Pascale Willem

Changing pattern of lymphoma subgroups at a tertiary academic complex in a high-prevalence HIV setting: a South African perspective.

Background:HIV infection has been associated with an increased risk of non-Hodgkin lymphoma, particularly in the first world. Despite the high burden of HIV infection in sub-Saharan regions, published data on HIV and malignancies are sparse from these areas. Materials and Methods:We recently published data on lymphomas diagnosed from January 2004 to December 2006, at a single center in Johannesburg, to serve as a baseline for long-term comparison during the period of highly active antiretroviral therapy rollout. We report a retrospective analysis of the follow-up data collected from January 2007 to December 2009 at the Johannesburg academic hospital complex (Gauteng, South Africa). Results:There were 2225 new diagnoses of lymphoproliferative disorders made during 2007-2009 as compared with 1897 cases diagnosed during 2004-2006. A significant increase in both high-grade B-cell lymphomas and Hodgkin lymphoma was documented during 2007-2009. This was associated with a statistically significant increase in HIV prevalence in those tested (from 44.3% in 2004-2006 to 62.0% in 2007-2009). HIV-positive patients presented at a statistically significantly younger median age and showed a relative overrepresentation of females when compared with HIV-negative patients. HIV-positive patients were diagnosed at later stages of HIV infection when compared with patients in the first world. Conclusions:The pattern of lymphoma subtypes and the demographics of the patients diagnosed have altered in association with significantly increased HIV prevalence. These changes have important public health implications. In particular, scale-up and earlier access to highly active antiretroviral therapy is essential with continued monitoring as access to therapy improves.

Translocation t(1;7) revisited: Report of three further cases and review☆

Three white patients, two with myelofibrosis and one with refractory anemia, presented with a t(1;7). The clinical and cytogenetic findings are discussed in the context of 45 cases already published. Rather than the specific association of t(1;7) with a particular hematologic disorder, a review of the literature strongly suggests correlation with therapeutic or environmental exposure to toxic substances. The proposed mechanisms to explain the origin of t(1;7) are briefly reviewed.

Dominant genetic aberrations and coexistent EBV infection in HIV-related oral plasmablastic lymphomas

We present common cytogenetic features in the largest cohort of plasmablastic lymphoma (PBL) of the oral cavity published to date. This cohort included 45 patients, 32 of whom had a known HIV status, of which 31 were HIV positive. Ninety eight per cent of all PBL cases were known to be EBV positive. In line with previous studies, we found that rearrangements of the MYC gene was the most common genetic abnormality seen in 60% of cases with the immunoglobulin heavy chain (IGH) locus as a partner in 51% of cases. Additional complex genetic aberrations were frequent, in particular, an increased copy number of the CCND1 gene was seen in 41% of cases with true amplification of CCND1 in 15% of cases. Aneuploidy was also observed for the BCL6 gene in 28% of cases. Interestingly, rearrangements of both IGH genes were detected in 16% of cases with t(14;18) and t(11;14) respectively involved in conjunction with a t(8;14) in two cases. These bi-allelic IGH rearrangements have not been described before in oral PBL. Our results reinforce the notion that EBV infection and MYC rearrangements are important events in the pathogenesis of oral PBL. The genetic diversity and complexity observed in these cases, underlines the importance to genetically characterise PBL patients at presentation as this may inform the choice of more effective treatment modalities.

Chromosomal radiosensitivity of HIV positive individuals

Purpose: Radiosensitivity in relation to the human immunodeficiency virus (HIV) status is important in South Africa as the prevalence of HIV infections is high. In this study the in vitro chromosomal radiosensitivity of HIV positive individuals was investigated and compared with that of HIV negative individuals. Materials and methods: Blood samples from 59 HIV positive and 39 HIV negative individuals were exposed in vitro to doses of 6MV X-rays ranging from 1–4 Gy. Chromosomal radiosensitivity was assessed with the micronucleus assay. Micronuclei are a measure of chromosomal damage and were quantified in at least 500 binucleated lymphoblasts (BN) per sample. Un-irradiated control samples from each donor were also analysed. Results: In 47% of HIV positive individuals difficulties with cell stimulation by adding phytohaemagglutinin (PHA) to blood cultures were noticed which resulted in insufficient yield of BN for microscopic analysis. Micronuclei frequencies were consistently higher in irradiated lymphocytes obtained from HIV positive individuals compared to that observed in cells from HIV negative donors. Data for both groups were fitted to the linear-quadratic equation Y = αD + βD2 where Y is the number of micronuclei in 500 binucleated cells and D is the dose in Gy. The fitted parameters for respectively HIV positive and HIV negative lymphocytes are α = 80.17 Gy−1, β = 14 Gy−2 and α = 54.5 Gy−1, β = 16.2 Gy−2. The confidence ellipses of these parameters are separated indicating that the increase in radiosensitivity is statistically significant. Conclusion: T-lymphocytes of HIV infected individuals were considerably more sensitive to X-rays compared to that of HIV negative donors. This may have implications for normal tissue tolerance during radiotherapy as well as for the radiological health of radiation workers.

