Novel Hypoxia-Associated Markers of Chemoresistance in Ovarian Cancer

Background:
Ovarian cancer is the fifth leading cause of cancer in women and has poor
long-term survival, in part, due to chemoresistance. Tumour hypoxia is associated with
chemoresistance in ovarian cancer. However, relatively little is known about the genes
activated in ovarian cancer which cause chemoresistance due to hypoxia. This study
aimed to firstly identify genes whose expression is associated with hypoxia-induced
chemoresistance, and secondly select hypoxia-associated biomarkers and evaluate their
expression in ovarian tumours.
Design:
Cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian cancer
cell lines were exposed to combinations of hypoxia and/or cisplatin as part of a matrix
designed to reflect clinically relevant scenarios. RNA was extracted and interrogated
on Affymetrix Human Gene arrays. Differential gene expression was analysed for cells
exposed to hypoxia and/or treated with cisplatin. Potential markers of chemoresistance
were selected for evaluation in a cohort of ovarian tumour samples by R
T-PCR.
Results:
A wide range of genes associated with chemoresistance were differentially
expressed in cells exposed to hypoxia and/or cisplatin. Selected genes [ANGPTL4,
HER3 and HIF-1
α
] were chosen for further validation in a cohort of ovarian tumour
samples. High expression of ANGPTL4 trended towards reduced progression-free and
overall survival. High expression of HER3 trended to increased progression-free but
reduced overall survival, while high expression of HIF-1
α
trended towards reduced
progression-free and increased overall survival.
Conclusions:
In conclusion, this study has further characterized the relationship between
hypoxia and chemoresistance in an ovarian cancer model. We have also identified many
potential biomarkers of hypoxia and platinum resistance and provided initial validation
of a subset of these markers in ovarian cancer tissues.

Increased expression of the RIα subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer

The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RIα subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RIα expression in patients with ovarian cancer. We have evaluated the expression of RIα in a panel of human ovarian tumours (n = 40) and five human ovarian cancer cell lines using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The human ovarian cell lines OAW42 and OTN14 express high endogenous levels of RIα mRNA and protein (at significantly higher mRNA levels than high tissue expressors, P < 0.05). The ovarian cell line A2780 expresses low endogenous levels of RIα mRNA and protein (also at higher mRNA levels than low tissue expressors, P < 0.05). Quantitative RT-PCR revealed no significant difference in RIα mRNA expression between different ovarian histological subtypes in this study. No associations were found between RIα mRNA expression and differentiation state. RIα mRNA expression was significantly associated with tumour stage (P = 0.0036), and this remained significant in univariate analysis (P = 0.0002). A trend emerged between RIα mRNA expression levels and overall survival in univariate analysis (P = 0.051), however, by multivariate analysis, stage remained the major determinant of overall survival (P = 0.0001). This study indicates that in ovarian epithelial tumours high RIα mRNA expression is associated with advanced stage disease. RIα expression may be of predictive value in ovarian cancer and may be associated with dysfunctional signalling pathways in this cancer type.

Highly accurate detection of ovarian cancer using CA125 but limited improvement with serum matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling

