[Show abstract][Hide abstract] ABSTRACT: Fluorescence nanoscopy provides means to discern the finer details of protein localization and interaction in cells by offering an order of magnitude higher resolution than conventional optical imaging techniques. However, these super resolution techniques put higher demands on the optical system as well as on the fluorescent probes, making multicolor fluorescence nanoscopy a challenging task. Here we present a new and simple procedure which exploits the photostability and excitation spectra of dyes to increase the number of simultaneous recordable targets in STED nanoscopy. We use this procedure to demonstrate four color STED imaging of platelets with ≤ 40 nm resolution and low crosstalk. Platelets can selectively store, sequester and release a multitude of different proteins, and in a manner specific for different physiological and disease states. By applying multicolor nanoscopy to study platelets, we can achieve spatial mapping of the protein organization with a high resolution, for multiple proteins at the same time and in the same cell. This provides a means to identify specific platelet activation states for diagnostic purposes and to understand the underlying protein storage and release mechanisms. We studied the organization of the pro- and anti-angiogenic proteins VEGF and PF-4 together with fibrinogen and filamentous actin, and found distinct features in their respective protein localization. Further, colocalization analysis revealed only minor overlap between the proteins VEGF and PF-4 indicating that they have separate storage and release mechanisms, corresponding well with their opposite roles as pro- and anti-angiogenic proteins, respectively.
[Show abstract][Hide abstract] ABSTRACT: Chromosomal aneuploidy has been identified as a prognostic factor in the majority of sporadic carcinomas. However, it is not known how chromosomal aneuploidy affects chromosome-specific protein expression in particular, and the cellular proteome equilibrium in general.
The aim was to detect chromosomal aneuploidy-associated expression changes in cell clones carrying trisomies found in colorectal cancer.
We used microcell-mediated chromosomal transfer to generate three artificial trisomic cell clones of the karyotypically stable, diploid, yet mismatch-deficient, colorectal cancer cell line DLD1 - each of them harboring one extra copy of either chromosome 3, 7 or 13. Protein expression differences were assessed by two-dimensional gel electrophoresis and mass spectrometry, compared to whole-genome gene expression data, and evaluated by PANTHER classification system and Ingenuity Pathway Analysis (IPA).
In total, 79 differentially expressed proteins were identified between the trisomic clones and the parental cell line. Up-regulation of PCNA and HMGB1 as well as down-regulation of IDH3A and PSMB3 were revealed as trisomy-associated alterations involved in regulating genome stability.
These results show that trisomies affect the expression of genes and proteins that are not necessarily located on the trisomic chromosome, but reflect a pathway-related alteration of the cellular equilibrium.
[Show abstract][Hide abstract] ABSTRACT: The extent of epithelial cellular material (ECM) occurring in venous blood samples after diagnostic core needle biopsy (CNB) was studied in 23 patients with CNB diagnosed prostate cancer without provable metastases and 15 patients without cancer. The data show a significant increase of ECM in the peripheral blood sampled 20 seconds or 30 minutes after the last of 10 CNB procedures compared to the number of ECM detectable in the blood samples taken before the performance of CNB. The data indicate that diagnostic CNB of prostate cancer causes an extensive tissue trauma with a potential risk of cancer cell dissemination.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to assess efficacy and safety of percutaneous ultrasound (US) guided preferential radiofrequency ablation (PRFA) in early breast carcinoma under local anesthesia and to evaluate a new assessment protocol. Eighteen breast cancer patients were enrolled in order to receive PRFA treatment three weeks prior to resection. Pain assessment was performed using the visual analoge scale. Analysis of treatment success was performed using magnetic resonance imaging (MRI) as well as histological assays for hematoxylin & eosin (H&E) and cytokeratine 8 (CK8). In a subset of patients contrast enhanced ultrasound (CEUS) was performed before and after treatment. MRI showed no residual tumor growth in 100% (18/18) of cases. Complete tumor devitalization was indicated in 83% (15/18) of patients as judged by H&E staining and in 89% (16/18) as judged by immunostaining for CK8. In 100% (18/18) at least one histologic method showed devitalization in the entire tumor. Treatment was well tolerated. Pain experienced during the procedure was mild. US-guided PRFA of small breast carcinoma is feasible under local anesthesia. MRI and CK8 have proven valuable additions to the RF breast tumor ablation protocol. CEUS shows potential as a modality for radiological follow-up.
