Concurrent RB1 loss and BRCA-deficiency predicts enhanced immunological response and long-term survival in tubo-ovarian high-grade serous carcinoma.

Background: Somatic loss of the tumour suppressor RB1 is a common event in tubo-ovarian high-grade serous carcinoma (HGSC), which frequently co-occurs with alterations in homologous recombination DNA repair genes including BRCA1 and BRCA2 (BRCA). We examined whether tumour expression of RB1 was associated with survival across ovarian cancer histotypes (HGSC, endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous carcinoma (LGSC)), and how co-occurrence of germline BRCA pathogenic variants and RB1 loss influences long-term survival in a large series of HGSC. Patients and methods: RB1 protein expression patterns were classified by immunohistochemistry in epithelial ovarian carcinomas of 7436 patients from 20 studies participating in the Ovarian Tumor Tissue Analysis consortium and assessed for associations with overall survival (OS), accounting for patient age at diagnosis and FIGO stage. We examined RB1 expression and germline BRCA status in a subset of 1134 HGSC, and related genotype to survival, tumour infiltrating CD8+ lymphocyte counts and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cell lines with and without BRCA1 mutations to model co-loss with treatment response. We also performed genomic analyses on 126 primary HGSC to explore the molecular characteristics of concurrent homologous recombination deficiency and RB1 loss. Results: RB1 protein loss was most frequent in HGSC (16.4%) and was highly correlated with RB1 mRNA expression. RB1 loss was associated with longer OS in HGSC (hazard ratio [HR] 0.74, 95% confidence interval [CI] 0.66-0.83, P = 6.8 ×10-7), but with poorer prognosis in ENOC (HR 2.17, 95% CI 1.17-4.03, P = 0.0140). Germline BRCA mutations and RB1 loss co-occurred in HGSC (P < 0.0001). Patients with both RB1 loss and germline BRCA mutations had a superior OS (HR 0.38, 95% CI 0.25-0.58, P = 5.2 ×10-6) compared to patients with either alteration alone, and their median OS was three times longer than non-carriers whose tumours retained RB1 expression (9.3 years vs. 3.1 years). Enhanced sensitivity to cisplatin (P < 0.01) and paclitaxel (P < 0.05) was seen in BRCA1 mutated cell lines with RB1 knockout. Among 126 patients with whole-genome and transcriptome sequence data, combined RB1 loss and genomic evidence of homologous recombination deficiency was correlated with transcriptional markers of enhanced interferon response, cell cycle deregulation, and reduced epithelial-mesenchymal transition in primary HGSC. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA mutation was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

Ultra-low dose computed tomography of the chest in an emergency setting: A prospective agreement study.

Ultra-low dose computed tomography (ULD-CT) assessed by non-radiologists in a medical Emergency Department (ED) has not been examined in previous studies. To (i) investigate intragroup agreement among attending physicians caring for ED patients (i.e., radiologists, senior- and junior clinicians) and medical students for the detection of acute lung conditions on ULD-CT and supine chest X-ray (sCXR), and (ii) evaluate the accuracy of interpretation compared to the reference standard. In this prospective study, non-traumatic patients presenting to the ED, who received an sCXR were included. Between February and July 2019, 91 patients who underwent 93 consecutive examinations were enrolled. Subsequently, a ULD-CT and non-contrast CT were performed. The ULD-CT and sCXR were assessed by 3 radiologists, 3 senior clinicians, 3 junior clinicians, and 3 medical students for pneumonia, pneumothorax, pleural effusion, and pulmonary edema. The non-contrast CT, assessed by a chest radiologist, was used as the reference standard. The results of the assessments were compared within each group (intragroup agreement) and with the reference standard (accuracy) using kappa statistics. Accuracy and intragroup agreement improved for pneumothorax on ULD-CT compared with the sCXR for all groups. Accuracy and intragroup agreement improved for pneumonia on ULD-CT when assessed by radiologists and for pleural effusion when assessed by medical students. In patients with acute lung conditions ULD-CT offers improvement in the detection of pneumonia by radiologists and the detection of pneumothorax by radiologists as well as non-radiologists compared to sCXR. Therefore, ULD-CT may be considered as an alternative first-line imaging modality to sCXR for non-traumatic patients who present to EDs.

