Peculiarities of data interpretation upon direct tissue analysis by Fourier transform ion cyclotron resonance mass spectrometry.

The importance of high-resolution mass spectrometry for the correct data interpretation of a direct tissue analysis is demonstrated with an example of its clinical application for an endometriosis study. Multivariate analysis of the data discovers lipid species differentially expressed in different tissues under investigation. High-resolution mass spectrometry allows unambiguous separation of peaks with close masses that correspond to proton and sodium adducts of phosphatidylcholines and to phosphatidylcholines differing in double bond number.

Impact and mechanistic role of oral contraceptive pills on the number and epithelial type of ovarian cortical inclusion cysts; a clinicopathology and immunohistochemical study.

BACKGROUND: Ovarian epithelial cancers are among the most lethal women's cancers. There is no doubt about the preventive role of oral contraceptive pills (OCPs) in development of ovarian cancers. But, there are limited numbers of studies to address the effect of these agents on the number of cortical inclusion cysts (CICs), their epithelial type and suppression of the metaplastic phenomenon by these pills. The aim of this study was to clarify the role of these agents in the prevention of these cyst formation and tubal metaplasia and also examine the mesenchymal-epithelial transition theory in this context by immunohistochemical methods. METHODS: The representative section(s) of ovarian cortex from a total number of 201 consecutive total abdominal hysterectomy with bilateral or unilateral salpingo-oophorectomy specimens were examined for mean number of CICs and their epithelial type between two groups of the patients. Group A included the patients who were on oral contraceptive pills for more than 5 years. All of the subjects with other contraceptive methods or a history of less than 5 years contraceptive pills usage were stratified in group B. Sections from 20 cases in which more than five inclusion cysts were found, were selected for IHC staining with calretinine and PAX8 as markers for mesothelium and mullerian epithelium respectively. RESULTS: The mean age of the patients was 51.67 years with no significant differences between two groups. The mean number of cysts were 1.27 and 3.23 in group A and B respectively (P =0.0001). Similarly the mean number of CICs, lined by tubal epithelium, was significantly different between two groups (0.65 vs 2.65, P =0.0001). In IHC staining 123 out of 150 CICs (82 %) were PAX+ while only 7 CICs (4.8 %) showed positive reaction for calretinin irrespective of type of epithelium. CONCLUSION: Our findings showed that the use of OCP for more than five years in women, significantly prevents development of cortical inclusion cysts in the ovaries which lined by tubal (PAX8 positive) type epithelium. These findings may explain the alternative mechanism of oral contraceptive pills or long time use of progesterone in suppression of tubal type overgrowth and subsequently prevention of ovarian epithelial cancers.

Transcriptional upregulation of HNF-1ß by NF-κB in ovarian clear cell carcinoma modulates susceptibility to apoptosis through alteration in bcl-2 expression.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a transcriptional factor that has an important role in endometriosis-ovarian clear cell carcinoma (OCCC) sequence by modulating cell kinetics and glucose metabolism. However, little is known about the detailed molecular mechanisms that govern its regulation and function. Herein, we focus on upstream and downstream regulatory factors of HNF-1ß in OCCCs. In clinical samples, HNF-1ß expression was positively correlated with the active form of NF-κB/p65 in OCCCs, and closely linked with a low nuclear grade and non-solid architecture. In cell lines, transfection of p65 resulted in increased HNF-1ß mRNA and protein expression in TOV-21G cells (OCCC cell line with endogenous HNF-1ß expression), in line with activation of the promoter, probably through interacting with the basic transcriptional machinery. Suppression of endogenous HNF-1ß expression by siRNA increased apoptosis in TOV-21G cells, while treatment of Hec251 cells (endometrial carcinoma cell line with extremely low endogenous HNF-1ß expression) stably overexpressing exogenous HNF-1ß with doxorubicin abrogated apoptosis of the cells, along with increased ratio of bcl-2 relative to bax. Moreover, overexpression of HNF-1ß led to upregulation of bcl-2 expression at the transcriptional level in TOV-21G cells, which provided evidence for a positive correlation between HNF-1ß and bcl-2 expression in OCCCs. These data, therefore, suggest that association between HNF-1ß and NF-κB signaling may participate in cell survival by alteration of apoptotic events, particularly in mitochondria-mediated pathways, through upregulation of bcl-2 expression in OCCCs.

Involvement of PAPP-A and IGFR1 in Cystic Ovarian Disease in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.

