Human Papillomavirus at Multiple Sites Associated with Anal Squamous Intraepithelial Lesions in HIV-Seropositive Individuals.

OBJECTIVE: HIV-Seropositive patients have higher risk of HPV infection even on anti-retroviral therapy. Infection with high-risk HPV genotypes can cause dysplasia leading to cancer. This study assessed HPV at different anatomical sites in HIV-seropositive individuals and factors associated with anal squamous intraepithelial lesions (ASIL). METHODS: Specimens were obtained from multiple anatomical sites for each participant in conjunction with routine screening for anal dysplasia. Female specimens included cervical and anal cytologies and oral wash. Male specimens included anal cytologies, oral wash, and exfoliated cells from penile head, penile shaft, scrotum, and from uncircumcised subjects, inner foreskin. Demographic and clinical characteristics were recorded. Following DNA extraction, HIV DNA copy was assessed by qPCR; HPV was genotyped. Statistical analyses included calculation of odds ratios (OR) and 95% confidence intervals (CI), t-tests or Mann-Whitney tests. RESULTS: Males were more likely to have ASIL: 29/50 (58%) compared to 1/11 females (9%) (OR=13.81, 95% CI: 1.64-116.32). HPV 6 or 11 in anal specimens was significantly associated with ASIL (OR= 6.29, 95% CI: 1.49-26.44). Number of HPV genotypes in anal specimens was also significant: ASIL+ (3.4 ± 3.1) versus ASIL- (1.6 ± 3.1) (p=0.009). Among 44 males, HPV was detected from at least one anatomical site for 33 participants (75%): 27 anus (61%), 19 oral wash (44%), 17 penile shaft (39%), 11 scrotum (26%), 10 penile head (23%), 0 foreskin. Detection of HPV in penile shaft specimens was significantly associated with ASIL (OR=6.79, 95% CI: 1.57-29.36) as was number of HPV genotypes in penile shaft specimens: ASIL+ (2.4 ± 4.0) versus ASIL- (0.6 ± 1.7) (p=0.025). Only 1/11 females had ASIL; only 1/11 females had cervical dysplasia: OR was not estimable due to small numbers. CONCLUSIONS: Males were more prone to ASIL than females. HPV at anal as well as non-anal sites may be indicative of ASIL.

HIV DNA reservoir increases risk for cognitive disorders in cART-naïve 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.

Effective use of small-interfering RNA to characterize residual B-cell non-Hodgkin lymphoma cells following chemotherapy.

BACKGROUND: Children diagnosed with non-Hodgkin lymphoma (NHL) respond well to therapy resulting in relatively good prognosis. The exceptions are those who continue to have minimal residual disease (MRD). MRD NHL cells have been characterized as having increased mitochondrial DNA (mtDNA) copy numbers with increased expression of citrate synthase and isocitrate dehydrogenase. A proof-of-concept was designed to use small-interfering RNA (siRNA) as a tool to elucidate the relationship between citrate synthase and isocitrate dehydrogenase with cancer cell integrity. METHODS: mtDNA copy number and lactate dehydrogenase activities were assessed in chemotherapy-exposed residual NHL cells after introduction of siRNA against citrate synthase and/or isocitrate dehydrogenase expression. RESULTS: There was a significant decrease in lactate production in cells transfected with citrate synthase siRNA (p=0.02). Citrate synthase-silenced cells had decreased mtDNA copy numbers (p=0.03) compared to isocitrate dehydrogenase-silenced cells or combined citrate synthase- and isocitrate dehydrogenase-silenced cells. CONCLUSION: Inhibition of citrate synthase expression in siRNA-treated NHL cells resulted in decreased mtDNA copy numbers and lactate dehydrogenase expression. This observation needs further validation to determine the role of citrate synthase in mtDNA integrity and if citrate synthase siRNA could be a potential therapeutic modality in eradicating residual B-NHL cells.

Minimal disease assessment in the treatment of children and adolescents with intermediate-risk (Stage III/IV) B-cell non-Hodgkin lymphoma: a children's oncology group report.