Systemic air embolism following computed-tomography-guided transthoracic needle biopsy of lung lesion - a systematic search of case reports and case series.

Background: Systemic air embolism is a rare, however potentially fatal, low incidence, complication to CT-guided transthoracic needle biopsy of lung lesions. Purpose: The purpose of this review of case reports and series was to pool data about this rare complication and glance for a pattern or similarities in the patients' initial symptoms and course, as well as the management of the patients in relation to current guidelines. Material and methods: PubMed was searched for case reports and case series about systemic air embolisms following CT-guided transthoracic needle biopsy of lung lesions from inception to November 2021. A reviewer screened the results for eligibility and included studies which reported at least two outcomes of interest. Data was extracted by one author and a descriptive analysis was conducted. Results: Of 1,136 studies screened, 83 were eligible for inclusion involving 97 patients. The mean age was 64.8±11.7 years and ≈60% of the patients were men. In 15 cases the outcome was fatal, and most of the fatal cases (n = 12) had cardiac arrest as the primary initial symptom. In addition to conventional oxygen therapy, 34 patients received hyperbaric oxygen therapy, and in 30 cases the physician in charge chose to change the patient from standard supine position to - most often - Trendelenburg position. Conclusion: No similarities were found that could lead to more rapid diagnosis or more correct management. The staff should keep systemic air embolisms in mind, when more common complications are ruled out, and consider hyperbaric oxygen therapy in case of suspicion.

Diagnostic potential of nanoparticle aided assays for MUC16 and MUC1 glycovariants in ovarian cancer.

Our study reports the discovery and evaluation of nanoparticle aided sensitive assays for glycovariants of MUC16 and MUC1 in a unique collection of paired ovarian cyst fluids and serum samples obtained at or prior to surgery for ovarian carcinoma suspicion. Selected glycovariants and the immunoassays for CA125, CA15-3 and HE4 were compared and validated in 347 cyst fluid and serum samples. Whereas CA125 and CA15-3 performed poorly in cyst fluid to separate carcinoma and controls, four glycovariants including MUC16MGL , MUC16STn , MUC1STn and MUC1Tn provided highly improved separations. In serum, the two STn glycovariants outperformed conventional CA125, CA15-3 and HE4 assays in all subcategories analyzed with main benefits obtained at high specificities and at postmenopausal and early-stage disease. Serum MUC16STn performed best at high specificity (90%-99%), but sensitivity was also improved by the other glycovariants and CA15-3. The highly improved specificity, excellent analytical sensitivity and robustness of the nanoparticle assisted glycovariant assays carry great promise for improved identification and early detection of ovarian carcinoma in routine differential diagnostics.

Diagnostic accuracy of ultra-low-dose chest computed tomography in an emergency department.

BACKGROUND: This study examined whether ultra-low-dose chest computed tomography (ULD-CT) could improve detection of acute chest conditions. PURPOSE: To determine (i) whether diagnostic accuracy of ULD-CT is superior to supine chest X-ray (sCXR) for acute chest conditions and (ii) the feasibility of ULD-CT in an emergency department. MATERIAL AND METHODS: From 1 February to 31 July 2019, 91 non-traumatic patients from the Emergency Department were prospectively enrolled in the study if they received an sCXR. An ULD-CT and a non-contrast chest CT (NCCT) scan were then performed. Three radiologists assessed the sCXR and ULD-CT examinations for cardiogenic pulmonary edema, pneumonia, pneumothorax, and pleural effusion. Resources and effort were compared for sCXR and ULD-CT to evaluate feasibility. Diagnostic accuracy was calculated for sCXR and ULD-CT using NCCT as the reference standard. RESULTS: The mean effective dose of ULD-CT was 0.05±0.01 mSv. For pleural effusion and cardiogenic pulmonary edema, no difference in diagnostic accuracy between ULD-CT and sCXR was observed. For pneumonia and pneumothorax, sensitivities were 100% (95% confidence interval [CI] 69-100) and 50% (95% CI 7-93) for ULD-CT and 60% (95% CI 26-88) and 0% (95% CI 0-0) for sCXR, respectively. Median examination time was 10 min for ULD-CT vs. 5 min for sCXR (P<0.001). For ULD-CT 1-2 more staff members were needed compared to sCXR (P<0.001). ULD-CT was rated more challenging to perform than sCXR (P<0.001). CONCLUSION: ULD-CT seems equal or better in detecting acute chest conditions compared to sCXR. However, ULD-CT examinations demand more effort and resources.

