| 11. |
- Huttenhain, Ruth, et al.
(författare)
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A targeted mass spectrometry strategy for developing proteomic biomarkers : a case study of epithelial ovarian cancer
- 2019
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Ingår i: Molecular and Cellular Proteomics. - 1535-9484. ; 18:9, s. 1836-1850
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Tidskriftsartikel (refereegranskat)abstract
- Protein biomarkers for epithelial ovarian cancer are critical for the early detection of the cancer to improve patient prognosis and for the clinical management of the disease to monitor treatment response and to detect recurrences. Unfortunately, the discovery of protein biomarkers is hampered by the limited availability of reliable and sensitive assays needed for the reproducible quantification of proteins in complex biological matrices such as blood plasma. In recent years, targeted mass spectrometry, exemplified by Selected Reaction Monitoring (SRM) has emerged as a method, capable of overcoming this limitation. Here, we present a comprehensive SRM-based strategy for developing plasma-based protein biomarkers for epithelial ovarian cancer and illustrate how the SRM platform, when combined with rigorous experimental design and statistical analysis, can result in detection of predictive analytes.Our biomarker development strategy first involved a discovery-driven proteomic effort to derive potential N-glycoprotein biomarker candidates for plasma-based detection of human ovarian cancer from a genetically engineered mouse model of endometrioid ovarian cancer, which accurately recapitulates the human disease. Next, 65 candidate markers selected from proteins of different abundance in the discovery dataset were reproducibly quantified with SRM assays across a large cohort of over 200 plasma samples from ovarian cancer patients and healthy controls. Finally, these measurements were used to derive a 5-protein signature for distinguishing individuals with epithelial ovarian cancer from healthy controls. The sensitivity of the candidate biomarker signature in combination with CA125 ELISA-based measurements currently used in clinic, exceeded that of CA125 ELISA-based measurements alone. The SRM-based strategy in this study is broadly applicable. It can be used in any study that requires accurate and reproducible quantification of selected proteins in a high-throughput and multiplexed fashion.
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| 12. |
- Huttenhain, Ruth, et al.
(författare)
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Reproducible Quantification of Cancer-Associated Proteins in Body Fluids Using Targeted Proteomics
- 2012
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Ingår i: Science Translational Medicine. - : American Association for the Advancement of Science (AAAS). - 1946-6242 .- 1946-6234. ; 4:142, s. 94-142
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Tidskriftsartikel (refereegranskat)abstract
- The rigorous testing of hypotheses on suitable sample cohorts is a major limitation in translational research. This is particularly the case for the validation of protein biomarkers; the lack of accurate, reproducible, and sensitive assays for most proteins has precluded the systematic assessment of hundreds of potential marker proteins described in the literature. Here, we describe a high-throughput method for the development and refinement of selected reaction monitoring (SRM) assays for human proteins. The method was applied to generate such assays for more than 1000 cancer-associated proteins, which are functionally related to candidate cancer driver mutations. We used the assays to determine the detectability of the target proteins in two clinically relevant samples: plasma and urine. One hundred eighty-two proteins were detected in depleted plasma, spanning five orders of magnitude in abundance and reaching below a concentration of 10 ng/ml. The narrower concentration range of proteins in urine allowed the detection of 408 proteins. Moreover, we demonstrate that these SRM assays allow reproducible quantification by monitoring 34 biomarker candidates across 83 patient plasma samples. Through public access to the entire assay library, researchers will be able to target their cancer-associated proteins of interest in any sample type using the detectability information in plasma and urine as a guide. The generated expandable reference map of SRM assays for cancer-associated proteins will be a valuable resource for accelerating and planning biomarker verification studies.