A semi-automated micronucleus-centromere assay to assess low-dose radiation exposure in human lymphocytes

Abstract Purpose: The in vitro micronucleus (MN) assay is a reliable method to assess radiation-induced chromosomal damage in human peripheral blood lymphocytes. It is used to evaluate in vivo radiation over-exposure and to assess in vitro chromosomal radiosensitivity. A limitation of the MN assay is the relatively high and variable spontaneous MN frequency that restricts low-dose estimation to doses of about 0.3 gray (Gy). As radiation-induced MN mainly contain acentric fragments and spontaneous MN originate from lagging chromosomes, both MN types can be distinguished from each other by using fluorescence in situ hybridisation (FISH) with a pan-centromeric probe. The aim of this study was to investigate if the sensitivity, reliability and processing time of the MN assay can be enhanced by combining the automated MN assay with pan-centromere scoring. Materials and methods: Blood samples from 10 healthy donors were irradiated in vitro with low doses of gamma-rays. Dose response curves were determined for fully-automated and semi-automated MN scoring and semi-automated scoring of centromere negative MN (MNCM−). Results: A good correlation was obtained between fully-automated and semi-automated MN scoring (r2 = 0.9973) and between fully automated MN scoring and semi-automated scoring of MNCM− (r2 = 0.998). With the Wilcoxon test, a significant p value was obtained between 0 and 0.2 Gy for the fully-automated MN analysis, between 0 and 0.1 Gy for semi-automated MN analysis and between 0 and 0.05 Gy for semi-automated scoring of MNCM−. Conclusion: The semi-automated micronucleus-centromere assay combines high-speed MN analysis with a more accurate assessment in the low-dose range which makes it of special interest for large-scale radiation applications.

Genomic imbalances in esophageal carcinoma cell lines involve Wnt pathway genes

AIM To identify molecular markers shared across South African esophageal squamous cell carcinoma (ESCC) cell lines using cytogenetics, fluorescence in situ hybridization (FISH) and single nucleotide polymorphism (SNP) array copy number analysis. METHODS We used conventional cytogenetics, FISH, and multicolor FISH to characterize the chromosomal rearrangements of five ESCC cell lines established in South Africa. The whole genome copy number profile was established from 250K SNP arrays, and data was analyzed with the CNAT 4.0 and GISTIC software. RESULTS We detected common translocation breakpoints involving chromosomes 1p11-12 and 3p11.2, the latter correlated with the deletion, or interruption of the EPHA3 gene. The most significant amplifications involved the following chromosomal regions and genes: 11q13.3 (CCND1, FGF3, FGF4, FGF19, MYEOV), 8q24.21(C-MYC, FAM84B), 11q22.1-q22.3 (BIRC2, BIRC3), 5p15.2 (CTNND2), 3q11.2-q12.2 (MINA) and 18p11.32 (TYMS, YES1). The significant deletions included 1p31.2-p31.1 (CTH, GADD45α, DIRAS3), 2q22.1 (LRP1B), 3p12.1-p14.2 (FHIT), 4q22.1-q32.1 (CASP6, SMAD1), 8p23.2-q11.1 (BNIP3L) and 18q21.1-q21.2 (SMAD4, DCC). The 3p11.2 translocation breakpoint was shared across four cell lines, supporting a role for genes involved at this site, in particular, the EPHA3 gene which has previously been reported to be deleted in ESCC. CONCLUSION The finding that a significant number of genes that were amplified (FGF3, FGF4, FGF19, CCND1 and C-MYC) or deleted (SFRP2 gene) are involved in the Wnt and fibroblast growth factor signaling pathways, suggests that these pathways may be activated in these cell lines.