Targeted Selected Reaction Monitoring Verifies Histology Specific Peptide Signatures in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (OC) is a disease with high mortality due to vague early clinical symptoms. Benign ovarian cysts are common and accurate diagnosis remains a challenge because of the molecular heterogeneity of OC. We set out to investigate whether the disease diversity seen in ovarian cyst fluids and tumor tissue could be detected in plasma. Using existing mass spectrometry (MS)-based proteomics data, we constructed a selected reaction monitoring (SRM) assay targeting peptides from 177 cancer-related and classical proteins associated with OC. Plasma from benign, borderline, and malignant ovarian tumors were used to verify expression (n = 74). Unsupervised and supervised multivariate analyses were used for comparisons. The peptide signatures revealed by the supervised multivariate analysis contained 55 to 77 peptides each. The predictive (Q2) values were higher for benign vs. low-grade serous Q2 = 0.615, mucinous Q2 = 0.611, endometrioid Q2 = 0.428 and high-grade serous Q2 = 0.375 (stage I-II Q2 = 0.515; stage III Q2 = 0.43) OC compared to benign vs. all malignant Q2 = 0.226. With targeted SRM MS we constructed a multiplexed assay for simultaneous detection and relative quantification of 185 peptides from 177 proteins in only 20 µL of plasma. With the approach of histology-specific peptide patterns, derived from pre-selected proteins, we may be able to detect not only high-grade serous OC but also the less common OC subtypes.

Analysis of blood group antigens on MUC5AC in mucinous ovarian cancer tissues using in situ proximity ligation assay.

MUC5AC has been indicated to be a marker for mucinous ovarian cancer (OC). We investigated the use of in situ proximity ligation assay (PLA) for blood group ABH expressing MUC5AC to differentiate between serous and mucinous OC, to validate preceding observations that also MUC5AC ABH expression is increased in mucinous OC. We developed PLA for anti-A, B, and H/anti-MUC5AC and a PLA using a combined lectin Ulex europaeus agglutinin I (UEA I)/anti-MUC5AC assay. The PLAs were verified with mass spectrometry, where mucinous OC secretor positive patients' cysts fluids containing ABH O-linked oligosaccharides also showed positive OC tissue PLA staining. A nonsecretor mucinous OC cyst fluid was negative for ABH and displayed negative PLA staining of the matched tissue. Using the UEA I/MUC5AC PLA, we screened a tissue micro array of 410 ovarian tissue samples from patients with various stages of mucinous or serous OC, 32 samples with metastasis to the ovaries and 34 controls. The PLA allowed differentiating mucinous tumors with a sensitivity of 84% and a specificity of 97% both against serous cancer but also compared to tissues from controls. This sensitivity is close to the expected incidence of secretor individuals in a population. The recorded sensitivity was also found to be higher compared to mucinous type cancer with metastasis to the ovaries, where only 32% were positive. We conclude that UEA 1/MUC5AC PLA allows glycospecific differentiation between serous and mucinous OC in patients with positive secretor status and will not identify secretor negative individuals with mucinous OC.

Consideration should be given to smoking, endometriosis, renal function (eGFR) and age when interpreting CA125 and HE4 in ovarian tumor diagnostics.

