High-throughput sequencing of the B-cell receptor in African Burkitt lymphoma reveals clues to pathogenesis.

Burkitt lymphoma (BL), the most common pediatric cancer in sub-Saharan Africa, is a malignancy of antigen-experienced B lymphocytes. High-throughput sequencing (HTS) of the immunoglobulin heavy (IGH) and light chain (IGK/IGL) loci was performed on genomic DNA from 51 primary BL tumors: 19 from Uganda and 32 from Ghana. Reverse transcription polymerase chain reaction analysis and tumor RNA sequencing (RNAseq) was performed on the Ugandan tumors to confirm and extend the findings from the HTS of tumor DNA. Clonal IGH and IGK/IGL rearrangements were identified in 41 and 46 tumors, respectively. Evidence for rearrangement of the second IGH allele was observed in only 6 of 41 tumor samples with a clonal IGH rearrangement, suggesting that the normal process of biallelic IGHD to IGHJ diversity-joining (DJ) rearrangement is often disrupted in BL progenitor cells. Most tumors, including those with a sole dominant, nonexpressed DJ rearrangement, contained many IGH and IGK/IGL sequences that differed from the dominant rearrangement by < 10 nucleotides, suggesting that the target of ongoing mutagenesis of these loci in BL tumor cells is not limited to expressed alleles. IGHV usage in both BL tumor cohorts revealed enrichment for IGHV genes that are infrequently used in memory B cells from healthy subjects. Analysis of publicly available DNA sequencing and RNAseq data revealed that these same IGHV genes were overrepresented in dominant tumor-associated IGH rearrangements in several independent BL tumor cohorts. These data suggest that BL derives from an abnormal B-cell progenitor and that aberrant mutational processes are active on the immunoglobulin loci in BL cells.

Linkage to HIV care and antiretroviral therapy by HIV testing service type in Central Mozambique: a retrospective cohort study.

BACKGROUND: Access to antiretroviral therapy (ART) has increased dramatically in resource-limited settings since its introduction a decade ago. However, ART coverage remains low in countries with the highest disease burden, which may be partially explained by poor testing to care linkages. HIV testing service may impact early attrition in the HIV treatment cascade. METHODS: A retrospective cohort study was conducted in 18 clinics in central Mozambique using routine patient data and monthly reports. Patients referred from voluntary counseling and testing (VCT) were compared with those referred from prevention of mother-to-child transmission (PMTCT) for 3 outcomes: (1) enrollment at an HIV clinic ≤30 days after testing HIV positive, (2) CD4 test ≤30 days after enrollment, and (3) ART initiation ≤90 days after first CD4 test. RESULTS: Patient retention in the HIV care system dropped at each step from HIV testing to ART initiation. Enrollment in HIV care was not significantly different between PMTCT and VCT [risk ratio (RR) = 0.84, 0.72 < RR < 1.02]. Women tested in PMTCT were less likely to have a CD4 test ≤30 days after enrollment when adjusting for age, education level, and marital status (adjusted RR = 0.84, 0.70 < RR < 1.00), and were less likely to initiate ART ≤90 days after their first CD4 test when adjusting for age, education, and marital status (adjusted RR = 0.56, 0.44 < RR < 0.71). CONCLUSIONS: Poor linkages between HIV testing and care hamper efforts to improve coverage for HIV care and treatment services. Increased loss to follow-up among women diagnosed in PMTCT relative to VCT is worrisome and merits further qualitative research and programmatic attention.

Enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay.

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 µl of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (version 8/2010). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).