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| 13. |
- Jansson, Malin, 1978-
(författare)
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The role of stroma-derived substances in breast cancer progression and their function as tumour markers
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: In 2020, more than 2,260,000 women were diagnosed with breast cancer. Most patients are cured with surgery and adjuvant treatment, but despite that, approximately 700,000 women die of the disease every year. The historical focus on breast cancer progression has been on the malignant epithelial cell. However, cancer cells do not grow in isolation. In recent years, the importance of the tumour microenvironment in cancer progression has been highlighted. Perlecan and type IV collagen are basement membrane (BM) proteins in the normal mammary gland, and type I collagen is the main fibrillar collagen in the interstitial extracellular matrix (ECM). In cancer development, perlecan and type IV collagen have multifunctional roles and when degraded from the BM, bioactive substances and other fragments are released in the circulation. Significant ECM changes also occur that lead to an accumulation of fibrillar collagens. Given their abundance in the ECM; perlecan, type IV and type I collagen are of interest for breast cancer progression and may be of importance as new biomarkers to monitor disease, predict patient outcome and the treatment effect.Aim: In this thesis, the protein and mRNA expression of perlecan, type IV and I collagen in breast cancer tissue is studied. The aim is to characterize the expression pattern of these proteins in breast cancer tissue and to see whether there is a correlation to known prognostic biomarkers and to the patient prognosis. Moreover, to evaluate circulating perlecan and type IV collagen as diagnostic and prognostic biomarkers in breast cancer patients.Methods: In this thesis project, eight different patient cohorts were used. In freshly frozen normal breast and breast cancer tissue, perlecan protein expression was visualized using immunofluorescence. Type IV and I collagen protein expression were studied with immunohistochemistry in formalin-fixed, paraffin embedded primary breast cancer tissue, and type IV collagen in metastatic breast cancer tissue. For gene expression analysis, mRNA and clinicopathological data were extracted from the Cancer Genome Atlas and cBioportal database. Circulating plasma levels of perlecan were analysed in breast cancer patients and controls, circulating levels of CA15-3 and type IV collagen in patients with primary and metastatic breast cancer as well as controls. Perlecan and type IV collagen were measured with ELISA assays, and CA15-3 were using an electrochemiluminescence immunoassay.Results: In breast cancer tissue, perlecan and type IV collagen protein expression in the epithelial BM was fragmented or completely lost, and perlecan and type IV collagen was expressed to varying extent in the tumour stroma. The mRNA analysis confirmed that type IV collagen mRNA was expressed in primary breast cancer tissue and highly expressed in metastatic tissue. Type I collagen was mostly highly expressed in the tumour stroma. Low type I collagen protein and mRNA expression correlated with biomarkers for aggressive breast cancer, but no effect on survival could be seen. Among patients receiving chemotherapy, low stromal type I collagen protein expression was associated with better survival compared to high expression, even after adjusting for other relevant factors. There was no correlation of perlecan or type IV collagen protein expression to clinically used prognostic biomarkers, but an oestrogen receptor dependent correlation between mRNA expression of perlecan and several matrix-degrading enzymes were found. Survival analysis showed that high stromal type IV collagen protein and mRNA expression in the primary tumour was significantly associated with a poorer survival, and high protein expression with a risk of developing distant metastasis. Metastatic breast cancer patients had higher levels of circulating type IV collagen compared to healthy controls and patients with primary breast cancer. High circulating type IV collagen levels correlated with poorer survival in metastatic breast cancer patients, and was superior to CA15-3 at detecting metastatic breast cancer.Conclusions: The protein expression pattern of perlecan, type IV collagen and type I collagen become abnormal during breast cancer development. Stromal type IV collagen protein and mRNA in the primary tumour correlates to poorer prognosis, most likely due to a higher risk of developing metastatic disease. Circulating type IV collagen can function as a biomarker for detecting metastatic disease in patients with primary breast cancer and is prognostic in patients with metastatic breast cancer. Low stromal type I collagen is a marker for an aggressive breast cancer disease and can predict chemotherapy response.
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| 14. |
- Kurbasic, Emila, et al.
(författare)
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Changes in glycoprotein expression between primary breast tumour and synchronous lymph node metastases or asynchronous distant metastases.
- 2015
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Ingår i: Clinical Proteomics. - : Springer Science and Business Media LLC. - 1559-0275 .- 1542-6416. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Breast cancer is a very heterogeneous disease and some patients are cured by the surgical removal of the primary tumour whilst other patients suffer from metastasis and spreading of the disease, despite adjuvant therapy. A number of prognostic and treatment predictive factors have been identified such as tumour size, oestrogen (ER) and progesterone (PgR) receptor status, human epidermal growth factor receptor type 2 (HER2) status, histological grade, Ki67 and age. Lymph node involvement is also assessed during surgery to determine if the tumour has spread which requires dissection of the axilla and adjuvant treatment. The prognostic and treatment predictive factors assessing the nature of the tumour are all routinely based on the status of the primary tumour.
