Experimental evidence that mutated-self peptides derived from mitochondrial DNA somatic mutations have the potential to trigger autoimmunity.

Autoimmune disease is a critical health concern, whose etiology remains enigmatic. We hypothesized that immune responses to somatically mutated self proteins could have a role in the development of autoimmune disease. IFN-γ secretion by T cells stimulated with mitochondrial peptides encoded by published mitochondrial DNA was monitored to test the hypothesis. Human peripheral blood mononuclear cells (PBMCs) of healthy controls and autoimmune patients were assessed for their responses to the self peptides and mutated-self peptides differing from self by one amino acid. None of the self peptides but some of the mutated-self peptides elicited an immune response in healthy controls. In some autoimmune patients, PBMCs responded not only to some of the mutated-self peptides, but also to some of the self peptides, suggesting that there is a breach of self-tolerance in these patients. Although PBMCs from healthy controls failed to respond to self peptides when stimulated with self, the mutated-self peptide could elicit a response to the self peptide upon re-stimulation in vitro, suggesting that priming with mutated-self peptides elicits a cross-reactive response with self. The data raise the possibility that DNA somatic mutations are one of the events that trigger and/or sustain T cell responses in autoimmune diseases.

miRNA profiling for clear cell renal cell carcinoma: biomarker discovery and identification of potential controls and consequences of miRNA dysregulation.

PURPOSE: Renal cell carcinoma is the most common neoplasm of the adult kidney. Currently to our knowledge there are no biomarkers for diagnostic, prognostic or predictive applications for renal cell carcinoma. miRNAs are nonprotein coding RNAs that negatively regulate gene expression and are potential biomarkers for cancer. MATERIALS AND METHODS: We analyzed 70 matched pairs of clear cell renal cell carcinoma and normal kidney tissues from the same patients by microarray analysis and validated our results by quantitative real-time polymerase chain reaction. We also performed extensive bioinformatic analysis to explore the role and regulation of miRNAs in clear cell renal cell carcinoma. RESULTS: We identified 166 miRNAs that were significantly dysregulated in clear cell renal cell carcinoma, including miR-122, miR-155 and miR-210, which had the highest over expression, and miR-200c, miR-335 and miR-218, which were most down-regulated. Analysis of previously reported miRNAs dysregulated in RCC showed overall agreement in the direction of dysregulation. Extensive target prediction analysis revealed that many miRNAs were predicted to target genes involved in renal cell carcinoma pathogenesis. In renal cell carcinoma miRNA dysregulation can be attributed in part to chromosomal aberrations, co-regulation of miRNA clusters and co-expression with host genes. We also performed a preliminary analysis showing that miR-155 expression correlated with clear cell renal cell carcinoma size. This finding must be validated in a larger independent cohort. CONCLUSIONS: Analysis showed that miRNAs are dysregulated in clear cell renal cell carcinoma and may contribute to kidney cancer pathogenesis by targeting more than 1 key molecule. We identified mechanisms that may contribute to miRNA dysregulation in clear cell renal cell carcinoma. Dysregulated miRNAs represent potential biomarkers for kidney cancer.

Overexpression of prothymosin alpha predicts poor disease outcome in head and neck cancer.

BACKGROUND: In our recent study, tissue proteomic analysis of oral pre-malignant lesions (OPLs) and normal oral mucosa led to the identification of a panel of biomarkers, including prothymosin alpha (PTMA), to distinguish OPLs from histologically normal oral tissues. This study aimed to determine the clinical significance of PTMA overexpression in oral squamous cell hyperplasia, dysplasia and head and neck squamous cell carcinoma (HNSCC). METHODOLOGY: Immunohistochemistry of PTMA protein was performed in HNSCCs (n = 100), squamous cell hyperplasia (n = 116), dysplasia (n = 50) and histologically normal oral tissues (n = 100). Statistical analysis was carried out to determine the association of PTMA overexpression with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. RESULTS: Our immunohistochemical analysis demonstrated significant overexpression of nuclear PTMA in squamous cell hyperplasia (63.8%), dysplasia (50%) and HNSCC (61%) in comparison with oral normal mucosa (p(trend)<0.001). Chi-square analysis showed significant association of nuclear PTMA with advanced tumor stages (III+IV). Kaplan Meier survival analysis indicated reduced disease free survival (DFS) in HNSCC patients (p<0.001; median survival 11 months). Notably, Cox-multivariate analysis revealed nuclear PTMA as an independent predictor of poor prognosis of HNSCC patients (p<0.001, Hazard's ratio, HR = 5.2, 95% CI = 2.3-11.8) in comparison with the histological grade, T-stage, nodal status and tumor stage. CONCLUSIONS: Nuclear PTMA may serve as prognostic marker in HNSCC to determine the subset of patients that are likely to show recurrence of the disease.

