Screening for Residual Disease in Pediatric Burkitt Lymphoma Using Consensus Primer Pools

Assessing molecular persistent or minimal residual disease (PD/MRD) in childhood Burkitt lymphoma (BL) is challenging because access to original tumor is usually needed to design patient-specific primers (PSPs). Because BL is characterized by rearranged immunoglobulin heavy chain (IgVH) genes, IgVH primer pools from IgVH1–IgVH7 regions were tested to detect PD/MRD, thus eliminating the need for original tumor. The focus of the current study was to assess the feasibility of using IgVH primer pools to detect disease in clinical specimens. Fourteen children diagnosed with B-NHL had follow-up repository specimens available to assess PD/MRD. Of the 14 patients, 12 were PD/MRD negative after 2 months of therapy and remained in remission at the end of therapy; 2/14 patients were PD/MRD positive at 2-3 months and later relapsed. PSP-based assays from these 14 patients showed 100% concordance with the current assay. This feasibility study warrants further investigation to assess PD/MRD using IgVH primer pools, which could have clinical significance as a real-time assessment tool to monitor pediatric BL and possibly other B-cell non-Hodgkin lymphoma therapy

HIV DNA Reservoir Increases Risk for Cognitive Disorders in cART-Nave Patients

Objectives Cognitive impairment remains frequent in HIV, despite combination antiretroviral therapy (cART). Leading theories implicate peripheral monocyte HIV DNA reservoirs as a mechanism for spread of the virus to the brain. These reservoirs remain present despite cART. The objective of this study was to determine if the level of HIV DNA in CD14+ enriched monocytes predicted cognitive impairment and brain injury. Methods We enrolled 61 cART-naïve HIV-infected Thais in a prospective study and measured HIV DNA in CD14+ enriched monocyte samples in a blinded fashion. We determined HAND diagnoses by consensus panel and all participants underwent magnetic resonance spectroscopy (MRS) to measure markers of brain injury. Immune activation was measured via cytokines in cerebrospinal fluid (CSF). Results The mean (SD) age was 35 (6.9) years, CD4 T-lymphocyte count was 236 (139) and log10 plasma HIV RNA was 4.8 (0.73). Twenty-eight of 61 met HAND criteria. The log10 CD14+ HIV DNA was associated with HAND in unadjusted and adjusted models (p = 0.001). There was a 14.5 increased odds ratio for HAND per 1 log-value of HIV DNA (10-fold increase in copy number). Plasma CD14+ HIV DNA was associated with plasma and CSF neopterin (p = 0.023) and with MRS markers of neuronal injury (lower N-acetyl aspartate) and glial dysfunction (higher myoinositol) in multiple brain regions. Interpretation Reservoir burden of HIV DNA in monocyte-enriched (CD14+) peripheral blood cells increases risk for HAND in treatment-naïve HIV+ subjects and is directly associated with CSF immune activation and both brain injury and glial dysfunction by MRS

Determining the role of HIV-DNA in relation to activated monocytes in the development of HIV-associated neurocognitive disorders

Ph.D. University of Hawaii at Manoa 2010.Includes bibliographical references.There has been an increase in the prevalence of HIV-associated neurocognitive disorders (HAND) in spite of effective antiretroviral therapy (ART). It is believed that activated monocytes, CD14+/16+, are involved in the HAND pathogenesis through trafficking of HIV into the central nervous system (CNS). Once inside, CD14+/16+ cells initiate inflammation resulting in neuronal damage. Monocytes are also able to harbor HIV and serve as viral reservoirs, which are identified by presence of HIV DNA. These concepts are supported by findings in our lab where individuals with HAND have higher HIV DNA copy numbers in circulating CD14+/16+ cells compared to other cell subsets. The goal of this study is to characterize HIV DNA in these cells from other compartments through experiments on cerebrospinal fluid (CSF) and archival CNS tissue. The overall hypothesis is HIV DNA levels in cell subsets correspond to observations in the periphery. We also posit that insertional mutagenesis may be involved in neuropathogenesis through disruption of genes that may drive inflammation and neuronal apoptosis. Thus, HIV integration studies were done to determine genes that within and around the integration sites in the various CSF subsets. Results of HIV DNA levels in the CSF cell subsets were in contrast to levels reported in the periphery. Non-activated monocytes, CD14+/16-, contained the highest levels of HIV DNA regardless of neurocognitive status. This suggests that CSF viral reservoirs may be present that serve as sanctuaries for HIV evolution and compartmentalization. Conversely, the highest levels of HIV DNA were found in CD14+/16+ in HIV-encephalitis brain sections, which may reflect ongoing inflammation that might be occurring. Furthermore, common CSF integration sites were found within genes that were involved in apoptosis, macrophage behavior, and transcriptional regulation; all of which could promote HAND. The studies included the use of a unique flow cytometry technique that was shown to be comparable to our current quantitative real-time PCR assay to detect HIV DNA. The information obtained through these studies sets the stage for the focus of future studies and provide additional molecular tools to monitor the involvement of monocytes throughout the progression of HAND

