Associations between IgG reactivity to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens and Burkitt lymphoma in Ghana and Uganda case-control studies.

BACKGROUND: Endemic Burkitt lymphoma (eBL) is an aggressive childhood B-cell lymphoma linked to Plasmodium falciparum (Pf) malaria in sub-Saharan Africa. We investigated antibody reactivity to several human receptor-binding domains of the Pf erythrocyte membrane protein 1 (PfEMP1) that play a key role in malaria pathogenesis and are targets of acquired immunity to malaria. METHODS: Serum/plasma IgG antibody reactivity was measured to 22 Pf antigens, including 18 to PfEMP1 CIDR domains between cases and controls from two populations (149 eBL cases and 150 controls from Ghana and 194 eBL cases and 600 controls from Uganda). Adjusted odds ratios (aORs) for case-control associations were estimated by logistic regression. FINDINGS: There was stronger reactivity to the severe malaria associated CIDRα1 domains than other CIDR domains both in cases and controls. eBL cases reacted to fewer antigens than controls (Ghana: p = 0·001; Uganda: p = 0·03), with statistically significant lower ORs associated with reactivity to 13+ antigens in Ghana (aOR 0·39, 95% CI 0·24-0·63; pheterogeneity = 0·00011) and Uganda (aOR 0·60, 95% CI 0.41-0·88; pheterogeneity = 0·008). eBL was inversely associated with reactivity, coded as quartiles, to group A variant CIDRδ1 (ptrend = 0·035) in Ghana and group B CD36-binding variants CIDRα2·2 (ptrend = 0·006) and CIDRα2·4 (ptrend = 0·033) in Uganda, and positively associated with reactivity to SERA5 in Ghana (ptrend = 0·017) and Uganda (ptrend = 0·007) and group A CIDRα1·5 variant in Uganda only (ptrend = 0·034). INTERPRETATION: eBL cases reacted to fewer antigens than controls using samples from two populations, Ghana and Uganda. Attenuated humoral immunity to Pf EMP1 may contribute to susceptibility to low-grade malaria and eBL risk. FUNDING: Intramural Research Program, National Cancer Institute and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services.

The changing clinical pattern of endemic Burkitt lymphoma in Western Africa: Experience from a tertiary center in Ghana.

BACKGROUND: Burkitt lymphoma (BL) is the most common childhood cancer in Ghana, where the endemic variant is the predominant subtype and historically presents as a highly chemo-sensitive jaw tumor. This study aimed to update the current epidemiological characteristics of childhood BL in our institution. PROCEDURE: Patient data for all children diagnosed with BL and seen at Korle Bu Teaching Hospital between January 2007 and December 2012 were retrospectively analyzed. RESULTS: BL was diagnosed in 173 children (<13 years) during the study period, with the abdomen as the most common tumor site (46%) followed by the jaw (31%). Abdominal tumors were associated with advanced/disseminated disease (P = 0.002), and were more likely to occur in females irrespective of tumor stage (relative risk = 1.56 [95% CI; 1.1-12.3]). Twenty-five percent (43/173) of the study cohort died and mortality was influenced by increasing age (P = 0.02) and advanced disease (P = 0.03). Treatment delay was experienced by nine in ten patients primarily due to familial financial constraint (75%). Treatment abandonment was observed as a first event in 94% of patients and two thirds of children in the study were eventually lost to follow-up. CONCLUSION: The predominance of primary abdominal tumors in our study cohort may indicate a changing epidemiological pattern of BL in Ghana. High rates of treatment delay and abandonment were evident and are likely to be contributing factors to the poor childhood cancer survival outcomes seen in resource-limited countries in Africa.

Frequency of EBV LMP-1 Promoter and Coding Variations in Burkitt Lymphoma Samples in Africa and South America and Peripheral Blood in Uganda.

Epstein-Barr virus (EBV) is linked to several cancers, including endemic Burkitt lymphoma (eBL), but causal variants are unknown. We recently reported novel sequence variants in the LMP-1 gene and promoter in EBV genomes sequenced from 13 of 14 BL biopsies. Alignments of the novel sequence variants for 114 published EBV genomes, including 27 from BL cases, revealed four LMP-1 variant patterns, designated A to D. Pattern A variant was found in 48% of BL EBV genomes. Here, we used PCR-Sanger sequencing to evaluate 50 additional BL biopsies from Ghana, Brazil, and Argentina, and peripheral blood samples from 113 eBL cases and 115 controls in Uganda. Pattern A was found in 60.9% of 64 BL biopsies evaluated. Compared to PCR-negative subjects in Uganda, detection of Pattern A in peripheral blood was associated with eBL case status (odds ratio [OR] 31.7, 95% confidence interval: 6.8⁻149), controlling for relevant confounders. Variant Pattern A and Pattern D were associated with eBL case status, but with lower ORs (9.7 and 13.6, respectively). Our results support the hypothesis that EBV LMP-1 Pattern A may be associated with eBL, but it is not the sole associated variant. Further research is needed to replicate and elucidate our findings.

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.

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration.

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.

Epstein-Barr virus from Burkitt Lymphoma biopsies from Africa and South America share novel LMP-1 promoter and gene variations.