OBJECTIVES: To evaluate the impact of different biologic, histopathologic and lifestyle factors on serum levels of human epididymis protein 4 (HE4) and Cancer antigen 125 (CA125) in the diagnostic work up of women with an ovarian cyst or pelvic tumor. METHODS: The statistical evaluation was performed on a population of 445 women diagnosed with a benign ovarian disease, included in a large Swedish multicenter trial (ClinicalTrials.gov NCT03193671). Multivariable logistic regression analyses were performed to distinguish between the true negatives and false positives through adjusting for biologic, histopathologic and lifestyle factors on serum samples of CA125 and HE4 separately. The likelihood ratio test was used to determine statistical significance and Benjamini-Hochberg correction to adjust for multiple testing. RESULTS: A total of 31% of the women had false positive CA125 but only 9% had false positive results of HE4. Smoking (OR 6.62 95% CI 2.93-15.12) and impaired renal function, measured by eGFR (OR 0.18 95% CI 0.08-0.39), were independently predictive of falsely elevated serum levels of HE4. Endometriosis was the only variable predictive of falsely elevated serum levels of CA125 (OR 7.96 95% CI 4.53-14.39). Age correlated with increased serum levels of HE4. CONCLUSIONS: Smoking, renal failure, age and endometriosis are factors that independently should be considered when assessing serum levels of HE4 and CA125 in women with an ovarian cyst or pelvic mass to avoid false indications of malignant disease.

Risk of epithelial ovarian cancer Type I and II after hysterectomy, salpingectomy and tubal ligation-A nationwide case-control study.

The proposed different origins and pathways to of the dualistic model of epithelial ovarian cancer (EOC) may affect and alter the potential risk reduction related to hysterectomy, salpingectomy and tubal ligation. The aim of our study was to analyze associations between hysterectomy, salpingectomy or tubal ligation and risk reduction of EOC Type I and II. In this nationwide register-based case-control study, women diagnosed with EOC, Fallopian tube or primary peritoneal cancer between 2008 and 2014 were included. Cases were classified into Type I and II according to histology and predefined criteria. The exposure variables: hysterectomy, salpingectomy and tubal ligation were identified from national registries. Conditional logistic regression analyses were performed to evaluate associations between Type I and II EOC and the exposure variables. Among 4669 registered cases, 4040 were eligible and assessed for subtyping resulting in 1033 Type I and 3007 Type II. Ten controls were randomly assigned to each case from the register of population. In regression analyses, women with previous salpingectomy had a significantly lower risk of EOC Type II (odds ratio [OR] 0.62; 95% confidence interval [95%CI] 0.45-0.85) but not Type I (OR 1.16; 95%CI 0.75-1.78). Hysterectomy was associated with a reduced risk of both EOC Type I (OR 0.71; 95%CI 0.52-0.99) and Type II (OR 0.81; 95%CI 0.68-0.96). Similar estimates were obtained for tubal ligation, although without statistical significance. The association between salpingectomy and reduced risk of EOC Type II supports the proposed theory of high-grade serous cancer originating from the tubal fimbriae.

Diagnostic accuracy of low-dose and ultra-low-dose CT in detection of chest pathology: a systematic review.

PURPOSE: Studies have evaluated imaging modalities with a lower radiation dose than standard-dose CT (SD-CT) for chest examination. This systematic review aimed to summarize evidence on diagnostic accuracy of these modalities - low-dose and ultra-low-dose CT (LD- and ULD-CT) - for chest pathology. METHOD: Ovid-MEDLINE, Ovid-EMBASE and the Cochrane Library were systematically searched April 29th-30th, 2019 and screened by two reviewers. Studies on diagnostic accuracy were included if they defined their index tests as 'LD-CT', 'Reduced-dose CT' or 'ULD-CT' and had SD-CT as reference standard. Risk of bias was evaluated on study level using the Quality Assessment of Diagnostic Accuracy Studies-2. A narrative synthesis was conducted to compare the diagnostic accuracy measurements. RESULTS: Of the 4257 studies identified, 18 were eligible for inclusion. SD-CT (3.17 ± 1.47 mSv) was used as reference standard in all studies to evaluate diagnostic accuracy of LD- (1.22 ± 0.34 mSv) and ULD-CT (0.22 ± 0.05 mSv), respectively. LD-CT had high sensitivities for detection of bronchiectasis (82-96%), honeycomb (75-100%), and varying sensitivities for nodules (63-99%) and ground glass opacities (GGO) (77-91%). ULD-CT had high sensitivities for GGO (93-100%), pneumothorax (100%), consolidations (90-100%), and varying sensitivities for nodules (60-100%) and emphysema (65-90%). CONCLUSION: The included studies found LD-CT to have high diagnostic accuracy in detection of honeycombing and bronchiectasis and ULD-CT to have high diagnostic accuracy for pneumothorax, consolidations and GGO. Summarizing evidence on diagnostic accuracy of LD- and ULD-CT for other chest pathology was not possible due to varying outcome measures, lack of precision estimates and heterogeneous study design and methodology.