Breast (Edinburgh, Scotland) 01/2014; · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Context: Biological material reflecting the in vivo composition of markers provides a high potential for biomarker discovery. Objective: We compared the serum proteome following heat- and nitrogen-preservation, with and without subsequent storage at room temperature. Materials and methods: Serum samples were collected, treated and analysed by two-dimensional gel electrophoresis. Protein spots were identified and confirmed by two mass spectrometry approaches (MALDI & ESI) and subjected to Ingenuity Pathway Analysis. Results: We revealed 24 differentially expressed proteins (p ≤ 0.05) between nitrogen and heat preservation, and 87 between nitrogen and heat preservation with subsequent storage for 120 h at room-temperature. Mass spectrometry identified 25 polypeptides. Pathway analysis resulted in networks maintaining Cellular Assembly and Organization, Movement and Maintenance. Conclusion: Heat-stabilization does not substantially change the short-term proteome composition of serum compared with nitrogen treatment. However, heat-stabilization alone seems insufficient for long-term sample preservation for serum samples. We identified transthyretin and apolipoprotein A-IV as sample quality markers.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND:: Malignant transformation in ulcerative colitis (UC) is associated with pronounced chromosomal instability, reflected by aneuploidy. Although aneuploidy can precede primary cancer diagnosis in UC for more than a decade, little is known of its cellular consequences. METHODS:: Whole-genome gene expression analysis was applied to noninflamed colon mucosa, mucosal biopsies of patients with UC, and UC-associated carcinomas (UCCs). DNA image cytometry was used to stratify samples into ploidy types. Differentially expressed genes (DEGs) were analyzed by Ingenuity Pathway Analysis and validated by real-time quantitative PCR. RESULTS:: Gene expression changes were more pronounced between normal mucosa and UC (2587 DEGs) than between UC and UCC (827 DEGs). Cytometry identified colitis patients with euploid or aneuploid mucosa biopsies, whereas all UCCs were aneuploid. However, 1749 DEGs distinguished euploid UC and UCCs, whereas only 15 DEGs differentiated aneuploid UC and UCCs. A total of 16 genes were differentially expressed throughout the whole sequence from normal controls to UCCs. Particularly, genes pivotal for chromosome segregation (e.g., SMC3 and NUF2) were differentially regulated along aneuploidy development. CONCLUSIONS:: The high number of DEGs between normal mucosa and colitis is dominated by inflammatory-associated genes. Subsequent acquisition of aneuploidy leads to subtle but distinct transcriptional alterations, revealing novel target genes that drive genomic instability and thus carcinogenesis. The gene expression signature of malignant phenotypes in aneuploid UC suggests that these lesions might need to be considered as severe as high-grade dysplasia.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: This study aimed to analyze the prognostic value of DNA content and biological markers for cell cycle regulation and invasion in primary carcinoma of the vagina (PCV). MATERIAL AND METHODS: Seventy-two consecutive patients with PCV, categorized as short-term (≤2 years) and long-term (≥8 years) survivors, were evaluated for DNA content by image cytometry, and for expression of p53, p21, cyclin A, Ki67, E-cadherin, and laminin-5γ2 chain by immunohistochemistry. The relationship between these biological markers and histopathological and clinical parameters was assessed. RESULTS: All PCV showed aneuploid DNA content. Most of the PCV patients showed no overexpression of p53 and high expression of p21, cyclin A, and Ki67. Loss or underexpression of E-cadherin was found in 94% (68/72) of PCV patients, and all patients showed immunopositivity for the laminin-5γ2 chain. Tumors with a vaginal longitudinal location in the lower third or in the entire vagina more often had overexpression of p53, high expression of Ki67 (P = 0.044), and underexpression of E-cadherin (P = 0.038), than tumors confined only to the upper third. Overexpression of p53 was significantly associated with short-term survival in the univariate analysis, but not in the multivariate analysis adjusted for age at diagnosis and tumor size. CONCLUSIONS: The expression level of some markers was related to tumor location, which might be indicative of different genesis. Overexpression of p53 was associated with short-term survival, but the only independent predictors of survival were age at diagnosis and tumor size.
International Journal of Gynecological Cancer 11/2012; · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dysregulation of how platelets store, sequester and release specific proteins seems to be implicated in many disease states, including cancer. Dual-color immunofluorescence stimulated emission depletion (STED) microscopy with 40 nm resolution is used to map pro-angiogenic VEGF, anti-angiogenic PF-4 and fibrinogen in >300 individual platelets. This reveals that these proteins are stored in a segmented, zonal manner within regional clusters, significantly smaller than the size of an α-granule. No colocalization between the different proteins is observed. Upon platelet activation by thrombin or ADP, the proteins undergo significant spatial rearrangements, including alterations in the size and number of the protein clusters, and specific for a certain protein and the type of activation induced. Following these observations, a simple assignment procedure is used to show that the three distinct states of platelets (non-, ADP- and thrombin-activated) can be identified based on the average size, number and peripheral localization profiles of the regional protein clusters within the platelets. Thus, high-resolution spatial mapping of specific proteins is a useful procedure to detect and characterize deviations in the selective storage, release and uptake of these proteins in the platelets. Since these deviations seem to be specific for, and may even underlie, certain patophysiological states, these findings may have interesting diagnostic and therapeutic implications.