Biochemical composition of the fluid of ovarian cysts and pre-ovulatory follicles compared to the serum in sows.

OBJECTIVE: The objective of this study was to compare the biochemical composition of follicular cysts, pre-ovulatory follicles and serum in sows. MATERIAL AND METHODS: The research involved multiparous sows (cysts-bearing sows, n = 21; non-cysts-bearing sows, n = 22). Concentration of glucose, protein, cholesterol (CHOL), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triacylglycerol (TAG) in the samples was determined. RESULTS: Glucose concentration in serum was higher than in cysts and follicles (p < 0.01) and glucose concentration in cysts was higher than in follicles (p < 0.01). Differences were also observed between the concentration of glucose in serum of cysts-bearing and non-cysts-bearing sows (p < 0.01). Protein concentration in cysts and follicles was lower than in serum (p < 0.01). Concentration of cholesterol in the serum of cysts-bearing sows and non-cysts-bearing sows was higher than the one in cysts and follicles (p < 0.01). Cholesterol concentration in the serum of cysts-bearing sows was higher than the one in non-cysts-bearing sows (p < 0.01). Concentration of HDL in serum of both cysts-bearing and non-cysts-bearing sows was also higher than the one in cysts and follicles (p < 0.01). Cysts-bearing sows had a higher concentration of HDL in the serum than non-cysts-bearing sows. Differences were also observed between the concentration of HDL in cysts and the one in follicles (p < 0.05). LDL was determined not to be present in either cysts or pre-ovulatory follicles. TAG concentration in the serum of cysts-bearing sows was higher than the one in the serum of non-cysts-bearing sows (p < 0.05). Differences were also detected between the TAG concentrations in cysts and in follicles (p < 0.01). CONCLUSION: The differences in the biochemical composition of the fluid in follicular cysts and pre-ovulatory follicles point to the variable intensification of the course of metabolic processes in pathological and physiological ovarian structures.

Is it possible to diagnose malignancy from fluid in cystic ovarian tumors?

OBJECTIVE: p53 gene mutations are frequently identified in ovarian cancer tissue. The aim of this study was to investigate whether wild type or mutated genomic DNA can be identified in ovarian cystic fluid specimens. STUDY DESIGN: Forty-eight Japanese patients with cystic ovarian tumors (30 benign cysts, 8 borderline malignant tumors, and 10 cancers) were investigated. Cystic fluid and tumor tissue were obtained during surgery. After DNA extraction from the cystic fluid, polymerase chain reaction (PCR) and sequence analysis for exons 4-9 of the p53 gene was performed. In two cases of mucinous cystic tumor of borderline malignancy and endometrioid adenocarcinoma, the p53 gene sequences were determined. Immunohistochemical staining for abnormal p53 gene product was also performed. RESULTS: DNA was successfully extracted from all cystic fluid specimens. Furthermore, exons 4-9 of the p53 gene could be identified by electrophoresis from all samples. In a mucinous cystic tumor of borderline malignancy, one point mutation was identified at codon 223 in exon 6 (CCT → CTT) of the p53 gene. Aberrant p53 gene product was also observed in the tumor cells by immunohistochemical staining. Moreover, in another case of endometrial adenocarcinoma, a point mutation at codon 245 in exon 7 (GGC → AGC) was detected by the direct sequencing of the amplified Exon. Notably, the mutation was not present in the peripheral blood (PB) sample and tissue specimens from the patient. CONCLUSION: In cystic ovarian tumors, cystic fluid may provide informative material for molecular studies since it reflects the p53 status of tumor tissue in the cyst wall. This system might help to identify ovarian malignancy without resection of the tumor tissues.

Ovarian cyst fluids are a cache of tumor biomarkers that include calgranulin A and calgranulin B isoforms.

SELDI-TOF MS analysis of cyst fluids identified 95 peaks that discriminate malignant, borderline, and benign ovarian tumors. Three prominent peaks, which correspond to calgranulin A (m/z 10847) and two isoforms of calgranulin B (m/z 12717 and 13294), have higher concentrations in borderline and malignant cyst fluids. Together, calgranulin A and B distinguish borderline and malignant tumors from benign tumors with 28.6% and 63.6% sensitivity for early stage disease, respectively, at 95% specificity and with 74.8% accuracy. Ovarian cyst fluids are useful for discovering discriminatory biomarkers, such as calgranulin, which may have utility for detecting, diagnosing, and biochemically classifying ovarian tumors.

