Gary J. Vanasse

Does an Index Composed of Clinical Data Reflect Effects of Inflammation, Coagulation, and Monocyte Activation on Mortality Among Those Aging With HIV?

BACKGROUND When added to age, CD4 count and human immunodeficiency virus type 1 (HIV-1) RNA alone (Restricted Index), hemoglobin, FIB-4 Index, hepatitis C virus (HCV), and estimated glomerular filtration rate improve prediction of mortality. Weighted and combined, these 7 routine clinical variables constitute the Veterans Aging Cohort Study (VACS) Index. Because nonroutine biomarkers of inflammation (interleukin 6 [IL-6]), coagulation (D-dimer), and monocyte activation (sCD14) also predict mortality, we test the association of these indices and biomarkers with each other and with mortality. METHODS Samples from 1302 HIV-infected veterans on antiretroviral therapy were analyzed. Indices were calculated closest to date of collection. We calculated Spearman correlations stratified by HIV-1 RNA and HCV status and measured association with mortality using C statistics and net reclassification improvement (NRI). RESULTS Of 1302 subjects, 915 had HIV-1 RNA <500 copies/mL and 154 died. The VACS Index was more correlated with IL-6, D-dimer, and sCD14 than the Restricted Index (P < .001). It was also more predictive of mortality (C statistic, 0.76; 95% confidence interval [CI], .72-.80) than any biomarker (C statistic, 0.66-0.70) or the Restricted Index (C statistic, 0.71; 95% CI, .67-.75). Compared to the Restricted Index alone, NRI resulted from incremental addition of VACS Index components (10%), D-dimer (7%), and sCD14 (4%), but not from IL-6 (0%). CONCLUSIONS Among HIV-infected individuals, independent of CD4, HIV-1 RNA, and age, hemoglobin and markers of liver and renal injury are associated with inflammation. Addition of D-dimer and sCD14, but not IL-6, improves the predictive accuracy of the VACS Index for mortality.

Prevalence of 25-hydroxyvitamin D deficiency in subgroups of elderly persons with anemia: association with anemia of inflammation

Anemia and vitamin D deficiency are conditions that both result in significant morbidity and increase with age. The potential relationship between them remains poorly understood, particularly in the elderly. We used the Third National Health and Nutrition Examination Survey to examine the association of vitamin D deficiency with anemia subtypes in persons aged ≥ 60 years. Vitamin D deficiency was defined as serum levels < 20 ng/mL, and anemia was defined according to World Health Organization criteria. Vitamin D deficiency was associated with anemia prevalence independent of age, sex, or race/ethnicity (odds ratio, 1.47; 95% confidence interval, 1.06-2.05; P = .02) and varied significantly by anemia subtype (P overall = .003). The prevalence of vitamin D deficiency was 33.3% in the nonanemic population, 56% in anemia of inflammation (AI; P = .008), and 33.0% in unexplained anemia (P = .55). Non-Hispanic blacks had a 7-fold increased risk of AI compared with whites, and this was partially attenuated after adjusting for vitamin D deficiency. These data show that vitamin D deficiency is associated with specific subtypes of anemia in the elderly, especially in those with AI. Vitamin D may suppress inflammatory pathways, and studies to determine whether vitamin D supplementation ameliorates AI are warranted.

Prevalence of polycythemia vera and essential thrombocythemia

Polycythemia vera (PV) and essential thrombocythemia (ET) are common types of myeloproliferative disorders (MPD), the prevalence of which has not been well documented in the United States. Recent breakthroughs in the molecular etiology of these disorders and the accelerated development of targeted pharmacotherapeutics to treat them underscore the need to define the affected population. In this study, we obtained health claims data from major commercial insurance payers in Connecticut and the Center for Medicare and Medicaid Services to estimate the prevalence of PV and ET. Specifically, logistic regression was utilized to develop algorithms to predict the probability that an individual with claims suggestive of MPD truly has PV or ET, and the algorithms were then applied to health claims to estimate the number of PV and ET patients in Connecticut. As of 2003, the age‐standardized prevalence was 22 per 100,000 and 24 per 100,000 for PV and ET, respectively, in Connecticut. Applying the age‐specific prevalence of PV and ET to the entire US population resulted in an estimated total of 65,243 patients with PV and 71,078 patients with ET in the United States in 2003. This study is the first to assess the prevalence of PV and ET in a large US population. Given the large number of individuals afflicted with these diseases and the fact that demographic changes alone will further increase the burden of these diseases in the foreseeable future, it is imperative to conduct more systematic research into the etiology and treatment of PV and ET. Am. J. Hematol., 2008.