Children/adolescents with mature B-cell non-Hodgkin lymphoma (B-NHL) have an excellent prognosis but relapses still occur. While chromosomal aberrations and/or clonal immunoglobulin (Ig) gene rearrangements may indicate risk of failure, a more universal approach was developed to detect minimal disease (MD). Children/adolescents with intermediate-risk B-NHL were treated with French-British-American/Lymphome Malins de Burkitt 96 (FAB/LMB96) B4 modified chemotherapy and rituximab. Specimens from diagnosis, end of induction (EOI), and end of therapy (EOT) were assayed for MD. Initial specimens were screened for IGHV family usage with primer pools followed by individual primers to identify MD. Thirty-two diagnostic/staging specimens screened positive with primer pools and unique IGHV family primers were identified. Two patients relapsed; first relapse (4 months from diagnosis) was MD-positive at EOI, the second (36 months from diagnosis) was MD-positive at EOT. At EOI, recurrent rates were similar between the MRD-positive and MRD-negative patients (P = 0·40). At EOT, only 13/32 patients had MRD data available with one relapse in the MRD-positive group and no recurrences in the MRD-negative group (P = 0·077). The study demonstrated molecular-disseminated disease in which IgIGHV primer pools could be used to assess MD. This feasibility study supports future investigations to assess the validity and significance of MD screening in a larger cohort of patients with intermediate-risk mature B-NHL.

Mitochondrial DNA in residual leukemia cells in cerebrospinal fluid in children with acute lymphoblastic leukemia.

UNLABELLED: This feasibility study was designed to assess the ability to measure mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) cells that contributed to minimal disease/persistent or residual disease (MD/PRD) from children with acute lymphoblastic leukemia (ALL). Increase in mtDNA copies in cancer cells has been suggested to play a role in MD/PRD. CSF as well as blood specimens from 6 children were assayed for MD/PRD and mtDNA copy numbers by quantitative real-time polymerase chain reaction. Of 7 MD/PRD-positive specimens, 6 had increased mtDNA copy numbers; while 11 MD/PRD-negative specimens had no increase in mtDNA copy numbers, p < 0.003. This is the first proof-of-concept study to measure mtDNA copy numbers in MD/PRD-positive CSF specimens from children with ALL. Increase of mtDNA copy numbers in MD/PRD childhood ALL cells and its significance as a mechanism for recurrence requires further investigation. KEYWORDS: Minimal residual disease; Acute lymphoblastic leukemia; Central nervous system; Cerebrospinal fluid; Mitochondria.

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 (IgV(H)) genes, IgV(H) primer pools from IgV(H1)-IgV(H7) 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 IgV(H) 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 IgV(H) 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.

Chemotoxicity recovery of mitochondria in non-Hodgkin lymphoma resulting in minimal residual disease.

BACKGROUND: The mechanisms responsible for resistant disease or recurrence of non-Hodgkin lymphoma (NHL) in children cover a wide spectrum from drug resistance to genetic mutations. A unique mechanism suggesting the role of mitochondria as the key energy source is studied following a clinical observation where pediatric Burkitt lymphoma (BL) specimens from patients on therapy were found to have increased copies of mitochondria DNA (mtDNA) in specimens which were shown to be positive for minimal residual disease and/or persistent disease (MRD/PD). This study hypothesized that the mitochondria play an important role in a cell's recovery from toxicity via a compensatory increase in mtDNA. PROCEDURE: BL specimens with MRD/PD were assayed for mtDNA. An in vitro model was then designed using Ramos cell lines by exposing the lymphoma cells to varying concentrations of doxorubicin and vincristine for 1 hr; and allowing for recovery in culture over 7 days. DNA was extracted from aliquots over several days to determine mtDNA copy numbers by real-time polymerase chain reaction (PCR). RESULTS: Increased mtDNA copy numbers were found in clinical specimens with MRD/PD as well as in recovering Ramos cells from chemotoxicity. CONCLUSIONS: The recovering lymphoma cells from the chemotoxic effects appeared to compensate by increasing mtDNA content, which may contribute to the clinical residual or resistant disease in some cases of childhood BL.