Lisa M. Fitzgerald

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer

Background:Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH).Methods:Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines.Results:Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10−11). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10−5 and 10−29), and identified breast and pancreatic cell lines with BRCA defects.Conclusion:The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

Expression of CD44 variant isoforms CD44v3 and CD44v6 is increased on T cells from patients with systemic lupus erythematosus and is correlated with disease activity

OBJECTIVE To quantify the expression of CD44 and variant isoforms CD44v3 and CD44v6 on T cells from patients with systemic lupus erythematosus (SLE), and to assess correlations of the level of expression of these molecules with disease manifestations. METHODS Information on clinical and demographic characteristics was collected, and blood samples were obtained from 72 patients with SLE and 32 healthy control subjects matched to the patients by sex, race, and age. Expression of CD44 and variants CD44v3 and v6 on T cell subsets was determined by flow cytometry, and Pearsons correlations of their expression levels with clinical variables, SLE Disease Activity Index (SLEDAI) scores, and presence of lupus nephritis were determined. Wilcoxons rank sum tests and conditional multivariable regression analyses were applied to identify differences in the expression of CD44 between patients with SLE and healthy controls. RESULTS Expression of CD44 was higher on CD4+ and CD8+ T cells from SLE patients compared with controls (P <or= 0.03). Expression of CD44v3 and CD44v6 was also higher on total T cells and CD4+ and CD8+ T cells from SLE patients compared with controls (P <or= 0.03). Cell surface levels of CD44v3 on total T cells, CD4+ T cells, and CD8+ T cells as well as cell surface expression of CD44v6 on total T cells and CD4+ T cells were correlated with the SLEDAI score (P < 0.05). The presence of lupus nephritis was associated with the expression of CD44v6 on total T cells, CD4+ T cells, and CD4-CD8- T cells (P < 0.05). Positivity for anti-double-stranded DNA antibodies was associated with the expression levels of CD44v6 on T cells (P < 0.05). CONCLUSION These results indicate that expression levels of CD44v3 and CD44v6 on T cells may represent useful biomarkers of SLE activity.

The catalytic subunit of protein phosphatase 2A (PP2Ac) promotes DNA hypomethylation by suppressing the phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/phosphorylated ERK/DNMT1 protein pathway in T-cells from controls and systemic lupus erythematosus patients.

Background: PP2A is a serine/threonine phosphatase with a central role in the pathogenesis of SLE. Results: Suppression of PP2A mediates increased DNA methylation through the MEK/ERK/DNMT1 pathway in normal and SLE T-cells. Conclusion: PP2A overexpression accounts for DNA hypomethylation in SLE T-cells. Significance: Here, we propose a link between increased PP2A expression and reduced DNA methylation in SLE. DNA hypomethylation is a characteristic feature of systemic lupus erythematosus (SLE) immune cells. Numerous reports have implicated the involvement of the MEK/ERK pathway in the reduction of DNA methyltransferase (DNMT) expression, hence inducing the transcription of methylation-sensitive genes in SLE patients. However, the molecular mechanisms involved remain unclear. Here, we investigated whether the catalytic subunit of protein phosphatase 2A (PP2Ac), which is overexpressed in SLE T-cells, contributes to reduced DNA methylation. We show that both chemical suppression and siRNA silencing of PP2Ac in T-cells resulted in sustained phosphorylation of MEK and ERK following stimulation with phorbol 12-myristate 13-acetate and ionomycin. Furthermore, PP2Ac suppression resulted in increased DNMT enzyme activity, DNA hypermethylation, and decreased expression of methylation-sensitive genes. Similarly, in SLE T-cells, suppression of PP2Ac resulted in increased MEK/ERK phosphorylation, enhanced DNMT1 expression and suppressed expression of the methylation-sensitive CD70 gene. Our results demonstrate that PP2A regulates DNA methylation by influencing the phosphorylation of MEK/ERK. We propose that enhanced PP2Ac in SLE T-cells may dephosphorylate and activate the signaling pathway upstream of DNMT1, thus disturbing the tight control of methylation-sensitive genes, which are involved in SLE pathogenesis.

