2 were significantly elevated in CCA patients, even those with normal CA125 levels. In terms of area under the receiver operating characteristic curve (AUC), TFPI2 was superior to CA125 in discriminating CCA patients from healthy women (AUC 0.97 for TFPI2 versus AUC 0.80 for CA125), or from patients with endometriosis (AUC 0.93 for TFPI2 versus 0.80 for CA125). This is the first evidence for TFPI2 as a serum biomarker of CCA. We propose that this biomarker may be useful for detection of CCA and for monitoring the transformation from endometriosis into CCA

Secretome-Based Identification of TFPI2, A Novel Serum Biomarker for Detection of Ovarian Clear Cell Adenocarcinoma

Of all of the epithelial ovarian cancers (EOC), clear cell adenocarcinoma (CCA) has the worst clinical prognosis. Furthermore, the conventional EOC biomarker CA125 is more often negative in CCA than in other subtypes of EOC. This study sought to discover a new diagnostic biomarker that would allow more reliable detection of CCA. Using mass spectrometry, we compared proteins in conditioned media from cell lines derived from CCA and other types of EOC. We identified 30 extracellular or released proteins specifically present in CCA-derived cell lines. Bioinformatics analyses identified a serine protease inhibitor, tissue factor pathway inhibitor 2 (TFPI2), as a potential biomarker for CCA. Real time RT-PCR and Western blot analyses revealed that TFPI2 was exclusively expressed in CCA-derived cell lines and tissues. For clinical validation, we measured levels of TFPI2 and CA125 in a set of sera from 30 healthy women, 30 patients with endometriosis, and 50 patients with CCA, using an automated enzyme-linked immunosorbent assay systems. Serum levels of TFPI2 were significantly elevated in CCA patients, even those with normal CA125 levels. In terms of area under the receiver operating characteristic curve (AUC), TFPI2 was superior to CA125 in discriminating CCA patients from healthy women (AUC 0.97 for TFPI2 versus AUC 0.80 for CA125), or from patients with endometriosis (AUC 0.93 for TFPI2 versus 0.80 for CA125). This is the first evidence for TFPI2 as a serum biomarker of CCA. We propose that this biomarker may be useful for detection of CCA and for monitoring the transformation from endometriosis into CCA

Expression rate of mesothelioma markers in some cancers.

<p>Intensity and proportion of staining in the cancer cells were evaluated in the entire microscopic field of each specimen. Cases were defined as positive if the Allred score of the marker was more than 3. In the immunostaining of calretinin or WT-1, staining in the nucleus, but not the cytoplasm, was designated as a positive sample. Calretinin or WT-1 was detected in the cytoplasm of 14% or 64% of cancers, respectively. The asterisk marks the sample from a mucus-producing cancer. AD, adenocarcinoma; BSS, biphasic synovial sarcoma; EAS, epithelioid angiosarcoma; EHE, epithelioid hemangioendothelioma; RCC, renal cell carcinoma; SCC, squamous cell carcinoma; UC, urothelial carcinoma.</p

Expression of intelectin-1 in normal tissues.

<p>Specimens were immunostained with anti-intelectin-1, and representative photographs are shown. A scale bar (100 µm) is shown in panel A, representatively. The scale bars of panel G (100 µm) and panel P (100 µm) are also shown. The closed black arrows indicate intelectin-1-positive cells. <b>A</b>, kidney collecting tubule; <b>B</b>, kidney cortex; <b>C</b>, intelectin-1-negative bladder; <b>D</b>, intelectin-1-positive bladder; <b>E</b>, bronchus. The open arrow shows the serous gland; <b>F</b>, bronchus. The open or closed arrow shows the serous or mucous gland, respectively; <b>G</b>, lung; <b>H</b>, intelectin-1-positive pleura; <b>I</b>, intelectin-1-positive peritoneum; <b>J</b>, intelectin-1-positive tunica vaginalis; <b>K</b>, pericardium; <b>L</b>, cardiac muscle and endocardium; <b>M</b>, vascular; <b>N</b>, S-100 protein staining of adipocytes in greater omentum; <b>O</b>, adipocytes in greater omentum; <b>P</b>, greater omentum with mesothelial cells.</p

Insignificant expression of intelectin-1 in non-MPM cancers that lack mucus production.