Anemia in Elderly Patients: An Emerging Problem for the 21st Century

Anemia is a significant problem in elderly patients. Although many anemic elderly patients can be diagnosed with nutritional deficiency, anemia of chronic inflammation or comorbid diseases that explain their decreased hematocrit, the etiology of anemia in a significant fraction remains obscure. There is evidence to suggest that the hematopoietic stem cell displays increasing erythropoietin (EPO) resistance with age. EPO levels rise in elderly, nonanemic patients, and it is hypothesized that there is an interplay between this rising demand for EPO and the decreasing ability of the aging kidney to produce adequate hormone to meet that need. There is further considerable evidence that aging is associated with increased proinflammatory cytokine expression and that many of these cytokines can contribute to EPO insensitivity. Consequently, genetic variation in the expression of these proinflammatory cytokines may influence the development of anemia in elderly patients, both through induction of hepcidin expression (anemia of inflammation) and through cytokine suppression of erythroid colony formation. The impact of inflammatory mediators, EPO insensitivity, and other factors that may act on the hematopoietic stem cell to decrease erythropoiesis are under active study and should serve to elucidate the pathophysiology of this important cause of morbidity and mortality in elderly individuals. A better understanding of the pathophysiology of anemia in elderly patients should provide critical entry points for interventions that will improve survival and quality of life in the aging population.

Design and validation of a high-throughput matrix-assisted laser desorption ionization time-of-flight mass spectrometry method for quantification of hepcidin in human plasma.

Disorders of iron metabolism affect over a billion people worldwide. The circulating peptide hormone hepcidin, the central regulator of iron distribution in mammals, holds great diagnostic potential for an array of iron-associated disorders, including iron loading (β-thalassemia), iron overload (hereditary hemochromatosis), and iron deficiency diseases. We describe a novel high-throughput matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry assay for quantification of hepcidin in human plasma. This assay involves enrichment using a functionalized MALDI chip, a novel solvent-detergent precipitation buffer, and quantification using a stable isotope labeled internal standard. The linear range of hepcidin in plasma was 1-120 nM, with a low limit of quantification (LOQ) (1 nM), high accuracy (<15% relative error (RE)), and high precision (intraday average 5.52-18.48% coefficient of variation (CV) and interday 9.32-14.83% CV). The assay showed strong correlation with an established hepcidin immunoassay (Spearman; R(2) = 0.839 n = 93 ethylenediaminetetraacetic acid (EDTA) plasma). A collection of normal healthy pediatric samples (range 3.8-32.5 ng/mL; mean 12.9 ng/mL; n = 119) showed significant differences from an adult collection (range 1.8-48.7 ng/mL; mean 16.1 ng/mL; n = 95; P = 0.0096). We discuss these preliminary reference ranges and correlations with additional parameters in light of the utility and limitations of hepcidin measurements as a stand-alone diagnostic and as a tool for therapeutic intervention.

Overexpression of SOCS3 is associated with decreased survival in a cohort of patients with de novo follicular lymphoma.

The prognostic significance of SOCS3 protein expression was determined in de novo follicular lymphomas (FL) with t(14;18) and bcl‐2 overexpression. Presentation lymph nodes from 82 FL patients for whom clinical information was available were immunohistochemically segregated into SOCS3‐positive (n = 42) or ‐negative (n = 40) cohorts, and overall survival (OS) was analysed. SOCS3‐positive FL patients had a median OS of 10 years compared with 22 years in SOCS3‐negative patients (P = 0·001, log rank test). After adjusting for Follicular Lymphoma International Prognostic Index subgroups, SOCS3 overexpression remained an independent predictor of decreased OS (P < 0·001). These findings suggest that overexpression of SOCS3 may be an independent poor prognostic variable in patients with de novo FL.

Therapy for Hepatitis C Virus Infection Increases Survival of Patients With Pretreatment Anemia

BACKGROUND & AIMS Individuals with chronic hepatitis C virus (HCV) infection and pretreatment anemia are less likely to begin and complete a full course of treatment for HCV. However, among those who are treated for HCV infection, the effect of treatment on mortality is not clear. METHODS We performed a retrospective analysis of 200,139 HCV-infected veterans using data from the Electronically Retrieved Cohort of Hepatitis C-Infected veterans (2001-2008). The effects of treatment and treatment duration on survival were compared based on data from 1820 treated and 27,690 untreated anemic HCV-infected veterans. The association between HCV treatment and mortality was estimated using the Cox proportional hazard models, with adjustments for potential confounders. The main outcome was all-cause mortality. RESULTS In multivariable analysis, pretreatment anemia was associated significantly with African American race (odds ratio [OR], 2.03; 95% confidence interval [CI], 1.95-2.11), chronic kidney disease (OR, 3.36; 95% CI, 3.23-3.51), and decompensated liver disease (OR, 3.69; 95% CI, 3.53-3.86). All-cause mortality for treated, anemic, HCV-infected veterans was lower (54.2 per 1000 person-years; 95% CI, 49.2-59.7 per 1000 person years) than for untreated, anemic HCV-infected veterans (146.8 per 1000 person-years; 95% CI, 144.2-149.4 per 1000 person-years). The adjusted hazard ratio for treatment of HCV in anemic veterans was 0.45 (95% CI, 0.39-0.51), which was reduced after exclusion of comorbidities (hazard ratio, 0.28; 95% CI, 0.22-0.37). CONCLUSIONS Based on a retrospective analysis of a veterans database, HCV therapy increases survival rates of individuals with pretreatment anemia. Additional studies are needed to determine strategies to increase rates of HCV therapy for this group.