Increased Diagnostic Accuracy of Adnexal Tumors with A Combination of Established Algorithms and Biomarkers.

Ovarian cancer is the most lethal gynecologic cancer. Pre-diagnostic testing lacks sensitivity and specificity, and surgery is often the only way to secure the diagnosis. Exploring new biomarkers is of great importance, but the rationale of combining validated well-established biomarkers and algorithms could be a more effective way forward. We hypothesized that we can improve differential diagnostics and reduce false positives by combining (a) risk of malignancy index (RMI) with serum HE4, (b) risk of ovarian malignancy algorithm (ROMA) with a transvaginal ultrasound score or (c) adding HE4 to CA125 in a simple algorithm. With logistic regression modeling, new algorithms were explored and validated using leave-one-out cross validation. The analyses were performed in an existing cohort prospectively collected prior to surgery, 2013-2016. A total of 445 benign tumors and 135 ovarian cancers were included. All presented models improved specificity at cut-off compared to the original algorithm, and goodness of fit was significant (p < 0.001). Our findings confirm that HE4 is a marker that improves specificity without hampering sensitivity or diagnostic accuracy in adnexal tumors. We provide in this study "easy-to-use" algorithms that could aid in the triage of women to the most appropriate level of care when presenting with an unknown ovarian cyst or suspicious ovarian cancer.

Univariate and classification analysis reveals potential diagnostic biomarkers for early stage ovarian cancer Type 1 and Type 2.

Biomarkers for early detection of ovarian tumors are urgently needed. Tumors of the ovary grow within cysts and most are benign. Surgical sampling is the only way to ensure accurate diagnosis, but often leads to morbidity and loss of female hormones. The present study explored the deep proteome in well-defined sets of ovarian tumors, FIGO stage I, Type 1 (low-grade serous, mucinous, endometrioid; n = 9), Type 2 (high-grade serous; n = 9), and benign serous (n = 9) using TMT-LC-MS/MS. Data are available via ProteomeXchange with identifier PXD010939. We evaluated new bioinformatics tools in the discovery phase. This innovative selection process involved different normalizations, a combination of univariate statistics, and logistic model tree and naive Bayes tree classifiers. We identified 142 proteins by this combined approach. One biomarker panel and nine individual proteins were verified in cyst fluid and serum: transaldolase-1, fructose-bisphosphate aldolase A (ALDOA), transketolase, ceruloplasmin, mesothelin, clusterin, tenascin-XB, laminin subunit gamma-1, and mucin-16. Six of the proteins were found significant (p < .05) in cyst fluid while ALDOA was the only protein significant in serum. The biomarker panel achieved ROC AUC 0.96 and 0.57 respectively. We conclude that classification algorithms complement traditional statistical methods by selecting combinations that may be missed by standard univariate tests. SIGNIFICANCE: In the discovery phase, we performed deep proteome analyses of well-defined histology subgroups of ovarian tumor cyst fluids, highly specified for stage and type (histology and grade). We present an original approach to selecting candidate biomarkers combining several normalization strategies, univariate statistics, and machine learning algorithms. The results from validation of selected proteins strengthen our prior proteomic and genomic data suggesting that cyst fluids are better than sera in early stage ovarian cancer diagnostics.

A multicenter clinical trial validating the performance of HE4, CA125, risk of ovarian malignancy algorithm and risk of malignancy index.