Epstein Barr virus (EBV) sequence variation is thought to contribute to Burkitt lymphoma (BL), but lack of data from primary BL tumors hampers efforts to test this hypothesis. We directly sequenced EBV from 12 BL biopsies from Ghana, Brazil, and Argentina, aligned the obtained reads to the wild-type (WT) EBV reference sequence, and compared them with 100 published EBV genomes from normal and diseased people from around the world. The 12 BL EBVs were Type 1. Eleven clustered close to each other and to EBV from Raji BL cell line, but away from 12 EBVs reported from other BL-derived cell lines and away from EBV from NPC and healthy people from Asia. We discovered 23 shared novel nucleotide-base changes in the latent membrane protein (LMP)-1 promoter and gene (associated with 9 novel amino acid changes in the LMP-1 protein) of the 11 BL EBVs. Alignment of this region for the 112 EBV genomes revealed four distinct patterns, tentatively termed patterns A to D. The distribution of BL EBVs was 48%, 8%, 24% and 20% for patterns A to D, respectively; the NPC EBV's were Pattern B, and EBV-WT was pattern D. Further work is needed to investigate the association between EBV LMP-1 patterns with BL.

Endemic Burkitt lymphoma is associated with strength and diversity of Plasmodium falciparum malaria stage-specific antigen antibody response.

Endemic Burkitt lymphoma (eBL) is linked to Plasmodium falciparum (Pf) infection geographically, but evidence from individual-level studies is limited. We investigated this issue among 354 childhood eBL cases and 384 age-, sex-, and location-matched controls enrolled in Ghana from 1965 to 1994. Immunoglobulin G1 (IgG1) and immunoglobulin G3 (IgG3) antibodies to antigens diagnostic of recent infection Pf histidine-rich protein-II (HRP-II) and 6NANP, Pf-vaccine candidates SE36 and 42-kDa region of the 3D7 Pf merozoite surface protein-1 (MSP-1), and tetanus toxoid were measured by indirect enzyme-linked immunoassay. Odds ratios (ORs) and 95% confidence intervals (CIs) for association with eBL were estimated using unconditional logistic regression. After adjustments, eBL was positively associated with HRP-IIIgG3 seropositivity (adjusted OR: 1.60; 95% CI 1.08-2.36) and inversely associated with SE36IgG1 seropositivity (adjusted OR: 0.37; 95% CI 0.21-0.64) and with tetanus toxoidIgG3 levels equal or higher than the mean (adjusted OR: 0.46; 95% CI 0.32-0.66). Anti-MSP-1IgG3 and anti-6NANPIgG3 were indeterminate. eBL risk was potentially 21 times higher (95% CI 5.8-74) in HRP-IIIgG3-seropositive and SE36IgG1-seronegative responders compared with HRP-IIIgG3-seronegative and SE36IgG1-seropositive responders. Our results suggest that recent malaria may be associated with risk of eBL but long-term infection may be protective.

Antibodies reactive to Plasmodium falciparum serine repeat antigen in children with Burkitt lymphoma from Ghana.

The role of protective immunity to Plasmodium falciparum (Pf) malaria in Burkitt lymphoma (BL) is unknown. We investigated the association between BL and antibodies reactive to SE36 antigen, a recombinant protein based on P. falciparum serine repeat antigen 5 gene, targeted by protective malaria immune responses. Cases were children (0-14 years) enrolled at the Korle-Bu Teaching Hospital, Accra, Ghana, during 1965-1994 with BL confirmed by histology or cytology (92% of cases). Controls were apparently healthy children enrolled contemporaneous to the cases from the nearest neighbor house to the case house and were age,- sex-frequency-matched to the cases. Anti-SE36 IgG antibodies were measured using enzyme-linked absorbent immunoassays (ELISAs). SE36 titers were estimated by extrapolating ELISA optical density readings to a standard fitting curve. Anti-SE36 titers were log-transformed for analysis. Odds ratios (ORs) and two-sided 95% confidence intervals (95% CIs) were estimated using unconditional logistic regression. The mean log endpoint dilution titers were 0.63 logs lower in cases than in controls (8.26 [SD 1.68] vs. 8.89 [SD 1.75], Student's t-test, p = 0.019). Lower titers were observed in cases than controls aged 0-4 years (p = 0.05) and in those aged 5-14 years (p = 0.06). Low and medium tertiles of anti-SE36 IgG antibodies were associated with increased OR for BL ([OR 1.67, 95% CI 1.21-2.31] and [OR 1.33, 95% CI 0.96-1.86], respectively, p(trend) = 0.002) in analyses adjusting for age, sex, calendar period and test plate. Our findings suggest that compared to similarly aged children enrolled from the same community, children with BL in Ghana have lower antibodies to SE36 antigen.

African Burkitt lymphoma: age-specific risk and correlations with malaria biomarkers.

African Burkitt lymphoma is an aggressive B-cell, non-Hodgkin lymphoma linked to Plasmodium falciparum malaria. Malaria biomarkers related to onset of African Burkitt lymphoma are unknown. We correlated age-specific patterns of 2,602 cases of African Burkitt lymphoma (60% male, mean ± SD age = 7.1 ± 2.9 years) from Uganda, Ghana, and Tanzania with malaria biomarkers published from these countries. Age-specific patterns of this disease and mean multiplicity of P. falciparum malaria parasites, defined as the average number of distinct genotypes per positive blood sample based on the merozoite surface protein-2 assessed by polymerase chain reaction, were correlated and both peaked between 5 and 9 years. This pattern, which was strong and consistent across regions, contrasted parasite prevalence, which peaked at 2 years and decreased slightly, and geometric mean parasite density, which peaked between 2 and 3 years and decreased sharply. Our findings suggest that concurrent infection with multiple malaria genotypes may be related to onset of African Burkitt lymphoma.

Osteomyelitis of the frontal bone.

SummaryOsteomyelitis of the skull is a rare clinical presentation. It usually occurs as a complication of trauma or sinusitis. Its complications can be life threatening though the initial symptoms and signs are subtle. Early diagnosis and appropriate management to prevent CNS complications reduce morbidity and mortality significantly. Intracranial complications of sinusitis, focal infections and meningitis remain a great challenge. Mortality from complications is 20-40%. The prevalence of skull osteomyelitis is about 1.5% of all osteomyelitis.