<p>Specimens were immunostained with MPM markers, and representative photographs are shown. A scale bar (100 µm) is shown in the bottom side of panel A, representatively. <b>A</b>, intelectin-1 staining of a colon adenocarcinoma (AD) lacking mucus production; <b>B</b>, intelectin-1 staining of mucus-producing colon AD; <b>C</b>, intelectin-1 staining of a gastric AD lacking mucus production; <b>D</b>, intelectin-1 staining of mucus-producing gastric AD; <b>E</b>, intelectin-1 staining of lung AD; <b>F</b>, calretinin staining of lung AD; <b>G</b>, intelectin-1 staining of lung squamous cell carcinoma (SCC); <b>H</b>, calretinin staining of lung SCC.</p

Clinical Significance of Tissue Factor Pathway Inhibitor 2, a Serum Biomarker Candidate for Ovarian Clear Cell Carcinoma - Fig 2

<p><b>ROC and AUC values for serum CA125 and TFPI2 levels in discrimination of CCC from other ovarian diseases (benign diseases, borderline, and non-CCC EOCs) (A), CCC versus borderline ovarian tumors (BD) and non-CCC EOCs (B), and CCC versus EMS (C).</b> Red line, TFPI2; black dotted line, CA125. Numbers indicate the calculated AUC values for TFPI2 and CA125.</p

Expression of marker antigens in MPM.

<p>The histologic type of MPM was classified by pathological diagnosis. The intensity and proportion of staining of MPM cells were evaluated in the entire microscopic field of the specimen. The individual data are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039889#pone.0039889.s002" target="_blank">Table S1</a>. Cases were defined as positive if the Allred score, which is the value of the intensity score plus the proportion score, of the marker was more than 3. In the immunostaining of calretinin or WT-1, staining in the nucleus, but not the cytoplasm, was designated as a positive sample. PD, poorly differentiated.</p

Expression of intelectin-1 in gastrointestinal goblet cells.

<p>Specimens were immunostained with anti-intelectin-1, and representative photographs are shown here. The scale bar (100 µm) is shown at the bottom of panel A, representatively. The scale bars of panel G (100 µm), panel H (100 µm), panel I (50 µm), and panel J (50 µm) are also shown. The arrows indicate paneth cells. <b>A</b>, duodenum; <b>B</b>, small intestine; <b>C</b>, colon; <b>D</b>, stomach; <b>E</b>, complete intestinal metaplasia (IM) in stomach; <b>F</b>, incomplete IM in stomach; <b>G</b>, hematoxylin–eosin (HE) staining of complete IM in stomach; <b>H</b>, complete IM in stomach; <b>I</b>, mucous granules in goblet cells; <b>J</b>, mucus-secreting goblet cells; <b>K</b>, liver and bile duct (open arrow); <b>L</b>, pancreas and pancreatic duct (open arrows).</p

Intelectin-1 expression in normal tissues.

<p>Expression scores were assigned by evaluating the intensity of intelectin-1 staining on the positive cells. The intensity of staining was defined by applying the Allred scoring as follows: 3+, strong staining; 2+, moderate staining; 1+, weak staining; –, no staining. The asterisk indicates the tissue in the digestive, urinary, respiratory, or reproductive system. IM, intestinal metaplasia.</p

ANXA4 expression visualized in surgically removed ovarian tumors using IHC.

<p>(A) Representative images for ANXA4 immunohistochemical (IHC) scores are shown. Each scale bar represents 200 µm. (B) IHC scores were significantly high in clear cell carcinoma compared with tumors with low malignant potential (LMP) and carcinomas (<i>P</i><0.05). ca, carcinoma; diff, differentiated.</p