Clinical disease activity and flare in SLE: Current concepts and novel biomarkers.

Systemic lupus erythematosus (SLE) is a complex and heterogeneous systemic autoimmune disease associated with innate and adaptive immune dysregulation. SLE occurs primarily in females of childbearing age, with increased prevalence and severity in minority populations. Despite improvements in treatment modalities, SLE patients frequently experience periods of heightened disease activity and flare that can lead to permanent organ damage, increased morbidity, and early mortality. Such outcomes impair quality of life and inflict a significant socioeconomic burden. Predicting changes in SLE disease activity could allow for closer monitoring and preemptive treatment, but existing clinical, demographic and serologic markers have been only modestly predictive. Novel, proactive approaches to clinical disease management are thus critically needed. Panels of blood biomarkers can detect a breadth of immune pathway dysregulation that captures SLE heterogeneity and disease activity. Alterations in the balance of pro-inflammatory and regulatory soluble mediators have been associated with changes in clinical disease activity and are detectable several weeks prior to clinical flare occurrence. A soluble mediator score has been highly predictive of impending flare in both European American and African American SLE patients, and this score does not require a priori knowledge of specific pathway activation in the patient. We review current concepts of disease activity and flare in SLE, focusing on the potential of novel blood biomarkers to characterize and predict changes in disease activity. Measuring the disordered immune response in SLE in this way promises to improve disease management and prevent organ damage in SLE.

Tracking of Systemic Lupus Erythematosus (SLE) Longitudinally Using Biosensor and Patient-Reported Data: A Report on the Fully Decentralized Mobile Study to Measure and Predict Lupus Disease Activity Using Digital Signals-The OASIS Study.

(1) Objective: Systemic lupus erythematosus (SLE) is a complex disease involving immune dysregulation, episodic flares, and poor quality of life (QOL). For a decentralized digital study of SLE patients, machine learning was used to assess patient-reported outcomes (PROs), QOL, and biometric data for predicting possible disease flares. (2) Methods: Participants were recruited from the LupusCorner online community. Adults self-reporting an SLE diagnosis were consented and given a mobile application to record patient profile (PP), PRO, and QOL metrics, and enlisted participants received smartwatches for digital biometric monitoring. The resulting data were profiled using feature selection and classification algorithms. (3) Results: 550 participants completed digital surveys, 144 (26%) agreed to wear smartwatches, and medical records (MRs) were obtained for 68. Mining of PP, PRO, QOL, and biometric data yielded a 26-feature model for classifying participants according to MR-identified disease flare risk. ROC curves significantly distinguished true from false positives (ten-fold cross-validation: p < 0.00023; five-fold: p < 0.00022). A 25-feature Bayesian model enabled time-variant prediction of participant-reported possible flares (P(true) > 0.85, p < 0.001; P(nonflare) > 0.83, p < 0.0001). (4) Conclusions: Regular profiling of patient well-being and biometric activity may support proactive screening for circumstances warranting clinical assessment.

A Flare Risk Index Informed by Select Immune Mediators in Systemic Lupus Erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is marked by immune dysregulation linked to varied clinical disease activity. Using a unique longitudinal cohort of SLE patients, this study sought to identify optimal immune mediators informing an empirically refined flare risk index (FRI) reflecting altered immunity prior to clinical disease flare. METHODS: Thirty-seven SLE-associated plasma mediators were evaluated by microfluidic immunoassay in 46 samples obtained in SLE patients with an imminent clinical disease flare (preflare) and 53 samples obtained in SLE patients without a flare over a corresponding period (pre-nonflare). SLE patients were selected from a unique longitudinal cohort of 106 patients with classified SLE (meeting the American College of Rheumatology 1997 revised criteria for SLE or the Systemic Lupus International Collaborating Clinics 2012 revised criteria for SLE). Autoantibody specificities, hybrid SLE Disease Activity Index (hSLEDAI) scores, clinical features, and medication usage were also compared at preflare (mean ± SD 111 ± 47 days prior to flare) versus pre-nonflare (99 ± 21 days prior to nonflare) time points. Variable importance was determined by random forest analysis with logistic regression subsequently applied to determine the optimal number and type of analytes informing a refined FRI. RESULTS: Preflare versus pre-nonflare differences were not associated with demographics, autoantibody specificities, hSLEDAI scores, clinical features, nor medication usage. Forward selection and backward elimination of mediators ranked by variable importance resulted in 17 plasma mediator candidates differentiating preflare from pre-nonflare visits. A final combination of 11 mediators best informed a newly refined FRI, which achieved a maximum sensitivity of 97% and maximum specificity of 98% after applying decision curve analysis to define low, medium, and high FRI scores. CONCLUSION: We verified altered immune mediators associated with imminent disease flare, and a subset of these mediators improved the FRI to identify SLE patients at risk of imminent flare. This molecularly informed, proactive management approach could be critical in prospective clinical trials and the clinical management of lupus.