OBJECTIVE: To validate, in a multicenter clinical trial, the performance of biomarkers and algorithms for differential diagnosis in a population of women diagnosed with an unknown ovarian cyst or pelvic tumor. METHODS: Six hospitals in Western Sweden consecutively enrolled 638 women from September 2013 to February 2016. Serum, transvaginal ultrasound data, and basic patient characteristics were collected preoperatively. Biomarker levels, risk of malignancy algorithm (ROMA), and risk of malignancy index (RMI) were calculated and compared with the final pathology report. RESULTS: Our sample of 638 patients had 445 benign, 31 borderline, and 162 malignant tumors recorded, and the overall incidence of epithelial ovarian cancer was 21%. In postmenopausal women, RMI (>200), ROMA (≥29.9), CA125 (>35 U/mL), and HE4 (>140 pmol/L) showed sensitivity at 89%, 91%, 92%, and 72%, respectively, and specificity at 80%, 77%, 80%, and 92%. In premenopausal women, sensitivity of RMI, ROMA (≥11.6), CA125, and HE4 (>70 pmol/L) was 87%, 87%, 96%, and 83%, respectively, and specificity was 90%, 81%, 60%, 91%. Diagnostic accuracy (ROC AUC) of RMI and ROMA in postmenopausal women was 0.85 and 0.84, and in premenopausal women, 0.90 and 0.81. CONCLUSION: Our results suggest that CA125 is superior to HE4 as a biomarker to identify women with ovarian cancer. HE4 more correctly identifies benign lesions, which may help in differential diagnoses to guide the level of care and decrease overtreatment. This study confirms prior results from single-center studies and suggests the implementation of HE4 measurement in daily practice.

Diagnostic potential of tumor DNA from ovarian cyst fluid.

We determined whether the mutations found in ovarian cancers could be identified in the patients' ovarian cyst fluids. Tumor-specific mutations were detectable in the cyst fluids of 19 of 23 (83%) borderline tumors, 10 of 13 (77%) type I cancers, and 18 of 18 (100%) type II cancers. In contrast, no mutations were found in the cyst fluids of 18 patients with benign tumors or non-neoplastic cysts. Though large, prospective studies are needed to demonstrate the safety and clinical utility of this approach, our results suggest that the genetic evaluation of cyst fluids might be able to inform the management of the large number of women with these lesions.

A novel diagnostic index combining HE4, CA125 and age may improve triage of women with suspected ovarian cancer - An international multicenter study in women with an ovarian mass.

AIM: To develop and validate a biomarker-based index to optimize referral and diagnosis of patients with suspected ovarian cancer. Furthermore, to compare this new index with the Risk of Malignancy Index (RMI) and Risk of Ovarian Malignancy Algorithm (ROMA). PATIENTS AND METHODS: A training study, consisting of patients with benign ovarian disease (n=809) and ovarian cancer (n=246), was used to develop the Copenhagen Index (CPH-I) utilizing the variables serum HE4, serum CA125 and patient age. Eight international studies provided the validation population; comprising 1060 patients with benign ovarian masses and 550 patients with ovarian cancer. RESULTS: Overall, 2665 patients were included. CPH-I was highly significant in discriminating benign from malignant ovarian disease. At the defined cut-off of 0.070 for CPH-I the sensitivity and specificity were 95.0% and 78.4% respectively in the training cohort and 82.0% and 88.4% in the validation cohort. Comparison of CPH-I, ROMA and RMI demonstrated area-under-curve (AUC) at 0.960, 0.954 and 0.959 respectively in the training study and 0.951, 0.953 and 0.935 respectively in the validation study. Using a sensitivity of 95.0%, the specificities for CPH-I, ROMA and RMI in the training cohort were 78.4%, 71.7% and 81.5% respectively, and in the validation cohort 67.3%, 70.7% and 69.5% respectively. CONCLUSION: All three indices perform well at the clinically relevant sensitivity of 95%, but CPH-I, unlike RMI and ROMA, is independent of ultrasound and menopausal status, and may provide a simple index to optimize referral of women with suspected ovarian cancer.

Early inflammatory response in epithelial ovarian tumor cyst fluids.