Splicing factor SF2/ASF rescues IL-2 production in T cells from systemic lupus erythematosus patients by activating IL-2 transcription

T cells from patients with systemic lupus erythematosus (SLE) produce insufficient amounts of the vital cytokine IL-2. We previously showed that SLE T cells express decreased levels of the T-cell receptor–CD3ζ chain and forced expression of CD3ζ into SLE T cells restores IL-2 production. We recently showed that the serine arginine protein splicing factor 2/alternative splicing factor (SF2/ASF) enhances the expression of CD3ζ chain by limiting the production of an unstable splice variant. Here we demonstrate that SF2/ASF levels are decreased in patients with SLE and more so in those with active disease. More importantly, we reveal a function of SF2/ASF, independent of T-cell receptor/CD3 signaling, whereby it is recruited to the IL-2 promoter, increases transcriptional activity, and enhances IL-2 production in SLE T cells. Our results demonstrate that SF2/ASF regulates IL-2 production and that decreased SF2/ASF expression in SLE T cells contributes to deficient IL-2 production.

High sensitivity, specificity and predictive value of the Connective Tissue Disease Screening Questionnaire among urban African-American women

The Connective Tissue Disease Screening Questionnaire (CSQ), developed to screen populations for SLE and other CTDs, has been validated in a predominantly Caucasian population with hospital-based controls. We aimed to test the performance characteristics of the CSQ in an urban, predominantly African-American population. The CSQ was administered by interview to women recruited for a study of environmental risk factors and SLE, including 99 cases with SLE validated by medical record review and 202 healthy controls recruited from the community. Overall, 88% of subjects had African heritage, 6% were Hispanic and 4% were non-Hispanic Caucasian. Controls were more likely to report African heritage than cases (91% versus 82%, P < 0.001). Sensitivity for detecting SLE was 88% and specificity was 91%. In this study, where the prevalence of SLE was 33%, predictive value of a positive CSQ was 82% and predictive value of a negative CSQ was 94%. The CSQ has slightly lower sensitivity but greater specificity for SLE in an urban, predominantly African-American population with community-based controls compared with a Caucasian population with hospital-based controls. These results suggest that the CSQ has adequate sensitivity and specificity and could be used in population studies to screen African-American women for SLE.

A systemic lupus erythematosus gene expression array in disease diagnosis and classification: a preliminary report.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease diagnosed on the presence of a constellation of clinical and laboratory findings. At the pathogenetic level, multiple factors using diverse biochemical and molecular pathways have been recognized. Succinct recognition and classification of clinical disease subsets, as well as the availability of disease biomarkers, remains largely unsolved. Based on information produced by the present authors’ and other laboratories, a lupus gene expression array consisting of 30 genes, previously claimed to contribute to aberrant function of T cells, was developed. An additional eight genes were included as controls. Peripheral blood was obtained from 10 patients (19 samples) with SLE and six patients with rheumatoid arthritis (RA) as well as 19 healthy controls. T cell mRNA was subjected to reverse transcription and PCR, and the gene expression levels were measured. Conventional statistical analysis was performed along with principal component analysis (PCA) to capture the contribution of all genes to disease diagnosis and clinical parameters. The lupus gene expression array faithfully informed on the expression levels of genes. The recorded changes in expression reflect those reported in the literature by using a relatively small (5 ml) amount of peripheral blood. PCA of gene expression levels placed SLE samples apart from normal and RA samples regardless of disease activity. Individual principal components tended to define specific disease manifestations such as arthritis and proteinuria. Thus, a lupus gene expression array based on genes previously claimed to contribute to immune pathogenesis of SLE may define the disease, and principal components of the expression of 30 genes may define patients with specific disease manifestations.