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| 15. |
- Niméus, Emma, et al.
(författare)
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Amplification of the cyclin D1 gene is associated with tumour subsite, DNA non-diploidy and high S-phase fraction in squamous cell carcinoma of the head and neck
- 2004
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Ingår i: Oral Oncology. - : Elsevier BV. - 1879-0593 .- 1368-8375. ; 40:6, s. 624-629
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Tidskriftsartikel (refereegranskat)abstract
- Amplification of CCND1 (cyclin D1 gene) in squamous cell carcinoma of the head and neck (SCCHN) is correlated to poor prognosis. The purpose of this study was to investigate whether CCND1 amplification is related to different subsites and also to DNA ploidy status and S-phase fraction (SPF). Biopsies from 67 patients with SCCHN were analysed for CCND1 amplification by fluorescence in situ hybridisation (FISH) and for ploidy status and SPF by flow cytometry (FCM). Twenty-one of 67 tumours (31%) showed CCND1 amplification and the frequencies differed significantly between different subsites (p = 0.01). Tumours from hypopharynx, larynx and oropharynx showed higher rates of amplification as compared to tumours from oral cavity and epipharynx. CCND1 amplification was also associated to DNA non-diploidy and high SPF (p = 0.002 and p = 0.002, respectively). In conclusion, the rate of CCND1 amplification differed between different subsites in SCCHN and was also associated to a more aggressive tumour phenotype, as defined by DNA non-diploidy and high SPF.
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| 16. |
- Niméus, Emma, et al.
(författare)
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Androgen Receptor in Stage I-II Primary Breast Cancer -Prognostic Value and Distribution in Subgroups
- 2017
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Ingår i: Anticancer research. - : Anticancer Research USA Inc.. - 1791-7530 .- 0250-7005. ; 37:12, s. 6845-6853
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND/AIM: The value of androgen receptor (AR) in breast cancer has gained renewed interest as a prognostic and treatment predictive biomarker. The aims of this work were to study the associations and the prognostic value of AR in patients from two clinical cohorts.MATERIALS AND METHODS: Cohort 1 included 208 premenopausal, node-negative patients of whom 87% had received no adjuvant medical treatment; cohort 2 consisted of 263 patients with stage II disease who had all received 2 years of adjuvant tamoxifen. A semi-quantitative assessment of nuclear AR expression divided into five groups (0-1%, 2-10%, 11-50%, 51-75%, and 76-100%) was performed. Survival analyses, stratified by cohort, were performed using both a trend-test and a cut-off of >10% for positivity.RESULTS: A total of 76% of all patients were AR+, and 89%, 48%, and 23% of the estrogen receptor-positive, negative, and triple-negative, respectively. In Cox regression, stratified by cohort, AR divided into five groups was a prognostic factor for 5-year distant disease-free survival with a hazard ratio of 0.86 per step in fraction score (p=0.018). With a predefined cut-off at 10%, moderate evidence of an effect remained (Hazard Ratio=0.67, p=0.077). In multivariable analysis, AR did not retain an independent prognostic value.CONCLUSION: AR is a weak, however, not independent prognostic factor for distant metastasis. Although the prognostic value of AR may be questionable, the study identified a subset of AR-positive triple-negative patients as being potential candidates for AR-directed therapy for which further studies are warranted.
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| 17. |
- Niméus, Emma, et al.
(författare)
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Cyclin B1 is a prognostic proliferation marker with a high reproducibility in a population-based lymph node negative breast cancer cohort
- 2010
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 127:4, s. 961-967
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Tidskriftsartikel (refereegranskat)abstract
- A large proportion of women with lymph node negative breast cancer treated with chemotherapy do not benefit from such treatment. Proliferation markers have been shown to recognize patients at high risk for recurrence. Ki67 has recently been included in the St Gallen guidelines. We investigated the prognostic importance of cyclin B1 in node negative breast cancer and included a study of reproducibility. In a population-based case-control study 190 women who died from breast cancer were defined as cases and 190 women alive at the time for the corresponding case's death as controls. Inclusion criteria were tumor size < 50 mm, no lymph node metastases, and no adjuvant chemotherapy. Tumor tissue was immunostained for cyclin B1. Two investigators evaluated the staining independently by counting approximately 100, 200, 500, and 1000 cells. Cyclin B1 was statistically significantly associated to breast cancer death, in both uni- and multivariate analyses (adjusted for tumor size, age, and endocrine therapy), with odds ratios 2-3 for both investigators. The agreement between the two investigators was good to very good, regardless of the number of counted cells (kappa values between 0.74 and 0.82).Cyclin B1 is a prognostic factor for breast cancer death in a population-based node negative patient cohort which can identify high-risk patients with a good to very good reproducibility. (c) 2009 UICC.