Mortality rates for epithelial ovarian cancer (EOC) are high, mainly due to late-stage diagnosis. The identification of biomarkers for this cancer could contribute to earlier diagnosis and increased survival rates. Given that chronic inflammation plays a central role in cancer initiation and progression, we selected and tested 15 cancer-related cytokines and growth factors in 38 ovarian cyst fluid samples. We used ovarian cyst fluid since it is found in proximity to the pathology and mined it for inflammatory biomarkers suitable for early detection of EOC. Immunoprecipitation and high-throughput sample fractionation were obtained by using tandem antibody libraries bead and mass spectrometry. Two proteins, monocyte chemoattractant protein-1 (MCP-1/CCL2) and interleucin-8 (IL-8/CXCL8), were significantly (P < 0.0001) higher in the malignant (n = 16) versus benign (n = 22) tumor cysts. Validation of MCP-1, IL-8, and growth-regulated protein-α (GROα/CXCL1) was performed with ELISA in benign, borderline, and malignant cyst fluids (n = 256) and corresponding serum (n = 256). CA125 was measured in serum from all patients and used in the algorithms performed. MCP-1, IL-8, and GROα are proinflammatory cytokines and promoters of tumor growth. From 5- to 100-fold higher concentrations of MCP-1, IL-8 and GROα were detected in the cyst fluids compared to the serum. Significant (P < 0.001) cytokine response was already established in borderline cyst fluids and stage I EOC. In serum a significant (P < 0.01) increase of IL-8 and GROα was found, but not until stage I and stage III EOC, respectively. These findings confirm that early events in tumorigenesis can be analyzed and detected in the tumor environment and we conclude that ovarian cyst fluid is a promising source in the search for new biomarkers for early ovarian tumors.

Diagnostic performance of the biomarkers HE4 and CA125 in type I and type II epithelial ovarian cancer.

OBJECTIVE: To evaluate the diagnostic performance of HE4 and CA125 in patients presenting with suspicious malignant ovarian cysts. We especially wanted to investigate the levels of HE4 and CA125 with regard to the gene and histology-unifying model of type I and type II epithelial ovarian cancer (EOC). METHODS: Plasma from 373 women presenting with a suspicious malignant ovarian cyst was collected prior to surgery. Histology, grade, and stage were determined according to FIGO-classification. HE4 and CA125 were analyzed using ELISA, and the markers were evaluated for significance separately and in combination. Receiver operating curves, the area under the curve, sensitivity and specificity were estimated. RESULTS: The combination of HE4 and CA125 resulted in the best diagnostic power in comparing benign tumors to EOC (ROC AUC 0.93, sensitivity 94.4% at 75% specificity) for type II. Diagnostic power in type I (ROC AUC 0.79, sensitivity 61.9% at 75% specificity) was less impressive. In particular, mucinous benign vs. malignant tumors could not significantly be separated by the dual marker combination. Impressively high ROC AUC 0.99 was found for the late stage type II EOC with 100% sensitivity at 75% specificity. CONCLUSIONS: HE4 and CA125 have a good ability to diagnose the more aggressive type II tumors but a poor diagnostic ability when patients are presenting with slow-growing type I in the early stage. Our results support the hypothesis that EOC should be looked upon as several different diseases, and that we lack biomarkers for sub-groups of EOC.

Potential tumor biomarkers identified in ovarian cyst fluid by quantitative proteomic analysis, iTRAQ.

BACKGROUND: Epithelial-derived ovarian adenocarcinoma (EOC) is the most deadly gynecologic tumor, and the principle cause of the poor survival rate is diagnosis at a late stage. Screening and diagnostic biomarkers with acceptable specificity and sensitivity are lacking. Ovarian cyst fluid should harbor early ovarian cancer biomarkers because of its closeness to the tumor. We investigated ovarian cyst fluid as a source for discovering biomarkers for use in the diagnosis of EOC. RESULTS: Using quantitative mass spectrometry, iTRAQ MS, we identified 837 proteins in cyst fluid from benign, EOC stage I, and EOC stage III. Only patients of serous histology were included in the study. Comparing the benign (n = 5) with the malignant (n = 10) group, 87 of the proteins were significantly (p < 0.05) differentially expressed. Two proteins, serum amyloid A-4 (SAA4) and astacin-like metalloendopeptidase (ASTL), were selected for verification of the iTRAQ method and external validation with immunoblot in a larger cohort with mixed histology, in plasma (n = 68), and cyst fluid (n = 68). The protein selections were based on either high significance and high fold change or abundant appearance and several peptide recognitions in the sample sets (p = 0.04, FC = 1.95) and (p < 0.001, FC = 8.48) for SAA4 and ASTL respectively. Both were found to be significantly expressed (p < 0.05), but the methods did not correlate concerning ASTL. CONCLUSIONS: Fluid from ovarian cysts connected directly to the primary tumor harbor many possible new tumor-specific biomarkers. We have identified 87 differentially expressed proteins and validated two candidates to verify the iTRAQ method. However several of the proteins are of interest for validation in a larger setting.