Objectives: Our objective was to test the performance of CA125 in classifying serum samples from a cohort of malignant and benign ovarian cancers and age-matched healthy controls and to assess whether combining information from matrix-assisted laser desorption/ionization (MALDI) time-of-flight profiling could improve diagnostic performance. Materials and Methods: Serum samples from women with ovarian neoplasms and healthy volunteers were subjected to CA125 assay and MALDI time-of-flight mass spectrometry (MS) profiling. Models were built from training data sets using discriminatory MALDI MS peaks in combination with CA125 values and tested their ability to classify blinded test samples. These were compared with models using CA125 threshold levels from 193 patients with ovarian cancer, 290 with benign neoplasm, and 2236 postmenopausal healthy controls. Results: Using a CA125 cutoff of 30 U/mL, an overall sensitivity of 94.8% (96.6% specificity) was obtained when comparing malignancies versus healthy postmenopausal controls, whereas a cutoff of 65 U/mL provided a sensitivity of 83.9% (99.6% specificity). High classification accuracies were obtained for early-stage cancers (93.5% sensitivity). Reasons for high accuracies include recruitment bias, restriction to postmenopausal women, and inclusion of only primary invasive epithelial ovarian cancer cases. The combination of MS profiling information with CA125 did not significantly improve the specificity/accuracy compared with classifications on the basis of CA125 alone. Conclusions: We report unexpectedly good performance of serum CA125 using threshold classification in discriminating healthy controls and women with benign masses from those with invasive ovarian cancer. This highlights the dependence of diagnostic tests on the characteristics of the study population and the crucial need for authors to provide sufficient relevant details to allow comparison. Our study also shows that MS profiling information adds little to diagnostic accuracy. This finding is in contrast with other reports and shows the limitations of serum MS profiling for biomarker discovery and as a diagnostic tool

Effect of total abdominal hysterectomy on ovarian blood supply in women of reproductive age

Objective. The purpose of this study was to evaluate the effect of total abdominal hysterectomy on ovarian blood supply using transvaginal color Doppler ultrasonography in women of reproductive age. Methods. This prospective study included 61 women aged 40 years or younger who were divided into 2 groups: group 1, comprising 31 patients who underwent total abdominal hysterectomy (TAH), and group 2, comprising 30 women with no abnormalities. Inclusion criteria included normal ovarian function at baseline, with basal follicle-stimulating hormone levels of less than 15 mUI/mL, normal body weight, no tobacco use, and no history of laparotomy or ovarian disease. Ovarian arterial blood supply by determination of the pulsatility index (PI) on Doppler analysis and ovarian volume on transvaginal ultrasonography were assessed at baseline and at 6 and 12 postoperative months. The Student t test, profile analysis, and Friedman and Mann-Whitney tests were used in the statistical analysis of data. Results. Statistical analysis of baseline data revealed that both groups were homogeneous. At months 6 and 12, greater ovarian volumes and lower PI values were observed in patients who underwent TAH (P < .05). By the end of the study, in 8 of the 31 patients who underwent TAH (25.5%), benign ovarian cysts were observed. in the control group, all the parameters studied remained unchanged. Conclusions. The reduced PI values observed on Doppler ultrasonography suggested a decrease in the resistance flow in the ovarian arteries in women of reproductive age who underwent TAH.

A comparison of CA125, HE4, risk ovarian malignancy algorithm (ROMA), and risk malignancy index (RMI) for the classification of ovarian masses

OBJECTIVE: Differentiation between benign and malignant ovarian neoplasms is essential for creating a system for patient referrals. Therefore, the contributions of the tumor markers CA125 and human epididymis protein 4 (HE4) as well as the risk ovarian malignancy algorithm (ROMA) and risk malignancy index (RMI) values were considered individually and in combination to evaluate their utility for establishing this type of patient referral system. METHODS: Patients who had been diagnosed with ovarian masses through imaging analyses (n = 128) were assessed for their expression of the tumor markers CA125 and HE4. The ROMA and RMI values were also determined. The sensitivity and specificity of each parameter were calculated using receiver operating characteristic curves according to the area under the curve (AUC) for each method. RESULTS: The sensitivities associated with the ability of CA125, HE4, ROMA, or RMI to distinguish between malignant versus benign ovarian masses were 70.4%, 79.6%, 74.1%, and 63%, respectively. Among carcinomas, the sensitivities of CA125, HE4, ROMA (pre-and post-menopausal), and RMI were 93.5%, 87.1%, 80%, 95.2%, and 87.1%, respectively. The most accurate numerical values were obtained with RMI, although the four parameters were shown to be statistically equivalent. CONCLUSION: There were no differences in accuracy between CA125, HE4, ROMA, and RMI for differentiating between types of ovarian masses. RMI had the lowest sensitivity but was the most numerically accurate method. HE4 demonstrated the best overall sensitivity for the evaluation of malignant ovarian tumors and the differential diagnosis of endometriosis. All of the parameters demonstrated increased sensitivity when tumors with low malignancy potential were considered low-risk, which may be used as an acceptable assessment method for referring patients to reference centers.