Resveratrol ameliorates TNFα-mediated suppression of erythropoiesis in human CD34(+) cells via modulation of NF-κB signalling.

Overexpression of pro‐inflammatory cytokines, including tumour necrosis factor alpha (TNFα), has been implicated in the pathogenesis of anaemia of inflammation. TNFα suppresses erythroid colony formation via both direct and indirect effects on haematopoietic progenitors, often involving activation of nuclear factor (NF)‐κB signalling resulting in downregulation of transcription factors critical for erythropoiesis. There is a dearth of effective and safe therapies for many patients with inflammatory anaemia. Resveratrol is a flavanol found in red wine grapes that possesses potent anti‐inflammatory properties, but studies of its impact on human erythropoiesis have proven contradictory. We investigated whether resveratrol ameliorates TNFα‐mediated suppression of erythropoiesis in human CD34+ haematopoietic progenitors. We found that resveratrol partially reverses the erythroid suppressive effects of TNFα, leading to significant recovery in burst forming unit‐erythroid colony formation in human CD34+ cells. CD34+ cells pre‐incubated with resveratrol for 72 h in the presence of TNFα inhibited NF‐κB activation via decreased NF‐κB nuclear localization without altering total NF‐κB protein levels and independent of IκB degradation. Resveratrol also significantly restored the baseline expression of erythroid transcription factors NFE2 and the GATA1/GATA2 ratio in CD34+ cells treated with TNFα. In conclusion, resveratrol may inhibit TNFα‐mediated NF‐κB activation and promote erythropoiesis in primary human CD34+ cells.

Single nucleotide polymorphisms in the human TNF gene are associated with anaemia and neutropenia in a cohort of patients with de novo myelodysplastic syndrome.