The oncofetal protein IMP3: a novel biomarker and triage tool for premalignant atypical endometriotic lesions.

OBJECTIVE: To determine whether the oncofetal protein IMP3 is detectable in endometriomas with or without histological atypia and whether IMP3 staining can be used as a triage tool to identify foci of atypical endometriosis in doubtful cases. DESIGN: Retrospective study. SETTING: Academic department and referral center for endometriosis. PATIENT(S): A consecutive series of 516 women who underwent excision of 874 endometriomas. INTERVENTION(S): Histological review by three expert pathologists and immunohistochemical staining for IMP3. MAIN OUTCOME MEASURE(S): Test performance of IMP3 immunohistochemistry versus first-round histology. RESULT(S): The prevalence of atypical endometriosis was 1.7% (95% confidence interval [CI], 0.9%-3.3%) based on the number of women and 1.0% (95% CI, 0.5%-1.9%) based on the number of cysts. Three cases of atypical endometriosis were identified at first-round histological examination. Immunohistochemistry detected seven of the eight cases diagnosed as preneoplastic atypia at second-round histology and one case diagnosed as reactive atypia at second-round histology. The sensitivity of first-round histology was 33.3%, compared with 88.9% of IMP3 immunohistochemistry. CONCLUSION(S): Immunohistochemical staining for IMP3 expression is a simple, inexpensive, and sensitive test that can be used in routine clinical practice as a triage tool to discriminate between cytological/structural atypia and confounding benign conditions.

Imbalance in serum soluble CD30/CD30L and CD40/CD40L systems are associated with ovarian tumors.

The aim of the study was to examine the concentrations of the soluble receptors and their ligands of CD30/CD30L and CD40/CD40L systems in the serum of women with ovarian tumor and in the ovarian cyst fluid of women with Cystadenoma serosum. The study included 120 women with ovarian tumors. As a control, sera were obtained from 60 healthy female volunteers. Concentrations of the sCD30, sCD30L, sCD40 and sCD40L in the serum and the ovarian cyst fluid were measured by ELISA enzyme-linked immunosorbent assay. Concentrations of both sCD30 and sCD30L in serum of women with ovarian tumors were significantly higher than in control (p < 0.0001). The highest serum receptor and its ligand levels were observed in women with ovarian cancer (p < 0.0001). Moreover, results showed significantly increased levels of sCD40 and sCD40L serum in women with ovarian tumors, as compared to the control group (p < 0.0001). The highest concentration of sCD40 in the serum of women with ovarian cancer and sCD40L in serum of women with Teratoma maturum (p < 0.0001) were observed. Impaired apoptosis among women with ovarian tumors is associated with the impairments of soluble CD30/CD30L and CD40/CD40L systems. Measurement of studied parameter concentrations in serum of women with ovarian tumors has been suggested to be a potential tool in monitoring of inflammatory. Evaluation of sCD30, sCD30L and sCD40 might be an early diagnostic marker in patients with the ovarian cancer. Concentrations of the studied parameters in the ovarian cyst fluid higher than the serum values suggest local suppression of the immune response. However, the final evaluation of the importance of measurement of serum levels of them requires further investigation.

Ovarian cystic tumor composed of Brenner tumor and struma ovarii.

Ovarian tumor composed only of Brenner tumor and struma ovarii is very rare; only 6 cases have been reported in the English literature, to the best of the author's knowledge. A 66-year-old woman underwent right oophorectomy because of torsion of right ovarian cyst. Macroscopically, the ovarian cyst was hemorrhagic and red. Cystic content was hemorrhagic fluid. Microscopically, the cyst walls were composed only of Brenner tumor (50% in area) and struma ovarii (50% in area). Hemorrhage and ischemic changes were seen. Other elements were not recognized. No malignant transformation was noted. These two elements were separately present, and no mergers between them were recognized. Immunohistochemically, the Brenner tumor element was positive for cytokeratins (AE1/3 and CAM5.2) and Ki67 (labeling=3%), but negative for thyroglobulin, TTF-1, p53, CA125, and vimentin. The struma ovarii element was positive for cytokeratins (AE1/3 and CAM5.2), thyroglobulin, TTF-1 and Ki67 (labeling=5%), but negative for p53, CA125 and vimentin. The findings suggests that there were cases of ovarian cyst composed only of Brenner tumor and struma ovarii, that such a case may be monodermal mature cystic teratoma or the Brenner tumor element was derived from surface epithelium in the preexisting struma ovarii, and that such a tumor manifest as cystic torsion.