Elevated glycodelin-A concentrations in serum and peritoneal fluid of women with ovarian endometriosis

The aim of this study was to evaluate serum and peritoneal fluid (PF) glycodelin-A concentrations in women with ovarian endometriosis. Ninety-nine matched pairs of serum and PF samples were included in our study. The case group comprised 57 women with ovarian endometriosis and the control group 42 healthy women undergoing sterilization or patients with benign ovarian cysts. Glycodelin-A concentrations were measured using ELISA. Endometriosis patients had significantly higher serum and PF glycodelin-A concentrations compared to controls, and this increase was observed in both proliferative and secretory cycle phases. Glycodelin-A concentrations were more than 10-fold higher in PF than in serum and correlated with each other. Intensity and frequency of menstrual pain positively correlated with glycodelin-A concentrations. Sensitivity and specificity of glycodelin-A as a biomarker for ovarian endometriosis were 82.1% and 78.4% in serum, and 79.7% and 77.5% in PF, respectively. These results indicate that Glycodelin-A has a potential role as a biomarker to be used in combination with other, independent marker molecules.

Increased levels of biglycan in endometriomas and peritoneal fluid samples from ovarian endometriosis patients

Abstract In our previous low-density-array gene-expression analysis we found an increased expression of biglycan gene in ovarian endometriosis patients. In the present study we evaluated biglycan expression at the protein level in tissue, serum and peritoneal fluid (PF) from ovarian endometriosis patients, patients with benign ovarian cysts and healthy women. Twenty samples of endometriomas and 27 of control tissues (benign ovarian cysts and eutopic endometrium of healthy women) were obtained laparoscopically or by curettage. Serum and PF samples were collected from 56 ovarian endometriosis patients and 40 controls (patients with benign cysts and healthy women). Tissue biglycan levels and serum and PF biglycan concentrations were determined by Western blotting and ELISA, respectively. Biglycan was detected in endometriomas and in benign cysts tissues but differed in glycosylation levels. The PF biglycan concentrations were significantly increased in ovarian endometriosis patients (mean ± SD = 220.3 ± 190.5 pg/mg protein) compared to the whole control group (101.9 ± 94.7 pg/mg protein, p < 0.001), while serum concentrations did not differ significantly. Biglycan appears to be involved in ovarian pathologies and probably has different roles in benign cysts as compared to ovarian endometriomas.

Phospholipase A2 group IIA is elevated in endometriomas but not in peritoneal fluid and serum of ovarian endometriosis patients.

Our previous gene expression analysis identified phospholipase A2 group IIA (PLA2G2A) as a potential biomarker of ovarian endometriosis. The aim of this study was to evaluate PLA2G2A mRNA and protein levels in tissue samples (endometriomas and normal endometrium) and in serum and peritoneal fluid of ovarian endometriosis patients and control women. One-hundred and sixteen women were included in this study: the case group included 70 ovarian endometriosis patients, and the control group included 38 healthy women and 8 patients with benign ovarian cysts. We observed 41.6-fold greater PLA2G2A mRNA levels in endometrioma tissue, compared to normal endometrium tissue. Using Western blotting, PLA2G2A was detected in all samples of endometriomas, but not in normal endometrium, and immunohistochemistry showed PLA2G2A-specific staining in epithelial cells of endometrioma paraffin sections. However, there were no significant differences in PLA2G2A levels between cases and controls according to ELISA of peritoneal fluid (6.0 ± 4.4 ng/ml, 6.6 ± 4.3 ng/ml; p = 0.5240) and serum (2.9 ± 2.1 ng/ml, 3.1 ± 2.2 ng/ml; p = 0.7989). Our data indicate that PLA2G2A is implicated in the pathophysiology of ovarian endometriosis, but that it cannot be used as a diagnostic biomarker.

Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis.

Endometriosis is a gynecologic disease that is characterized by nonspecific symptoms and invasive diagnostics. To date, there is no adequate noninvasive method for the diagnosis of endometriosis. Although more than 100 potential biomarkers have been investigated in blood and/or peritoneal fluid, none of these has proven useful in clinical practice. The aim to find a suitable panel of biomarkers that would allow noninvasive diagnosis thus remains of interest. We evaluated the concentrations of 16 cytokines and other secretory proteins in serum and peritoneal fluid of 58 women with ovarian endometriosis (cases) and 40 healthy women undergoing sterilization or patients with benign ovarian cysts (controls) using multiplexed double fluorescence-based immunometric assay platform and enzyme-linked immunosorbent assay. Significantly higher concentrations of glycodelin-A were shown in serum, and significantly higher levels of glycodelin-A, IL-6, and IL-8, and lower levels of leptin were measured in the peritoneal fluid of cases versus controls. In serum, the best performance was shown by models that included the ratio of leptin/glycodelin-A and the ratio of ficolin 2/glycodelin-A, whereas in the peritoneal fluid the best models included the ratio of biglycan/leptin, regulated on activation normal T-cell expressed and secreted/IL-6 and ficolin-2/glycodelin-A, and IL-8 per milligram of total protein, all in combination with age. The models using serum and peritoneal fluid distinguished between ovarian endometriosis patients and controls regardless of the menstrual cycle phase with relatively high sensitivity (72.5% to 84.2%), specificity (78.4% to 91.2%), and area under the curve (0.85 to 0.90).

Selenium binding protein 1 in ovarian cancer

Selenium binding protein I (SELENBP1) was identified to be the most significantly down-regulated protein in ovarian cancer cells by a membrane proteome profiling analysis. SELENBP1 expression levels in 4 normal ovaries, 8 benign ovarian tumors, 12 borderline ovarian tumors and 141 invasive ovarian cancers were analyzed with immunohistochemical assay. SELENBP1 expression was reduced in 87% cases of invasive ovarian cancer (122/141) and was significantly reduced in borderline tumors and invasive cancers (p < 0.001). Cox multivariate analysis within the 141 invasive cancer tissues showed that SELENBP1 expression score was a potential prognostic indicator for unfavorable prognosis of ovarian cancer (hazard ratio [HR], 2.18; 95% CI = L22-190; p = 0.009). Selenium can disrupt the androgen pathway, which has been implicated in modulating SELENBP1 expression. We investigated the effects of selenium and androgen on normal human ovarian surrace epithelial (HOSE) cells and cancer cells. Interestingly, SELENBP1 mRNA and protein levels were reduced by androgen and elevated by selenium treatment in the normal HOSE cells, whereas reversed responses were observed in the ovarian cancer cell lines. These results suggest that changes of SELENBP1 expression in malignant ovarian cancer are an indicator of aberration of selenium/androgen pathways and may reveal prognostic information of ovarian cancer. (c) 2005 Wiley-Liss, Inc.