Human Papillomavirus-16 DNA Quantitation Differentiates High-Grade Anal Neoplasia

Background: Due to their higher rates of anal dysplasia/cancer, human immunodeficiency virus (HIV)-positive individuals are recommended to undergo anal dysplasia screening, which consists of anal cytology (AC) and high resolution anoscopy (HRA) with anal biopsy (AB) after abnormal AC result. However, AC variability limits its usefulness. Our objective was to evaluate human papillomavirus (HPV)-16 DNA quantitation as part of the screening algorithm. Methods: HPV-16 was detected in AC specimens from 75 HIV-positive participants using quantitative real-time polymerase chain reaction. AB results were available from 18/44 patients who had abnormal AC. Statistical tests included Mann-Whitney U, Kruskal-Wallis, receiver operating characteristic (ROC) analysis and Kappa coefficient tests. Results: HPV-16 copy numbers differed significantly across AC (p = 0.001) and AB grades (p = 0.009). HPV-16 ≥ 65 copies/cell predicted high-grade AB (p = 0.04). Using this cut-off in comparison to AB, it had better specificity (1.00) than AC (0.75) and specificity (0.77) than qualitative HPV-16 detection (0.38). Also, the Kappa coefficient of the cut-off (κ = 0.649) was higher than AC (κ = 0.557) and qualitative HPV-16 detection (κ = 0.258) to AB. Conclusion: Higher HPV-16 copy numbers corresponded to higher AC and AB grades, suggesting the importance of HPV burden on disease stage. Furthermore, HPV-16 ≥ 65 copies/cell distinguished high-grade disease and demonstrated better sensitivity, specificity, and agreement with AB than AC or qualitative HPV-16 detection. These results support the potential use of HPV quantitation in conjunction with AC in anal dysplasia screening

Brief Report

Monocytes play a vital role in HIV-associated neurocognitive disorder (HAND), postulated to transport HIV into the brain and secrete pro-inflammatory cytokines. We analyzed cytokines released by cultured peripheral blood mononuclear cells enriched with the CD14 marker isolated from HIV-infected individuals with HAND and normal cognition (NC) in combination antiretroviral therapy naive and after 1 year on treatment. Interleukin-8 and monocyte chemoattractant protein-1 levels were higher in HAND compared with NC at baseline (P = 0.002 and P < 0.0001). These cytokines remained higher in HAND patients 1 year after combination antiretroviral therapy and were significant when NC patients who were initially HAND were excluded (P = 0.012 and P = 0.002). Both correlated with baseline CD14 peripheral blood mononuclear cell HIV DNA levels supporting the role of HIV DNA reservoir size and monocyte cytokines in HAND persistence

HIV DNA in CD14+ reservoirs is associated with regional brain atrophy in patients naive to combination antiretroviral therapy.

ObjectiveTo examine associations between regional brain volumes and HIV DNA in peripheral CD14 cells (monocytes) among HIV-infected individuals naive to combination antiretroviral therapy (cART).DesignA prospective study of HIV-infected Thai individuals who met Thai national criteria for cART initiation. Enrolment was stratified by HIV DNA in a blinded fashion.MethodsCD14 cells were isolated from peripheral mononuclear cells to high purity (median 91.4% monocytes by flow cytometry), and HIV DNA was quantified by multiplex real-time PCR. Baseline regional brain volumes obtained by T1-weighted 1.5-Tesla MRI were compared between HIV DNA groups using analysis of covariance (ANCOVA).ResultsWe studied 60 individuals with mean (SD) age of 34.7 (7.0) years, CD4 T-lymphocyte count of 232 (137) cells/μl and log10 plasma HIV RNA of 4.8 (0.73). Median (interquartile range, IQR) HIV DNA copy number per 10 CD14 cells was 54 (102). Using our previously determined optimal cut-point of 45 copies/10 cells for this cohort, a threshold value above which CD14 HIV DNA identified HIV-associated neurocognitive disorders (HANDs), we found that CD14 HIV DNA  ≥ 45 copies/10 cells was associated with reduced volumes of the nucleus accumbens (P=0.021), brainstem (P=0.033) and total gray matter (P=0.045) independently of age, CD4 cell count and intracranial volume.ConclusionHIV DNA burden in CD14 monocytes is directly linked to brain volumetric loss. Our findings implicate peripheral viral reservoirs in HIV-associated brain atrophy and support their involvement in the neuropathogenesis of HAND, underscoring the need for therapies that target these cells