Relationships between ovarian cysts and morphological and hormonal state of ovarian cortex in sows.

The aim of this study was to evaluate the relationship between ovarian cysts and concentrations of ovarian steroid hormones: 17beta-estradiol (E(2)), progesterone (P(4)), testosterone (T), and androstendione (A(4)) both in blood plasma and in cysts and morphological state of the ovarian cortex in sows. Females were divided into three groups: PCO (sows with polycystical ovaries), OO (sows with oligocystic ovaries) and control (sows without ovarian cysts). The ovaries for evaluations were collected after slaughtering of 18 multiparous sows. Between the PCO and OO animals, statistically significant differences in numbers of the follicular cysts (FC) (8.6 vs. 1.5), follicular theca-lutein cysts (FTLC) (8.0 vs. 2.0), follicular lutein cysts (FLC) (4.5 vs. 2.0) and corpus luteum cysts (CLC) (1.7 vs. 0.4) (P< or =0.01) were noted. In the PCO sows the most common kinds of cysts were FC and FTLC (8.6 and 8.0) whilst in OO sows the cysts occurred on their ovaries on a similar level (FC - 1.6, FTLC - 2.0, FLC - 2.0). Existence of more than 10 ovarian cysts in the sows significantly decreases the frequency of physiological ovarian follicles (primary, growing and maturing) and significantly increases the pathological process of atresia on all stages of ovarian follicles development (P< or =0.01). The study did not reveal any effect of growing or decreasing number of ovarian cyst on concentrations of E(2) and P(4) in blood plasma of sows. Polycystical ovaries significantly decreased concentrations of A(4) but increased the concentration of T in blood plasma (P< or =0.01). The general presence of ovarian cysts considerably positively correlated with concentrations of E(2), T and A(4) from cysts' fluid, of all kinds of ovarian cysts and atresia of primary follicles (a correlation coefficient r from 0.72 up to 0.97, P< or =0.05). The phenomenon of ovarian cysts significantly negatively correlated with all generations of ovarian follicles (P< or =0.05).

The balance between extracellular cathepsins and cystatin C is of importance for ovarian cancer.

BACKGROUND: A major step in cancer formation involves the degradation of the extracellular matrix, mediated by multiple degradative actions of (lysosomal) proteases. Extracellular release of lysosomal proteases (cathepsins) and their inhibitors has been associated with the development and progression of several types of cancer. We investigated whether cathepsins in ovarian cyst fluid (oCF) were associated with disease outcome in patients with epithelial ovarian cancer (EOC). MATERIALS AND METHODS: The levels of cathepsin B (CatB), H (CatH), L (CatL) and X (CatX) and their most abundant extracellular inhibitor cystatin C (CysC) were determined in oCF of 50 EOC patients by quantitative ELISAs. The cathepsin levels and ratios between cathepsins and CysC were related to clinicopathological parameters (Mann-Whitney U and Kruskal-Wallis tests) and survival (Cox Regression analysis). RESULTS: Median (25th-75th percentile) levels of cathepsin B, H, L, X and CysC in oCF were 97 (42-203), 18 (12-32), 61 (37-108), 20 (13-47) and 657 (501-805) ng mL(-1) respectively. Ratio of CysC/CatB was significantly lower for patients with metastatic compared with localised EOC (P = 0.025). Ratios of CysC/CatH and CysC/CatX differed significantly between histological subtypes (P = 0.012 and P = 0.035 respectively) and were significantly higher for high-grade tumours compared with low-grade tumours (P = 0.031 and P = 0.039 respectively). Neither cathepsins nor their ratios were significant predictors of survival for EOC patients. CONCLUSIONS: Ratios between CysC and cathepsins in oCF differed significantly between important clinicopathological subgroups. We believe that a complex cascade of proteolytic events, in which cathepsins play different roles, might be responsible for progression and metastasis in EOC.

Ovarian cyst fluid of serous ovarian tumors contains large quantities of the brain amino acid N-acetylaspartate.