Defects in tricarboxylic acid cycle enzymes Fumarate hydratase and Succinate dehydrogenase in cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a recently characterized cancer syndrome which predisposes to cutaneous and uterine leiomyomas as well as renal cell carcinoma (RCC). Uterine leiomyosarcoma (ULMS) has also been observed in certain Finnish HLRCC families. The predisposing gene for this syndrome, fumarate hydratase (FH), was identified in 2002. The well-known function of FH is in the tricarboxylic acid cycle (TCAC) in the energy metabolism of cells. As FH is a novel cancer gene, the role of FH mutations in tumours is in general unknown. Similarly, the mechanisms through which defective FH is associated with tumourigenesis are unclear. The loss of a wild type allele has been observed in virtually all HLRCC patients tumours and the FH enzyme activities are either totally lost or remarkably reduced in the tissues of mutation carrier patients. Therefore, FH is assumed to function as a tumour suppressor. Mutations in genes encoding subunits of other TCAC enzyme SDH have also been reported recently in tumours: mutations in SDHB, SDHC, and SDHD genes predispose to paraganglioma and pheochromocytoma. In the present study, mutations in the SDHB gene were observed to predispose to RCC. This was the first time that mutations in SDHB have been detected in extra-paraganglial tumours. Two different SDHB mutations were observed in two unrelated families. In the first family, the index patient was diagnosed with RCC at the age of 24 years. Additionally, his mother with a paraganglioma (PGL) of the heart and his maternal uncle with lung cancer were both carriers of the mutation. The RCC of the index patient and the PGL of his mother showed LOH. In the other family, an SDHB mutation was detected in two siblings who were both diagnosed with RCC at the ages of 24 and 26 years. Both of the siblings also suffered PGL. All these tumours showed LOH. Therefore, we concluded that mutations in SDHB predispose also for RCC in certain families. Several tumour types were analysed for FH mutations to define the role of FH mutations in these tumour types. In addition, patients with a putative cancer phenotype were analysed to identify new HLRCC families. Three FH variants were detected, of which two were novel. One of the variants was observed in a patient diagnosed with ULMS at the age of 41 years. However, LOH was not detected in the tumour tissue. The FH enzyme activity of the mutated protein was clearly reduced, being 43% of the activity of the normal protein. Together with the results from an earlier study we calculated that the prevalence of FH mutations in Finnish non-syndromic ULMS is around 2.4%. Therefore, FH mutations seem to have a minor role in the pathogenesis on non-syndromic ULMS. Two other germline variants were detected in a novel tumour type, ovarian mucinous cystadenoma. However, tumour tissues of the patients were not available for LOH studies and therefore LOH status remained unclear. Therefore, it is possible that FH mutations predispose also for ovarian tumours but further studies are needed to verify this result. A novel variant form of the FH gene (FHv) was identified and characterized in more detail. FHv contains an alternative first exon (1b), which appeared to function as 5 UTR sequence. The translation of FHv is initiated in vitro from exons two and three. The localization of FHv is both cytosolic and nuclear, in contrast to the localization of FH in mitochondria. FHv is expressed at low levels in all human tissues. Interestingly, the expression was induced after heat shock treatment and in chronic hypoxia. Therefore, FHv might have a role e.g. in the adaptation to unfavourable growth conditions. However, this remains to be elucidated.

Mitotic Arrest Deficiency Protein 2 (MAD2) and Histone Deacetylase 6 (HDAC6) Present a Complex Relationship in Their Regulation and Expression and Subsequent Impact on Chemoresponsiveness