MTHFR C677T and postmenopausal breast cancer risk by intakes of one-carbon metabolism nutrients: a nested case-control study.

INTRODUCTION: The C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene has been hypothesized to increase breast cancer risk. However, results have been inconsistent, and few studies have reported the association by menopausal status or by intakes of nutrients participating in one-carbon metabolism. Our aims were to investigate whether MTHFR C677T was associated with postmenopausal breast cancer risk and whether this relation was modified by intakes of folate, methionine, vitamins B(2), B(6), and B(12), and alcohol. METHODS: We studied 318 incident breast cancer cases and 647 age- and race-matched controls participating in a nested case-control study of postmenopausal women within the VITamins And Lifestyle (VITAL) cohort. Genotyping was conducted for MTHFR C677T and dietary and supplemental intakes were ascertained from a validated questionnaire. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression. RESULTS: We observed a 62% increased risk of breast cancer among postmenopausal women with the TT genotype (OR = 1.62; 95% CI: 1.05 to 2.48). Women with a higher number of variant T alleles had higher risk of breast cancer (P for trend = 0.04). Evidence of effect-modification by intakes of some B vitamins was observed. The most pronounced MTHFR-breast cancer risks were observed among women with the lowest intakes of dietary folate (P for interaction = 0.02) and total (diet plus supplemental) vitamin B(6) (P for interaction = 0.01), with no significant increased risks among women with higher intakes. CONCLUSIONS: This study provides support that the MTHFR 677TT genotype is associated with a moderate increase in risk of postmenopausal breast cancer and that this risk may be attenuated with high intakes of some one-carbon associated nutrients.

Polymorphisms in genes involved in sex hormone metabolism, estrogen plus progestin hormone therapy use, and risk of postmenopausal breast cancer.

Hormone therapy, estrogen plus progestin (E+P) particularly, is associated with increased risk of breast cancer. Functionally relevant polymorphisms in genes involved in sex hormone metabolism may alter exposure to exogenous sex hormones and affect risk of postmenopausal breast cancer. We evaluated associations of common polymorphisms in genes involved in estrogen and/or progesterone metabolism, E+P use, and their interactions with breast cancer risk in a case-control study of postmenopausal women (324 cases; 651 controls) nested within the VITAL cohort. None of the polymorphisms studied was, by itself, statistically significantly associated with breast cancer risk. E+P use was significantly associated with increased breast cancer risk (> or =10 years versus never; odds ratio, 1.9; 95% confidence interval, 1.3-2.8; P(trend) = 0.0002). Statistically significant interactions between CYP1A1 Ile(462)Val (P(interaction) = 0.04), CYP1A1 MspI (P(interaction) = 0.003), CYP1B1 Val(432)Leu (P(interaction) = 0.007), CYP1B1 Asn(453)Ser (P(interaction) = 0.04) and PGR Val(660)Leu (P(interaction) = 0.01), and E+P use were observed. The increased risk of breast cancer associated with E+P use was greater among women with at least one rare allele of the CYP1A1 Ile(462)Val, CYP1A1 MspI, CYP1B1 Asn(453)Ser, and PGR Val(660)Leu polymorphisms than among women homozygous for the common allele of these polymorphisms. Risk of breast cancer increased little with increasing years of E+P use among women with at least one CYP1B1 Val(432) allele; a large increase in risk was seen among women homozygous for CYP1B1 Leu(432). Our results support the hypothesis that specific polymorphisms in genes involved in sex hormone metabolism may modify the effect of E+P use on breast cancer risk.

Enhanced motility of KGF-transfected breast cancer cells.