[Show abstract][Hide abstract] ABSTRACT: To study elderly women > or = 60 years of age diagnosed with breast cancer and analyze this cohort according to death from breast cancer and death due to comorbidities. Patients aged 60-69 years of age were included in routine mammography screening, but not women aged > or = 70 years. This enabled a comparison between the 2 groups regarding screening effect, tumor size and survival in breast cancer.
A consecutive sample of 311 breast cancer patients > or = 60 years of age from 1991 were analyzed according to tumor size at diagnosis, frequencies of lymph node metastasis, tumor histological grade and stage, ploidy, proliferation index, stem-line-scatter index and survival rate in breast cancer and other causes of death. Tumor size was compared to a patient group aged 60-69 from 1987, before the introduction of mammography screening in Sweden.
In the screening group a significant reduction in tumor size was found at diagnosis compared to the sample from 1987 (p < 0.001) and to the older group > or = 70 years (p < 0.02). In the latter group a higher death rate appeared for breast cancer.
Older women would have a better outcome if included in the mammography screening program.
Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 08/2012; 34(4):189-94. · 0.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epithelial ovarian carcinoma has in general a poor prognosis since the vast majority of tumors are genomically unstable and clinically highly aggressive. This results in rapid progression of malignancy potential while still asymptomatic and thus in late diagnosis. It is therefore of critical importance to develop methods to diagnose epithelial ovarian carcinoma at its earliest developmental stage, that is, to differentiate between benign tissue and its early malignant transformed counterparts. Here we present a shotgun quantitative proteomic screen of benign and malignant epithelial ovarian tumors using iTRAQ technology with LC-MALDI-TOF/TOF and LC-ESI-QTOF MS/MS. Pathway analysis of the shotgun data pointed to the PI3K/Akt signaling pathway as a significant discriminatory pathway. Selected candidate proteins from the shotgun screen were further confirmed in 51 individual tissue samples of normal, benign, borderline or malignant origin using LC-MRM analysis. The MRM profile demonstrated significant differences between the four groups separating the normal tissue samples from all tumor groups as well as perfectly separating the benign and malignant tumors with a ROC-area of 1. This work demonstrates the utility of using a shotgun approach to filter out a signature of a few proteins only that discriminates between the different sample groups.
Journal of Proteome Research 04/2012; 11(5):2876-89. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thyroid proteomics is a new direction in thyroid cancer research aiming at etiological understanding and biomarker identification for improved diagnosis.
Two-dimensional electrophoresis was applied to cytosolic protein extracts from frozen thyroid samples (ten follicular adenomas, nine follicular carcinomas, ten papillary carcinomas, and ten reference thyroids). Spots with differential expression were revealed by image and multivariate statistical analyses, and identified by mass spectrometry.
A set of 25 protein spots significant for discriminating between the sample groups was identified. Proteins identified for nine of these spots were studied further including 14-3-3 protein beta/alpha, epsilon, and zeta/delta, peroxiredoxin 6, selenium-binding protein 1, protein disulfide-isomerase precursor, annexin A5 (ANXA5), tubulin alpha-1B chain, and α1-antitrypsin precursor. This subset of protein spots carried the same predictive power in differentiating between follicular carcinoma and adenoma or between follicular and papillary carcinoma, as compared with the larger set of 25 spots. Protein expression in the sample groups was demonstrated by western blot analyses. For ANXA5 and the 14-3-3 proteins, expression in tumor cell cytoplasm was demonstrated by immunohistochemistry both in the sample groups and an independent series of papillary thyroid carcinomas.
The proteins identified confirm previous findings in thyroid proteomics, and suggest additional proteins as dysregulated in thyroid tumors.
European Journal of Endocrinology 01/2012; 166(4):657-67. · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA aneuploidy has been identified as a prognostic factor in the majority of epithelial malignancies. We aimed at identifying ploidy-associated protein expression in endometrial cancer of different prognostic subgroups. Comparison of gel electrophoresis-based protein expression patterns between normal endometrium (n = 5), diploid (n = 7), and aneuploid (n = 7) endometrial carcinoma detected 121 ploidy-associated protein forms, 42 differentially expressed between normal endometrium and diploid endometrioid carcinomas, 37 between diploid and aneuploid endometrioid carcinomas, and 41 between diploid endometrioid and aneuploid uterine papillary serous cancer. Proteins were identified by mass spectrometry and evaluated by Ingenuity Pathway Analysis. Targets were confirmed by liquid chromatography/mass spectrometry. Mass spectrometry identified 41 distinct polypeptides and pathway analysis resulted in high-ranked networks with vimentin and Nf-κB as central nodes. These results identify ploidy-associated protein expression differences that overrule histopathology-associated expression differences and emphasize particular protein networks in genomic stability of endometrial cancer.