Anaemia is the most common haematological abnormality in the myelodysplastic syndromes (MDS), contributing to morbidity and mortality via reduced oxygen carrying capacity, transfusional iron overload, and a reduced quality of life (Delforge, 2003). Infection also remains a challenging complication and the most common cause of death in MDS patients (Kouides & Bennett, 1997). The expression of several proinflammatory cytokines, including tumour necrosis factoralpha (TNFa), is increased in MDS patients versus normal subjects (Gersuk et al, 1998). The TNF gene, which encodes TNFa, contains functional single nucleotide polymorphisms (SNPs) that have been linked to cytokine hypersecretion (Imahara & O’ Keefe, 2004). The high-expressing TNF -308A/ A genotype was found to be overrepresented 14-fold in a cohort of 21 MDS patients (Powers et al, 2007), but the extent to which TNF SNPs affect the phenotype or natural history of MDS remains to be elucidated. We postulated that underlying host factors including proinflammatory mediators are genetic modifiers of disease severity in MDS, influencing the degree of cytopenias, severity of infections, and disease progression. We therefore examined whether functional TNF polymorphisms altered the MDS phenotype. Our studies revealed that high-expressing SNPs in TNF are independently associated with National Cancer Institutes Grade II or greater anaemia and with neutropenia in our cohort of treatment naı̈ve, de novo MDS patients, supporting their role as disease modifiers in MDS. This study was approved by Dana-Farber Cancer Institute’s (DFCI) Investigational Review Board. DNA samples isolated from the bone marrows of 328 individuals with de novo MDS with accompanying clinical annotation and from the blood of 150 normal healthy subjects were generously provided by Dr. Benjamin Ebert and the DFCI tissue repository, respectively. MDS patients were defined according to FrenchAmerican-British (FAB) criteria. DNA was genotyped for the )308G/A and )238G/A polymorphisms of TNF by polymerase chain reaction (PCR) amplification with primer pairs: TNF 308G/A [5¢-AATAGGTTTTGAGGGCCATG-3¢ (forward) and 5¢-ATCTGGAGGAAGCGGTAGTG-3¢ (reverse)]; TNF -238G/ A [5¢-AGAAGACCCCCCTCGGAACC-3¢ (forward) and 5¢-ATCTGGAGGAAGCGGTAGTG-3¢ (reverse)]. PCR amplification conditions were as follows: 94 C for 5 min; 10 cycles of 94 C for 30 s, 65 C for 30 s (decreasing 1 C each cycle), and 72 C for 30 s (decreasing 1 C each cycle); 30 cycles of 94 C for 30 s, 55 C for 30 s, 72 C for 30 s; terminal extension at 72 C for 7 min. PCR products were digested with MspI (New England Biolabs Inc., Beverly, MA, USA) for 6 h at 37 C, and restriction fragment length polymorphism products analyzed by gel electrophoresis using 5-20% Tris-HCl polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA). Differences in allele frequencies were analyzed by chi-square testing. Logistic regression was used to assess whether TNF polymorphisms were associated with the following outcomes: age, FAB classification, International Prognostic Scoring System (IPSS) score, karyotype, blast percentage, survival, anaemia [haemoglobin (Hb) <130 g/l or <120 g/l for men and women, respectively], neutropenia [absolute neutrophil count (ANC) <1Æ5 · 10/l], thrombocytopenia (platelet count < 150 · 10/l), requirement for red blood cell (RBC) or platelet transfusions, and therapy with erythropoiesis stimulating agents (ESAs). Data was analyzed using the Statistical Package for the Social Sciences (spss) for Windows, version 15.0. MDS cases were classified by FAB criteria: Refractory Anaemia (n = 155), Refractory Anaemia with Ringed Sideroblasts (n = 30), Refractory Anaemia with Excess Blasts (n = 110), and Refractory Anaemia with Excess Blasts in Transformation (n = 33). Our MDS cohort was predominantly treatment naı̈ve, with only 20/328 patients having received ESA therapy as their only form of disease management at the time clinical data were collected. Within our MDS cohort, multivariate analysis of TNF -308 genotypes revealed that the AA genotype (high-expressing) was independently associated with neutropenia at ANC levels of less than 1Æ5 · 10/l (P = 0Æ04, Logistic regression) (Table I). Multivariate analysis of TNF 238 genotypes in our MDS cohort revealed that the presence of a high-expressing genotype, either GA or AA, was independently associated with the presence of NCI Grade II or greater anaemia (Hb < 100 g/l) (P = 0Æ035, Logistic regression) (Table I), which retained statistical significance after controlling for RBC transfusions and ESA therapy (P = 0Æ03). Furthermore, whereas the A allele in our MDS cohort was present in 14% of anaemic individuals (n = 41), no patients with a normal Hb level (n = 26) carried an A allele (P = 0Æ03, Chi-Square Test), which also retained statistical significance after controlling for RBC transfusions or ESA therapy (P = 0Æ02). TNF -308 or TNF -238 genotypes were not significantly associated with other outcomes analyzed. The frequencies of the GG, GA, and AA genotypes at both correspondence

Hepatitis C virus treatment and survival in patients with hepatitis C and human immunodeficiency virus co-infection and baseline anaemia

The impact of pretreatment anaemia on survival in individuals with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co‐infection is not known. Moreover, HCV treatment is offered less frequently to individuals with anaemia, due to haematological side effects of the treatment regimen. This study aimed to determine the effect of HCV treatment on survival among HCV/HIV co‐infected individuals with pretreatment anaemia using the Electronically Retrieved Cohort of HCV‐Infected Veterans (ERCHIVES). Individuals with HCV/HIV co‐infection were included in current analyses. Participants were considered treated if they were prescribed ≥4 weeks of HCV treatment. All‐cause mortality data were obtained using record linkage. Survival analyses were performed using Cox proportional hazard models. Among 5000 HCV/HIV co‐infected individuals, 1671 (33.4%) had pretreatment anaemia. In a follow‐up period of up to 7 years (19 500 person‐years), individuals with anaemia had significantly higher mortality rate compared with those without anaemia [144.2 (95% CI: 134.5–154.7) vs 47.5 (44.0–51.2) per 1000 person‐years, respectively]. Among individuals with anaemia, HCV treatment was associated with significantly lower mortality rate [66.6 (44.3–100.2) vs 149.6 (139.2–160.5) per 1000 person‐years, for treated vs untreated, respectively]. Treatment remained associated with substantial survival benefit after taking into account the effect of multiple comorbidities (hazards ratio: 0.34, 95% CI: 0.21–0.62). These data suggest that HCV/HIV co‐infected individuals with pretreatment anaemia have significantly higher mortality compared with those without anaemia. HCV treatment is associated with substantial survival benefit in this group. Additional studies are needed to determine strategies to improve HCV treatment rates among this group.