BACKGROUND: In a previous study, we reported that keratinocyte growth factor (KGF) produced a rapid increase in the motility of ER-positive breast cancer cells. Others have demonstrated that KGF treatment in rodent species produces rapid mammary ductal hyperplasia. Epithelial cells do not produce KGF; thus, in the present study, MCF-7 cells were stably transfected with a KGF-expressing vector and the motility and morphology of the transfected, non-transfected and empty vector cell lines compared. MATERIALS AND METHODS: A mammalian expression vector containing a KGF cDNA was transfected into MCF-7/beta cells, and two stable clones (MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9) were identified. Western blotting of conditioned medium from these clones was used to confirm the expression of KGF. The motility of wild-type and KGF-transfected MCF-7 cells was compared using time-lapse videomicroscopy and a cell culture wounding model which examined cell migration over a period of 1-3 days. RESULTS: The Western blots demonstrated that the expression of KGF in both the MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9 cell lines was higher than the wild-type and MCF-7/beta cell lines. The cell proliferation and migration distance was significantly greater for both KGF-transfected MCF-7 cell lines than the wild-type and MCF-7/beta cell lines under the same experimental conditions. Further, changes in motile morphology were observed in both the MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9 cell lines. In addition, the MCF-7/beta/KGF-T8 clone was found to produce much larger tumors than both the MCF-7/beta/KGF-T9 and EV clones in mouse xenografts. These results indicated that autocrine production of KGF in the KGF-transfected MCF-7 cell lines enhanced cell migration, migration-related morphology and xenograft tumor growth. CONCLUSION: KGF-transfected MCF-7 cells displayed a much greater motility than non-transfected cells, confirming the KGF motility enhancement effect which we previously reported. The use of KGF-transfected breast cancer cells in the xenograft model may help to study the mechanism of KGF-mediated cell motility and to identify specific KGF antagonists that may be used to prevent or impede KGF-mediated metastatic progression.

Tumor suppression by the prohibitin gene 3'untranslated region RNA in human breast cancer.

Prohibitin is a candidate tumor suppressor gene located on human chromosome 17q21, a region of frequent loss of heterozygosity in breast cancers. We showed previously that microinjection of RNA encoded by the prohibitin gene 3'untranslated region (3'UTR) blocks the G(1)-S transition causing cell cycle arrest in several human cancer cell lines, including MCF7. Two allelic forms (C versus T) of the prohibitin 3'UTR exist, and carriers of the less common variant (Tallele) with a family history of breast cancer exhibited an increased risk of breast cancer. In the present study, we examined the tumor suppressor activity of the prohibitin 3'UTR in human breast cancer cells. Stable clones of MCF7 cells expressing either the C allele or the T allele RNA under the control of the cytomegalovirus promoter were isolated and compared with empty vector clones. Clones expressing the C allele RNA (UTR/C) exhibited significant suppression of growth in cell proliferation assays, inhibition of colony formation in soft agar assays, and suppression of xenograft tumor growth when implanted on nude mice, compared with either T allele expressing or empty vector clones. Immunohistochemical analyses with Ki67 staining confirmed a significant reduction in proliferation of UTR/C tumors. Thus, the C allele of prohibitin 3'UTR produces a functional RNA, whereas a single nucleotide polymorphism creates a null allele (T allele) of which the RNA product has lost activity. Our data demonstrate for the first time that an RNA molecule functions as a tumor suppressor in human breast cancer.

Diverse gene expression pattern during 5-fluorouridine-induced apoptosis.

The purpose of this study was to establish experimental conditions to produce apoptosis by the fluorinated pyrimidine 5-fluorouridine and to examine the changes in gene expression that occurred during cell death. HCT-116 colorectal carcinoma cells were exposed to 10 microM 5-fluorouridine alone or in the presence of 1 mM uridine, 30 microM thymidine or both uridine and thymidine. A time-dependent increase in the percentage of apoptotic cells and a decrease in the percentage of viable cells were observed when the cells were treated with 5-fluorouridine in the absence of uridine (p < 0.001) but not in the presence of uridine. cDNA microarray analysis was used to study the expression of 1,200 different genes during apoptosis by 5-flurouridine. The expression of 33 genes was upregulated by 5-fold or greater at 16 and 24 h of 5-fluorouridine exposure. The largest cluster of upregulated genes included a group of genes classified as growth factors, cytokines and chemokines (e.g. interleukin-3, interleukin-4, B-cell growth factor 1 and stem cell growth factor). The expression of MIC-1 increased up to 100-fold during 5-flurouridine exposure. One hundred and twenty-four genes were downregulated by 5-fold or greater following exposure to 5-fluorouridine. The downregulated genes were distributed throughout the six different classifications on the array. Our data demonstrate a diverse pattern of gene expression during the fluorouridine-induced apoptosis and suggest that mechanisms besides a global inhibition of RNA synthesis/ processing contribute to the RNA-directed cytotoxicity of fluoropyrimidines.

Expression of prohibitin 3' untranslated region suppressor RNA alters morphology and inhibits motility of breast cancer cells.