BACKGROUND: In humans, N-acetyl L-aspartate (NAA) has not been detected in other tissues than the brain. The physiological function of NAA is yet undefined. Recently, it has been suggested that NAA may function as a molecular water pump, responsible for the removal of large amounts of water from the human brain. Ovarian tumors typically present as large cystic masses with considerable fluid accumulation. METHODOLOGY AND PRINCIPAL FINDINGS: Using Gas Chromatography-Mass Spectrometry, we demonstrated that NAA was present in a high micromolar concentration in oCF of epithelial ovarian tumors (EOTs) of serous histology, sometimes in the same range as found in the extracellular space of the human brain. In contrast, oCF of EOTs with a mucinous, endometrioid and clear cell histological subtype contained a low micromolar concentration of NAA. Serous EOTs have a cellular differentiation pattern which resembles the lining of the fallopian tube and differs from the other histological subtypes. The NAA concentration in two samples of fluid accumulation in the fallopian tube (hydrosalpinx) was in the same ranges as NAA found in oCF of serous EOTs. The NAA concentration in oCF of patients with serous EOTs was mostly 10 to 50 fold higher than their normal serum NAA concentration, whereas in patients with other EOT subtypes, serum and cyst fluid NAA concentration was comparable. CONCLUSIONS AND SIGNIFICANCE: The high concentration of NAA in cyst fluid of serous EOTs and low serum concentrations of NAA in these patients, suggest a local production of NAA in serous EOTs. Our findings provide the first identification of NAA concentrations high enough to suggest local production outside the human brain. Our findings contribute to the ongoing research understanding the physiological function of NAA in the human body.

Prognostic value of CA 125 in ovarian cyst fluid of patients with epithelial ovarian cancer.

Most ovarian tumors contain ovarian cyst fluid (oCF) which can be easily obtained during surgery. This is the first study that explored if CA 125 in oCF could be of prognostic value for patients with epithelial ovarian cancer (EOC). Of 54 patients with primary EOC, oCF and preoperative serum were collected and clinicopathological data were retrospectively obtained. CA 125 was measured with the commercially available CA 125 assay. CA 125 in oCF (n=54, median: 55,500 U/ml, range: 590-10,200,000 U/ml) was always higher than in the corresponding serum (n=51, median: 179 U/ml, range: 13-11,000 U/ml) (p<0.001) and values were moderately correlated (R=0.337, p=0.016). CA 125 in oCF was associated with histology (p<0.001) and tumor grade (p=0.038). High levels of oCF CA 125 (>median) were significantly associated with a poor disease-free survival (DFS) (log-rank p=0.002 and p=0.005 univariate Cox-regression). Other factors associated with a poor DFS in univariate analysis were advanced FIGO stage, suboptimal debulking (both p<0.001), high tumor grade (p=0.025), serous histology (p=0.003) and high serum (>media) CA 125 (p=0.009). In multivariate analysis, only FIGO stage was of independent predictive value. These findings indicate that, although high levels of oCF CA 125 were significantly associated with a poor survival of EOC patients, CA 125 in oCF was not of independent predictive value and might therefore not be useful as a prognostic biomarker for EOC.

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads.

High activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, is typically present in rapidly proliferating normal and malignant cells. The mitotically inactive steroidogenic cells in rodent testis and ovaries, however, also display high ODC activity. The activity of ODC in these cells responds to luteinizing hormone, and inhibition of ODC reduces the production of steroid hormones. Polyamines and ODC also control proliferation of germ cells and spermiogenesis. The activity of ODC, especially in proliferating cells, is regulated by antizyme inhibitor (AZIN). This protein displaces ODC from a complex with its inhibitor, antizyme. We have previously identified and cloned a second AZIN, i.e. antizyme inhibitor 2 (AZIN2), which has the highest levels of expression in brain and in testis. In the present study, we have used immunohistochemistry and in situ hybridization to localize the expression of AZIN2 in human gonads. We found a robust expression of AZIN2 in steroidogenic cells: testicular Leydig cells and Leydig cell tumors, in ovarian luteinized cells lining corpus luteum cysts, and in hilus cells. The results suggest that AZIN2 is not primarily involved in regulating the proliferation of the germinal epithelium, indicating a different role for AZIN1 and AZIN2 in the regulation of ODC. The localization of AZIN2 implies possible involvement in the gonadal synthesis and/or release of steroid hormones.

GSTP1-1 in ovarian cyst fluid and disease outcome of patients with ovarian cancer.