Ovarian cyst fluid is a rich proteome resource for detection of new tumor biomarkers.

BACKGROUND: We aimed to investigate the use of ovarian cyst fluid as a source for biomarker discovery and to find novel biomarkers for use in the diagnosis of epithelial ovarian tumors. RESULTS: Ovarian cyst fluids from 218 women were collected and 192 (benign n = 129, malignant n = 63) were analyzed using mass spectrometry. 1180 peaks were detected, 221 of which were differently expressed between benign and malignant ovarian tumors. Seventeen peaks had receiver operating curve and area under the curve values >0.70; the majority of these represented peaks for apolipoproteins C-III and C-I (ApoC-I), transthyretin (TTR), serum amyloid A4 (SAA4), and protein C inhibitor (PCI). ApoC-III, PCI, and serum CA125, with an ROC AUC 0.94 was the best combination for diagnosing epithelial ovarian cancer. ApoC-III and PCI was analyzed with ELISA in the original cohort (n = 40) and in 40 new cyst fluid samples for confirmation with an independent method and validation. Results from MS and ELISA for ApoC-III correlated well (p = 0.04). In the validation set, ApoC-III was significantly (p = 0.001) increased in the malignant epithelial ovarian cancers. CONCLUSIONS: Fluid from ovarian cysts connected directly to the primary tumor harbor many possible new tumor-specific biomarkers. Biomarkers found in ovarian cyst fluid may be used as molecular imaging targets for early diagnostics and prediction of therapy. Plasma abundant proteins are also influencing the cystic fluid proteome. Methods for isolating less frequent cyst fluid proteins are needed.

Evaluation of ovarian cancer biomarkers HE4 and CA-125 in women presenting with a suspicious cystic ovarian mass.

OBJECTIVE: Women presenting with a large or complex ovarian cyst are referred to extensive surgical staging to ensure the correct diagnosis and treatment of a possible epithelial ovarian cancer. We hypothesized that measurement of the biomarkers HE4 and CA-125 preoperatively would improve the assignment of these patients to the correct level of care. METHODS: Patients diagnosed with a cystic ovarian mass and scheduled for an operation at our center of excellence for ovarian cancer surgery from 2001 to 2010 were prospectively included (n=394) and plasma was collected consecutively. Cut-off for HE4 was calculated at 75% specificity (85 pM and 71.8 pM for post and premenopausal women). For CA-125, 35 U/mL cut-off was used. The study population included women with malignant (n=114), borderline (n=45), and benign (n=215) ovarian tumors. RESULTS: Receiver operator characteristic (ROC) area under the curve (AUC) in the benign versus malignant cohorts was 86.8% for CA-125 and 84.4% for HE4. Negative predictive value was 91.7% when at least one of the biomarkers was positive, with only early stage epithelial ovarian cancer showing false negative results. Sensitivity at set specificity (75%) was 87% for risk of ovarian malignancy algorithm (ROMA) in the postmenopausal cohort (cut-off point, 26.0%) and 81% in the premenopausal cohort (cut-off point, 17.3%). ROC AUC in the benign versus stage I epithelial ovarian cancer was only 72% for HE4 and 76% for CA-125. CONCLUSION: In our study, population HE4 did not outperform CA-125. Based on our data a prospective trial with patients already diagnosed with an ovarian cyst may be conducted.