The prohibitin 3' untranslated region (3'UTR) belongs to a novel class of non-coding regulatory RNAs. It arrests cell cycle progression by blocking G1-S transition in breast and other cancers. Our previous studies comparing MCF7 derived clones constitutively expressing a common allelic form of prohibitin RNA (UTR/C) to various controls demonstrated that it functions as a tumor suppressor. Here, we further characterized the morphology and motility of these transgenic breast cancer cells when grown in cell culture and on nude mice. In contrast to empty vector (EV) cells, UTR/C cells were observed to grow in an organized manner with more cell-cell contact and differentiate into structures with a duct-like appearance. Computer assisted cytometry to evaluate differences in nuclear morphology was performed on UTR/C and EV tissues from nude mice. Receiver operator curve areas generated using a logistic regression model were 0.8, indicating the ability to quantitatively distinguish UTR/C from EV tissues. Keratinocyte growth factor-induced motility experiments showed that migration of UTR/C cells was significantly reduced (80-90%) compared to EV cells. Together, these data indicate that this novel 3'UTR influences not only the tumorigenic phenotype but also may play a role in differentiation and migration of breast cancer cells.

Increased expression of alpha-1-antitrypsin, glutathione S-transferase pi and vascular endothelial growth factor in human pancreatic adenocarcinoma.

BACKGROUND: This study was designed to investigate abnormalities in gene expression in ductal adenocarcinoma of the pancreas using cDNA arrays. METHODS: Gene expression in pancreatic ductal adenocarcinoma was compared with normal pancreatic tissue controls. Specimens from 5 patients with pancreatic adenocarcinoma were taken fresh at operation and analyzed using commercially prepared cDNA arrays evaluating approximately 2,000 genes. Immunohistochemical staining was used to confirm protein expression of selected genes. RESULTS: Alpha-1-antitrypsin (A1AT) and glutathione S-transferase pi (GSTP) were significantly up-regulated in all 5 tumors. Vascular endothelial growth factor (VEGF) was up-regulated in 4 of the 5 patients. Immunohistochemical staining verified the overexpression of each of these genes. CONCLUSIONS: A1AT, GSTP, and VEGF are overexpressed in human pancreatic adenocarcinoma specimens taken fresh at operation. To our knowledge, this is the first study of human pancreatic ductal adenocarcinoma demonstrating the up-regulation of these genes using gene expression arrays.

A steroid metabolizing gene variant in a polyfactorial model improves risk prediction in a high incidence breast cancer population.

BACKGROUND: We have combined functional gene polymorphisms with clinical factors to improve prediction and understanding of sporadic breast cancer risk, particularly within a high incidence Caucasian population. METHODS: A polyfactorial risk model (PFRM) was built from both clinical data and functional single nucleotide polymorphism (SNP) gene candidates using multivariate logistic regression analysis on data from 5022 US Caucasian females (1671 breast cancer cases, 3351 controls), validated in an independent set of 1193 women (400 cases, 793 controls), and reassessed in a unique high incidence breast cancer population (165 cases, 173 controls) from Marin County, CA. RESULTS: The optimized PFRM consisted of 22 SNPs (19 genes, 6 regulating steroid metabolism) and 5 clinical risk factors, and its 5-year and lifetime risk prediction performance proved significantly superior (~ 2-fold) over the Gail model (Breast Cancer Risk Assessment Tool, BCRAT), whether assessed by odds (OR) or positive likelihood (PLR) ratios over increasing model risk levels. Improved performance of the PFRM in high risk Marin women was due in part to genotype enrichment by a CYP11B2 (-344T/C) variant. CONCLUSIONS AND GENERAL SIGNIFICANCE: Since the optimized PFRM consistently outperformed BCRAT in all Caucasian study populations, it represents an improved personalized risk assessment tool. The finding of higher Marin County risk linked to a CYP11B2 aldosterone synthase SNP associated with essential hypertension offers a new genetic clue to sporadic breast cancer predisposition.

Estrogen receptor-dependent and independent mechanisms of breast cancer carcinogenesis.

Long term exposure to estrogens is associated with an increased risk of breast cancer. The precise mechanisms responsible for estrogen mediated carcinogenesis are not well understood. The most widely accepted theory holds that estradiol (E(2)), acting through estrogen receptor alpha (ERα), stimulates cell proliferation and initiates mutations arising from replicative errors occurring during pre-mitotic DNA synthesis. The promotional effects of E(2) then support the growth of cells harboring mutations. Over a period of time, sufficient numbers of mutations accumulate to induce neoplastic transformation. Laboratory and epidemiological data also suggest that non-receptor mediated mechanisms resulting from the genotoxic effects of estrogen metabolites are involved in breast cancer development. This manuscript critically reviews existing data implicating both ER-dependent and -independent mechanisms. The weight of evidence supports the possibility that both mechanisms are involved in the carcinogenic process. In addition, estrogen metabolites likely modulate stem cell functionality and cancer progression. The roles of ER dependent and independent actions in the carcinogenic process are pertinent to the consideration of breast cancer preventative agents as anti-estrogens block only receptor mediated pathways whereas the aromatase inhibitors block both.