Cytotoxicity associated with electrospun polyvinyl alcohol

Polyvinyl alcohol (PVA) is a synthetic, water-soluble polymer, with applications in industries ranging from textiles to biomedical devices. Research on electrospinning of PVA has been targeted toward optimizing or finding novel applications in the biomedical field. However, the effects of electrospinning on PVA biocompatibility have not been thoroughly evaluated. In this study, the cytotoxicity of electrospun PVA (nPVA) which was not crosslinked after electrospinning was assessed. PVA polymers of several molecular weights were dissolved in distilled water and electrospun using the same parameters. Electrospun PVA materials with varying molecular weights were then dissolved in tissue culture medium and directly compared against solutions of nonelectrospun PVA polymer in human coronary artery smooth muscle cells and human coronary artery endothelial cells cultures. All nPVA solutions were cytotoxic at a threshold molar concentration that correlated with the molecular weight of the starting PVA polymer. In contrast, none of the nonelectrospun PVA solutions caused any cytotoxicity, regardless of their concentration in the cell culture. Evaluation of the nPVA material by differential scanning calorimetry confirmed that polymer degradation had occurred after electrospinning. To elucidate the identity of the nPVA component that caused cytotoxicity, nPVA materials were dissolved, fractionated using size exclusion columns, and the different fractions were added to HCASMC and human coronary artery endothelial cells cultures. These studies indicated that the cytotoxic component of the different nPVA solutions were present in the low-molecular-weight fraction. Additionally, the amount of PVA present in the 3-10 kg/mol fraction was approximately sixfold greater than that in the nonelectrospun samples. In conclusion, electrospinning of PVA resulted in small-molecular-weight fractions that were cytotoxic to cells. This result demonstrates that biocompatibility of electrospun biodegradable polymers should not be assumed on the basis of success of their nonelectrospun predecessors.

Abstract 3116: Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in ovarian cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: BRCA1 and BRCA2 are key members of the homologous recombination (HR) pathway. Mutations in these genes and other HR pathway defects have potential therapeutic relevance when used to support agents that introduce or exploit double-strand DNA breaks. This study examines the association between HR deficiency and genomic patterns of loss of heterozygosity (LOH). Methods: Ovarian tumors from two independent datasets were characterized for germline and somatic defects in BRCA1 and BRCA2. Whole genome LOH profiles were generated using Affymetrix SNP arrays. Publically available data was downloaded from the TCGA website for a third independent ovarian cancer dataset. RAD51C promoter methylation was assayed in two of the datasets. Comprehensive profiling of BRCA1 and BRCA2 defects, and genome wide LOH was also performed on approximately 70 breast, ovarian, colon and pancreatic cell lines. Results: Examination of the pattern of LOH within ovarian tumors with BRCA1, BRCA2, or RAD51C defects compared to tumors without defects in these genes has resulted in the development of a homologous recombination deficiency (HRD) score that has highly significant association with HRD (p=9*10-11). An intermediate class of LOH sizes (>15 Mb but less than a whole chromosome) is highly positive correlated with defective HR, suggesting this class of LOH exists due to double strand DNA break formation and requires repair by HR. The HRD score was validated in two independent ovarian cancer datasets (p=2*10-7 and 9*10-29), and successfully identified breast and pancreatic cell lines with BRCA defects, suggesting it will be effective across multiple tumor types. Conclusions: BRCA1 or BRCA2 mutation carriers have improved outcomes following treatment with DNA damaging agents such as platinum salts, and preclinical studies have demonstrated PARP inhibitor efficacy in BRCA1 or BRCA2 deficient cells. HR deficiency in ovarian cancer is not solely due to germline BRCA1 and BRCA2 mutations, and HR deficiency is not unique to ovarian tumors. Each type of cancer is likely to have a unique spectrum of genetic variants resulting in HRD. The HRD score appears capable of detecting HRD regardless of etiology or mechanism. This score could have clinical utility in breast and ovarian cancer, and could be used to target the use of PARP inhibitors and platinum salts in other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3116. doi:1538-7445.AM2012-3116