Accurate molecular classification of kidney cancer subtypes using microRNA signature.

BACKGROUND: Renal cell carcinoma (RCC) encompasses different histologic subtypes. Distinguishing between the subtypes is usually made by morphologic assessment, which is not always accurate. OBJECTIVE: Our aim was to identify microRNA (miRNA) signatures that can distinguish the different RCC subtypes accurately. DESIGN, SETTING, AND PARTICIPANTS: A total of 94 different subtype cases were analysed. miRNA microarray analysis was performed on fresh frozen tissues of three common RCC subtypes (clear cell, chromophobe, and papillary) and on oncocytoma. Results were validated on the original as well as on an independent set of tumours, using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis with miRNA-specific primers. MEASUREMENTS: Microarray data were analysed by standard approaches. Relative expression for qRT-PCR was determined using the ΔΔC(T) method, and expression values were normalised to small nucleolar RNA, C/D box 44 (SNORD44, formerly RNU44). Experiments were done in triplicate, and an average was calculated. Fold change was expressed as a log(2) value. The top-scoring pairs classifier identified operational decision rules for distinguishing between different RCC subtypes and was robust under cross-validation. RESULTS AND LIMITATIONS: We developed a classification system that can distinguish the different RCC subtypes using unique miRNA signatures in a maximum of four steps. The system has a sensitivity of 97% in distinguishing normal from RCC, 100% for clear cell RCC (ccRCC) subtype, 97% for papillary RCC (pRCC) subtype, and 100% accuracy in distinguishing oncocytoma from chromophobe RCC (chRCC) subtype. This system was cross-validated and showed an accuracy of about 90%. The oncogenesis of ccRCC is more closely related to pRCC, whereas chRCC is comparable with oncocytoma. We also developed a binary classification system that can distinguish between two individual subtypes. CONCLUSIONS: MiRNA expression patterns can distinguish between RCC subtypes.

Altered spectrum of somatic hypermutation in common variable immunodeficiency disease characteristic of defective repair of mutations.

Pathogenic common variable immunodeficiency diseases (CVID) are genetic, usually inherited diseases for which a limited number of genetic defects have been implicated. As CVID presents with a wide range of clinical characteristics, there are likely diverse and for the most part unidentified genetic causes. In some individuals, defects in somatic hypermutation (SHM) have been suggested as the underlying cause of CVID. To address the mechanisms of SHM defects in CVID, we conducted a comprehensive mutational analysis of immunoglobulin heavy chain sequences from CVID patients. We identified several remarkably specific alterations in the spectra of SHM in comparison to healthy individuals. We provide evidence that some CVID cases are associated with defective repair of AID-induced mutations by the DNA mismatch repair (MMR) machinery. Our findings together with reports of increased chromosomal radiosensitivity and associated lymphoproliferative disorders amongst CVID patients, suggest that altered DNA damage repair may be a cause of CVID.

Nuclear S100A7 is associated with poor prognosis in head and neck cancer.

BACKGROUND: Tissue proteomic analysis of head and neck squamous cell carcinoma (HNSCC) and normal oral mucosa using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and liquid chromatography-mass spectrometry, led to the identification of a panel of biomarkers including S100A7. In the multi-step process of head and neck tumorigenesis, the presence of dysplastic areas in the epithelium is proposed to be associated with a likely progression to cancer; however there are no established biomarkers to predict their potential of malignant transformation. This study aimed to determine the clinical significance of S100A7 overexpression in HNSCC. METHODOLOGY: Immunohistochemical analysis of S100A7 expression in HNSCC (100 cases), oral lesions (166 cases) and 100 histologically normal tissues was carried out and correlated with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. Overexpression of S100A7 protein was significant in oral lesions (squamous cell hyperplasia/dysplasia) and sustained in HNSCC in comparison with oral normal mucosa (p(trend)<0.001). Significant increase in nuclear S100A7 was observed in HNSCC as compared to dysplastic lesions (p = 0.005) and associated with well differentiated squamous cell carcinoma (p = 0.031). Notably, nuclear accumulation of S100A7 also emerged as an independent predictor of reduced disease free survival (p = 0.006, Hazard ratio (HR = 7.6), 95% CI = 1.3-5.1) in multivariate analysis underscoring its relevance as a poor prognosticator of HNSCC patients. CONCLUSIONS: Our study demonstrated nuclear accumulation of S100A7 may serve as predictor of poor prognosis in HNSCC patients. Further, increased nuclear accumulation of S100A7 in HNSCC as compared to dysplastic lesions warrants a large-scale longitudinal study of patients with dysplasia to evaluate its potential as a determinant of increased risk of transformation of oral premalignant lesions.