Vitamin D receptor polymorphisms and breast cancer risk in a high-incidence population: a pilot study.

BACKGROUND: Marin County, CA has very high incidence of breast cancer. Traditional risk factors, such as those included in the Gail model, do not effectively stratify breast cancer in this population. This retrospective case-control pilot study evaluates DNA from volunteers from a previous Marin County breast cancer epidemiology study. A polyfactorial risk model (OncoVue; InterGenetics Incorporated) that incorporates 22 polymorphisms in 19 genes and 5 clinical risk factors was used to stratify risk in Marin County women. STUDY DESIGN: DNA genotyping was performed on 164 Caucasian women diagnosed with primary breast cancer in Marin County from 1997 to 1999 and 174 age- and ethnicity-matched control subjects. Individual lifetime risks were determined using the polyfactorial risk model and genotype frequencies in women at elevated risk were compared with the overall genotypes. RESULTS: The vitamin D receptor VDR ApaI A2/A2 (rs7975232) homozygous polymorphism was present in high frequency in elevated-risk women. Sixty-four percent of elevated-risk women had the VDR Apa1 A2/A2 genotype compared with only 34% in the overall study, a statistically significant 1.9-fold difference (p = 0.0003). VDR Apa1 A2/a1 and a1/a1 genotypes were also present, but in lower frequencies. CONCLUSIONS: The high frequency of the VDR Apa1 A2/A2 homozygous polymorphism in women designated as elevated risk for breast cancer by the polyfactorial risk model might be related to the high incidence rates of breast cancer in Marin County, CA. Vitamin D supplementation could modify risk of breast cancer in this population.

Age-specific association of steroid hormone pathway gene polymorphisms with breast cancer risk.

BACKGROUND: Breast cancer (BC) is a complex disease, and the incidence rates for BC increase with age. Both environmental factors and genetics have an impact on the risk of BC. Although the effects of environmental factors may vary with age, it has been assumed generally that the penetrance of single nucleotide polymorphisms (SNPs) is constant throughout life. In the current study, the results demonstrated that certain SNPs exhibit BC risk associations that vary considerably with age. METHODS: SNPs in 12 steroid hormone pathway genes were investigated for associations with BC risk in white women who were enrolled in an age-matched, case-control (1:2 for cases and controls, respectively) study that consisted of a discovery set (n = 5000 women) and an independent validation set (n = 1583 women). RESULTS: Significant age-related trends were identified and confirmed for SNPs in 4 genes associated with BC risk. The cytosine/cytosine (C/C) genotype of cytochrome P450 XIB2 (CYP11B2) was associated with decreased risk at younger ages (ages 30-44 years) but an increased risk at older ages (ages 55-69 years). The homozygous cytosine-guanine (CG/CG) genotype of uridine phosphorylase glycosyltransferase 1A7 (UGT1A7) was associated with increased risk at younger ages but decreased risk at older ages. Associations in cytochrome P450 19 (CYP19) and progesterone receptor (PGR) were confined to middle age (ages 45-54 years). CONCLUSIONS: The identification of age-specific genetic associations may have profound implications for future etiologic studies of BC and for the use of SNP genotyping to accurately predict the risk of BC in women.

Oligogenic combinations associated with breast cancer risk in women under 53 years of age.

Common, but weakly penetrant, functional polymorphisms probably account for most of the genetic risk for breast cancer in the general population. Current polygenic risk models assume that component genes act independently. To test for potential gene-gene interactions, single nucleotide polymorphisms in ten genes with known or predicted roles in breast carcinogenesis were examined in a case-control study of 631 Caucasian women diagnosed with breast cancer under the age of 53 years and 1,504 controls under the age of 53 years. Association of breast cancer risk with individual genes and with two- and three-gene combinations was analyzed. Sixty-nine oligogenotypes from 37 distinct two- and three-gene combinations met stringent criteria for significance. Significant odds ratios (ORs) covered a 12-fold range: 0.5-5.9. Of the observed ORs, 17% differed significantly from the ORs predicted by a model of independent gene action, suggesting epistasis, i.e., that these genes interact to affect breast cancer risk in a manner not predictable from single gene effects. Exploration of the biological basis for these oligogenic interactions might reveal etiologic or therapeutic insights into breast cancer and other cancers.