Detoxification enzymes, especially glutathione S-transferase P1-1 (GSTP1-1), have been implicated in resistance to platinum-based chemotherapy. We studied GSTP1-1 levels in ovarian cyst fluid (oCF), obtained during surgery before chemotherapy, of patients with epithelial ovarian cancer and clinical outcomes were correlated. GSTP1-1 was determined by ELISA in oCF of 56 patients with epithelial ovarian cancer and 109 noncancer controls (21 borderline and 88 benign ovarian tumors). Differences in median GSTP1-1 between clinicopathologic subgroups were studied using Mann-Whitney U and Kruskal Wallis tests. Differences in disease-free (DFS) and overall survival (OS) between groups were analyzed by applying Kaplan-Meyer estimates and log-rank tests. Univariate and multivariate analysis were done using Cox proportional hazard model. Significantly higher levels of GSTP1-1 were found in the oCF of malignant (median, 383; range, 10-32,695 ng/mL) compared with benign (median, 20; range, 0-1,128 ng/mL) ovarian tumors (P < 0.01). Significantly higher GSTP1-1 levels were found in patients with advanced International Federation of Gynaecologists and Obstetricians stage (P = 0.01), high-grade tumors (P = 0.44), and/or high levels of preoperative CA 125 (P = 0.01). Of patients who received chemotherapy (stage, >or=Ic; n = 30), high GSTP1-1 levels were significantly associated with a poor DFS and OS (log-rank P = 0.047 and P = 0.033, respectively). International Federation of Gynaecologists and Obstetricians stage was the only independent predictor for DFS. GSTP1-1 was the only independent predictor for OS.

Identification of blood group A/A-Leb/y and B/B-Leb/y active glycotopes co-expressed on the O-glycans isolated from two distinct human ovarian cyst fluids.

Although the individual human blood group A and B determinants are well defined, their co-expression pattern on a particular glycan carrier in individuals of blood group AB status has not been delineated. To address this issue, complex O-glycans were isolated from two distinct sources of human ovarian cyst glycoproteins (HOC 89 and Cyst 19) and profiled by advanced MS analyses, in conjunction with defining their binding characteristics against a panel of lectins and monoclonal antibodies. The major O-glycans of HOC 89 were found to correspond to sialyl Tn, mono- and di-sialyl T structures, whereas those of Cyst 19 were apparently more heterogeneous and extended to larger sizes. A minimal structure that carries both A and B determinants on the same molecule was identified, in which the A epitope is attached directly to the core GalNAc, whereas the B epitope is preferentially located on the six arms of a core 2 structure. Both arms can be further extended with internal fucosylation that appears to be restricted to those non-sialylated chains already carrying the terminal ABH determinants, thus giving rise to rather prominent A/B-Le(b/y) glycotopes on larger O-glycans.

N-acetyl resonances in in vivo and in vitro NMR spectroscopy of cystic ovarian tumors.

An unassigned and prominent resonance in the region from delta 2.0-2.1 ppm has frequently been found in the in vivo MR spectra of cancer patients. We demonstrated the presence of this resonance with in vivo MRS in the cyst fluid of a patient with an ovarian tumor. (1)H-NMRS on the aspirated cyst fluid of this patient confirmed the observation. A complex of resonances was observed between 2.0 and 2.1 ppm. It was also present in 11 additional ovarian cyst fluid samples randomly chosen from our biobank. The resonance complex was significantly more prominent in samples from mucinous tumors than in samples from other histological subtypes. A macromolecule (>10 kDa) was found responsible for this complex of resonances. A correlation spectroscopy (COSY) experiment revealed cross peaks of two different types of bound sialic acid suggesting that N-glycans from glycoproteins and/or glycolipids cause this resonance complex. In the literature, plasma alpha-1 acid glycoprotein (AGP), known for its high content of N-linked glycans, has been suggested to contribute to the delta 2.0-2.1 spectral region. The AGP cyst fluid concentration did not correlate significantly with the peak height of the delta 2.0-2.1 resonance complex in our study. AGP may be partly responsible for the resonance complex but other N-acetylated glycoproteins and/or glycolipids also contribute. After deproteinization of the cyst fluid, N-acetyl-L-aspartic acid (NAA) was found to contribute significantly to the signal in this spectral region in three of the 12 samples. GC-MS independently confirmed the presence of NAA in high concentration in the three samples, which all derived from benign serous tumors. We conclude that both NAA and N-acetyl groups from glycoproteins and/or glycolipids may contribute to the delta 2.0-2.1 ppm resonance complex in ovarian cyst fluid. This spectral region seems to contain resonances from biomarkers that provide relevant clinical information on the type of ovarian tumor.