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| 18. |
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| 19. |
- Niméus, Emma, et al.
(författare)
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Gene expression profilers and conventional clinical markers to predict distant recurrences for premenopausal breast cancer patients after adjuvant chemotherapy.
- 2006
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Ingår i: European Journal of Cancer. - : Elsevier BV. - 1879-0852 .- 0959-8049. ; 42:16, s. 2729-2737
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Tidskriftsartikel (refereegranskat)abstract
- A large proportion of breast cancer patients are treated with adjuvant chemotherapy after the primary operation, but some will recur in spite of this treatment. In order to achieve an improved and more individualised therapy, our knowledge in mechanisms for drug resistance needs to be increased. We have investigated to what extent cDNA microarray measurements could distinguish the likelihood of recurrences after adjuvant CMF (cyclophosphamide, methotrexate and 5-fluorouracil) treatment of premenopausal, lymph node positive breast cancer patients, and have also compared this with the corresponding performance when using conventional clinical variables. We tried several gene selection strategies, and built classifiers using the resulting gene lists. The best performing classifier with odds ratio (OR) = 6.5 (95% confidence interval (CI) = 1.4-62) did not outperform corresponding classifiers based on clinical variables. For the clinical variables, calibrated on the samples, either using all the clinical parameters or the Nottingham Prognostic Index (NPI) parameters, the areas under the receiver operating characteristics (ROC) curve were 0.78 and 0.79, respectively. The ORs at 90% sensitivity were 15 (95% CI = 3.1-140) and 10 (95% CI = 2.1-97), respectively. Our data have provided evidence for a comparable prediction of clinical outcome in CMF-treated breast cancer patients using conventional clinical variables and gene expression based markers. (c) 2006 Elsevier Ltd. All rights reserved.
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| 20. |
- Niméus, Emma, et al.
(författare)
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Gene expression profiling in primary breast cancer distinguishes patients developing local recurrence after breast-conservation surgery, with or without postoperative radiotherapy
- 2008
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Ingår i: Breast Cancer Research. - : Springer Science and Business Media LLC. - 1465-5411 .- 1465-542X. ; 10:2
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Tidskriftsartikel (refereegranskat)abstract
- IntroductionSome patients with breast cancer develop local recurrence after breast-conservation surgery despite postoperative radiotherapy, whereas others remain free of local recurrence even in the absence of radiotherapy. As clinical parameters are insufficient for identifying these two groups of patients, we investigated whether gene expression profiling would add further information.MethodsWe performed gene expression analysis (oligonucleotide arrays, 26,824 reporters) on 143 patients with lymph node-negative disease and tumor-free margins. A support vector machine was employed to build classifiers using leave-one-out cross-validation.ResultsWithin the estrogen receptor-positive (ER+) subgroup, the gene expression profile clearly distinguished patients with local recurrence after radiotherapy (n = 20) from those without local recurrence (n = 80 with or without radiotherapy). The receiver operating characteristic (ROC) area was 0.91, and 5,237 of 26,824 reporters had a P value of less than 0.001 (false discovery rate = 0.005). This gene expression profile provides substantially added value to conventional clinical markers (for example, age, histological grade, and tumor size) in predicting local recurrence despite radiotherapy. Within the ER- subgroup, a weaker, but still significant, signal was found (ROC area = 0.74). The ROC area for distinguishing patients who develop local recurrence from those who remain local recurrence-free in the absence of radiotherapy was 0.66 (combined ER+/ER-).ConclusionA highly distinct gene expression profile for patients developing local recurrence after breast-conservation surgery despite radiotherapy has been identified. If verified in further studies, this profile might be a most important tool in the decision making for surgery and adjuvant therapy.
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