Introduction:
Ovarian cancer patients presenting with advanced stage (III/IV)
canceraretreatedwithcarboplatinumincombinationwithpaclitaxel.Despitea
significant initial response rate, fewer than 20% of patients become long-term
survivors. We have published that low MAD2 expression levels associate with
reduced progression free survival (PFS) in patients with high-grade serous
epithelial ovarian cancer (EOC). Moreover, we have demonstrated that MAD2
expressionisdown-regulatedbythemicroRNAmiR-433(
Furlong et al., 2011
).
Interestingly, miR-433 also down-regulates HDAC6 (
Simon et al., 2010
), which
uniquely deacetylates
a
-tubulin prior to HDAC6s binding to
b
-tubulin.
In vitro
studies have shown that HDAC6 inhibition in combination with paclitaxel
treatment enhances chemoresistant cancer cell death. To date, an interaction
between MAD2 and HDAC6 has not been reported.
Experimental design:
MAD2 and HDAC6 immunohistochemistry (IHC) and
Western blot analyses were performed to investigate the role of HDAC6 and
MAD2 in chemoresistance to paclitaxel in high-grade serous EOC.
Results and Discussion:
In vitro
experiments demonstrated that overex-
pression of pre-miR-433, which targets MAD2, resulted in down-regulation
of HDAC6 in EOC cell lines. High levels of HDAC6 are co-expressed with
MAD2 in the paclitaxel resistant UPN251 and OVCAR7 cell lines. While, all
4 paclitaxel resistant EOC cell lines express higher levels of miR-433 than
the paclitaxel sensitive A2780 cells, only ovca432 and ovca433 demonstrated
down-regulation of both HDAC6 and MAD2. Paclitaxel binds to
b
-tubulin and
causesmicrotubulepolymerizationinpaclitaxelsensitivecellsasdemonstrated
by tubulin acetylation in A2780 cells. However, paclitaxel failed to cause a
significant acetylation of
a
-tubulin and microtubule stabilisation in the resistant
UPN251 cells. Therefore resistance in this cell line may be mediated by
aberrantly high HDAC6 activity. We have previously shown that MAD2 knock-
down cells are resistant to paclitaxel (
Furlong F., et al., 2011; Prencipe M.,
et al., 2009
). We measured HDAC6 protein expression in MAD2 knockdown
cells and showed that MAD2 knockdown is associated with concomitant
up-regulation of HDAC6. We hypothesise that the up-regulation of HDAC6
by MAD2 knockdown renders cancer cells more resistant to paclitaxel and
increases the invasive potential of these cells. On-going experiments will test
this hypothesis. Lastly we have observed differential MAD2 and HDAC6 IHC
staining intensity in formalin fixed paraffin embedded EOC samples.
In conclusion
, we have reported on a novel interaction between MAD2 and
HDAC6 which may have important consequences for paclitaxel resistant EOC.
Moreover, understanding chemo-responsiveness in ovarian tumours will lead
to improved patient management and treatment options for women diagnosed
with this disease

Femoral exostosis causing vastus medialis pain in an active young lady: a case report.

BACKGROUND:
Musculoskeletal conditions are a common reason for consultation to General Practitioners (GPs)/family physicians in primary care. Osteochondromas are the most common benign bone tumours and usually occur in the metaphyseal region of long bones. Despite the distal femur being the commonest location to find these benign bone tumours, this is the first case report in the literature specifically describing vastus medialis muscle pain as the presenting symptom due to underlying bursa formation secondary to local pressure effects.

CASE PRESENTATION:
Twenty nine year old female of white British ethnic origin, presenting to a primary care clinic with a three year history of intermittent left distal medial thigh pain.

CONCLUSION:
The benign bone tumour, femoral exostosis/osteochondroma, was diagnosed via Magnaetic Resonance Imaging (MRI) and treated conservatively, with surgical excision an option if not resolving. GPs/family physicians need to be aware of this diagnosis and that femoral exostosis/osteochondroma can present to primary care physicians, particularly within the second decade of life.

BRCA1, a 'complex' protein involved in the maintenance of genomic stability

BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression.

Supervivencia cáncer específica posterior a nefrectomía radical frente a nefrectomía parcial en carcinoma renal t2: revisión sistemática

Introducción: El tratamiento estándar para los tumores renales localizados es la nefrectomía radical, sin embargo debido a la variación el tamaño del tumor renal en el momento del diagnóstico, se ha reemplazado en algunos casos por la nefrectomía parcial. Objetivo: Este estudio busca comparar el resultado oncológico de la nefrectomía parcial en términos de supervivencia cáncer específica, respecto a la nefrectomía radical, en pacientes mayores de 50 años con carcinoma renal estadio II (T2N0M0) Métodos: Se realizó una revisión sistemática de la literatura, con inclusión de estudios de casos y controles, cohortes y experimentos clínicos aleatorizados incluidos en las bases de datos de MEDLINE , EMBASE y CENTRAL Resultados: La búsqueda inicial emitió un total de 101 resultados, 11 artículos fueron preseleccionados y sólo un artículo cumplió con los criterios de selección; éste se clasificó como nivel de evidencia II. Conclusión: No fue posible concluir su equivalencia oncológica de la nefrectomía radical con la nefrectomía parcial, dado que no hay diseños de estudios que permitan llegar a esta conclusión.