Heterogeneous ribonucleoprotein K is a marker of oral leukoplakia and correlates with poor prognosis of squamous cell carcinoma.

Oral leukoplakia is a heterogeneous lesion with risk of cancer development; there are no biomarkers to predict its potential of malignant transformation. Tissue proteomic analysis of oral leukoplakia using iTRAQ labeling liquid chromatography-mass spectrometry showed overexpression of heterogeneous ribonucleoprotein K (hnRNP K), a transformation-related RNA-binding protein, in leukoplakia in comparison with normal tissue. Herein, we investigated the clinical significance of hnRNP K in identification of oral leukoplakic lesions in early stages and as a prognostic marker in head-and-neck/oral squamous cell carcinomas (HNOSCCs). Immunohistochemical analysis of hnRNP K was performed in 100 HNOSCCs, 199 leukoplakias and 55 nonmalignant tissues and correlated with clinicopathologic parameters and disease prognosis over 6 years for HNOSCCs. hnRNP K nuclear expression increased from normal tissues to leukoplakia, and frank malignancy (p < 0.001). Cytoplasmic hnRNP K increased significantly from leukoplakia to HNOSCCs (p < 0.001) and was associated with poor prognosis of HNOSCCs (p = 0.011) by Kaplan-Meier analysis. The most important finding of our follow-up study is that cytoplasmic hnRNP K is an independent predictor of disease recurrence in HNOSCC patients. In conclusion, nuclear hnRNP K may serve as a potential marker for early diagnosis, whereas its cytoplasmic accumulation can help to identify a subgroup of HNOSCC patients with poor prognosis, suggesting its putative utility in clinical management of HNOSCC.

Verification of endometrial tissue biomarkers previously discovered using mass spectrometry-based proteomics by means of immunohistochemistry in a tissue microarray format.

Verification of candidate protein biomarkers is a necessary step in moving from the initial discovery to application. Here, we report results of a verification exercise involving six candidate endometrial cancer biomarkers previously discovered using mass-tagging and multidimensional liquid chromatography/tandem mass spectrometry (DeSouza L., et al. J. Proteome Res. 2005, 4, 377-386) on a cohort of 148 patient samples by means of immunohistochemistry on a tissue microarray format. A panel of the three best-performing biomarkers, chaperonin 10, pyruvate kinase M2, and alpha-1-antitrypsin, achieved a sensitivity of 0.85, specificity of 0.93, predictive value of 0.90, and positive predictive value of 0.88 in discriminating malignant from benign endometrium. The ruggedness of this panel of biomarkers was verified in a 2/3-training-set-1/3-test-set cross-validation analysis by randomly splitting the cohort in 10 ways. The roles of chaperonin 10 and pyruvate kinase M2 in tumorigenesis confirm them as credible cancer biomarkers.

Endometrial carcinoma biomarker discovery and verification using differentially tagged clinical samples with multidimensional liquid chromatography and tandem mass spectrometry.

The utility of differentially expressed proteins discovered and identified in an earlier study (DeSouza, L., Diehl, G., Rodrigues, M. J., Guo, J., Romaschin, A. D., Colgan, T. J., and Siu, K. W. M. (2005) Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cleavable ICAT with multidimensional liquid chromatography and tandem mass spectrometry. J. Proteome Res. 4, 377-386) to discriminate malignant and benign endometrial tissue samples was verified in a 40-sample iTRAQ (isobaric tags for relative and absolute quantitation) labeling study involving normal proliferative and secretory samples and Types I and II endometrial cancer samples. None of these proteins had the sensitivity and specificity to be used individually to discriminate between normal and cancer samples. However, a panel of pyruvate kinase, chaperonin 10, and alpha1-antitrypsin achieved the best results with a sensitivity, specificity, predictive value, and positive predictive value of 0.95 each in a logistic regression analysis. In addition, three new potential markers were discovered, whereas two other proteins showed promising trends but were not detected in sufficient numbers of samples to permit statistical validation. Differential expressions of some of these candidate biomarkers were independently verified using immunohistochemistry.

High-definition macromolecular composition of yeast RNA-processing complexes.

A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins. To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae. We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization. Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules. Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described. Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript. This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.