Cellular and Molecular Life Sciences CMLS 07/2011; 69(2):325-33. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies. Further understanding of the translation of DNA aneuploidy into protein expression will help to define novel biomarkers to improve therapies and prognosis. DNA ploidy was assessed by image cytometry. Comparison of gel-electrophoresis-based protein expression patterns of three diploid and four aneuploid colorectal cancer cell lines detected 64 ploidy-associated proteins. Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in two overlapping high-ranked networks maintaining Cellular Assembly and Organization, Cell Cycle, and Cellular Growth and Proliferation. CAPZA1, TXNL1, and HDAC2 were significantly validated by Western blotting in cell lines and the latter two showed expression differences also in clinical samples using a tissue microarray of normal mucosa (n=19), diploid (n=31), and aneuploid (n=47) carcinomas. The results suggest that distinct protein expression patterns, affecting TXNL1 and HDAC2, distinguish aneuploid with poor prognosis from diploid colorectal cancers.
Cellular and Molecular Life Sciences CMLS 02/2011; 68(19):3261-74. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Twenty percent of cancer patients experience adverse effects after radiotherapy. The therapeutic doses are adjusted to the most sensitive individuals, resulting in a suboptimal dose for many patients. At present there is no screening system available to predict individual radiosensitivity. The main aim of this study is to investigate differences in protein expression pathways induced by low doses of radiation in whole blood obtained from radiosensitive and non-radiosensitive patients. To address this aim, a protocol for exposure condition, dose and analysis of whole blood was developed. The conditions for handling blood samples were optimised. The optimal doses and post irradiation conditions were determined. We found that 1 mGy and 150 mGy significantly changed protein expression in leukocytes collected from the two donors. The result shows that the protocol developed is suitable for characterisation of proteomic profiles associated with low dose exposure of leukocytes.
Int. J. of Low Radiation. 01/2011; 8(5/6):374 - 387.
[Show abstract][Hide abstract] ABSTRACT: Dissemination of tumour cells occurring both spontaneously or caused by diagnostic biopsy procedures is the most serious complication of solid malignancies. In the present work we focus on local tumour spread and how this complication of cancer disease can be counteracted.
From a cohort of 864 breast cancer patients we selected those who died of their primary cancer and those who died because of a simultaneously existing cardio-cerebral-vascular disease (CCVD) and were exposed to anticoagulants.
The study was based on breast cancer patients diagnosed at Karolinska University Hospital during 1991 (n = 519) and 1997-1998 (n = 345).
Axillary lymph node metastasis (ALNM) and survival of breast cancer patients with concurrent CCVD.
Breast cancer patients belonging to the group that died of CCVD showed ALNM at the time of tumour diagnosis in 27% of the cases compared with 68% diagnosed in the group that died of their breast cancer (p < 0.0001). Likewise we observed a highly significant (p < 0.0001) difference in mean survival time with an average of 102 months in the group of breast cancer patients who died of CCVD and an average of 61 months in the group who died of breast cancer.
The data presented herein indicate that breast cancer patients regularly involved in treatment with anticoagulants because of simultaneously existing CCVD develop ALNM significantly less frequently and have an increased average survival time compared with breast cancer patients not suffering from CCVD.
[Show abstract][Hide abstract] ABSTRACT: In addition to clinical characteristics, DNA aneuploidy has been identified as a prognostic factor in epithelial malignancies in general and in endometrial cancers in particular. We mapped ploidy-associated chromosomal aberrations and identified corresponding gene and protein expression changes in endometrial cancers of different prognostic subgroups.
DNA image cytometry classified 25 endometrioid cancers to be either diploid (n = 16) or aneuploid (n = 9), and all uterine papillary serous cancers (UPSC) to be aneuploid (n = 8). All samples were subjected to comparative genomic hybridization and gene expression profiling. Identified genes were subjected to Ingenuity pathway analysis (IPA) and were correlated to protein expression changes.
Comparative genomic hybridization revealed ploidy-associated specific, recurrent genomic imbalances. Gene expression analysis identified 54 genes between diploid and aneuploid endometrioid carcinomas, 39 genes between aneuploid endometrioid cancer and UPSC, and 76 genes between diploid endometrioid and aneuploid UPSC to be differentially expressed. Protein profiling identified AKR7A2 and ANXA2 to show translational alterations consistent with the transcriptional changes. The majority of differentially expressed genes and proteins belonged to identical molecular functions, foremost Cancer, Cell Death, and Cellular Assembly and Organization.
We conclude that the grade of genomic instability rather than the histopathological subtype correlates with specific gene and protein expression changes. The identified genes and proteins might be useful as molecular targets for improved diagnostic and therapeutic intervention and merit prospective validation.
Molecular Cancer 01/2011; 10:132. · 5.13 Impact Factor