A novel approach to simultaneously scan genes at fragile sites

BackgroundFragile sites are regions of the genome sensitive to replication stress and to exposure to environmental carcinogens. The two most commonly expressed fragile sites FRA3B and FRA16D host the histidine triad (FHIT) and WW domain containing oxidoreductase (WWOX) genes respectively. There is growing evidence that both genes contribute to cancer development and they are frequently altered by allelic and homozygous deletions in a variety of tumors. Their status is linked to prognosis in several malignancies and they are thought to be involved in early tumorigenesis.The loci for FHIT and WWOX both span over a megabase but the genes encode for small transcripts. Thus the screening of intragenic deletion can be difficult and has relied on loss of heterozygosity LOH assays, or genomic arrays.MethodsMultiplex ligation dependent probe amplification MLPA, allows for the detection of deletions/duplications and relative quantification of up to 40 specific probes in a single assay. A FHIT/WWOX MLPA assay was designed, applied and validated in five esophageal squamous cell carcinoma ESCC, cell lines established in South Africa where this cancer is of high prevalence. Sixteen probes covered all FHIT exons and 7 probes covered WWOX.ResultsBoth homozygous and hemizygous deletions were detected in FHIT, in four of the cell lines with a preferential deletion of exons 5 and 4. Chromosome 3 short arm was present in normal copy number indicating that deletions were site specific. In contrast WWOX was not altered in any cell lines. RT-PCR expression pattern paralleled the pattern of deletions. Ten primary ESCC tumor specimens were subsequently screened with this assay. FHIT exon deletions were found in four of them.ConclusionThis method offers an alternative to loss of heterozygosity studies. Simultaneous scanning of FHIT and WWOX exons in the context of early tumorigenesis and tumor progression, may help clarify the mechanistic events related to cancer development which are not revealed by imuno histochemistry assays. The presence of site specific deletions of FHIT in these cell lines and primary tumors support its possible role in South African ESCC and justifies a wider screening.

Plasmablastic lymphoma versus diffuse large B cell lymphoma with plasmablastic differentiation: proposal for a novel diagnostic scoring system

High-grade non-Hodgkin’s lymphomas with plasmablastic differentiation are frequently encountered in HIV-positive patients. Differentiating between diffuse large B cell lymphoma (DLBCL) with plasmablastic differentiation and true cases of plasmablastic lymphoma (PBL) is sometimes challenging, particularly as a substantial overlap in immunphenotype exists between late-stage B cell neoplasms and PBL. This study sought to develop an immunohistochemical panel to more reliably distinguishing between PBL and DLBCL with plasmablastic differentiation. Thirty-nine CD20-negative, ALK-negative, HHV8-negative non-Hodgkin’s lymphomas with plasmablastic differentiation defined by their morphological features, high proliferation index and positivity for MUM1/IRF4 and PRDM/Blimp1 protein expression were compared regarding their protein expression profiles, viral status and c-MYC-gene aberrations. These lymphomas were subsequently divided in two groups utilising CD10 and Pax5. Tumours without reactivity for either of these markers exhibited higher expression of CD138 and CD117 frequently used as the plasma cell (PC) markers, whilst tumours with reactivity for one or both markers showed a significantly higher expression of CD38 and MYC-gene aberrations. A novel diagnostic scoring system which includes the immunohistochemical expression of CD10 and Pax5 is proposed to differentiate between DLBCL with plasmablastic differentiation and true cases of PBL.

Hepatosplenic T-cell lymphoma: A case series

Hepatosplenic T-cell lymphoma (HSTCL) is a rare type of Non-Hodgkin Lymphoma (NHL), grouped under the mature or peripheral T-cell lymphomas. It is characterised by extranodal infiltration and proliferation of malignant T-cells within the sinusoids of the liver, sinuses and red pulp of the spleen, and the bone marrow. The tumour cells express CD2 and CD3, but are CD4, CD5 and CD8 negative and express a clonally restricted gamma-delta (or less commonly alpha-beta) T-cell receptor. The disease has an aggressive clinical course associated with a poor prognosis. We highlight and report three patients from South Africa with HSTCL, all of whom had hepatosplenomegaly and cytopaenias, and despite being HIV seronegative and immunocompetent, had a poor outcome, with a mean survival of 7.5 months in the two evaluable patients. This rare entity has not previously been reported from South Africa and as yet needs to be adequately characterised in a population where lymphoma is the most common haematological malignancy in adults, and where approximately two thirds of the adult lymphoma population are HIV seropositive.

Derivative (11)t(7;11)(q11;q24) in a child with acute megakaryoblastic leukemia

We document the clinical, morphologic, and cytogenetic findings in a 2 1/2-year-old patient with acute megakaryoblastic leukemia. Cytogenetic analysis of peripheral blood leukemic cells revealed the presence of a der(11)t(7;11)(q11;q24). Chromosome 7 involvement is observed in nearly half of all cases of megakaryoblastic leukemias, however, the type of abnormality differs widely from one case to another. The der(11)t(7;11)(q11;q24) described here is the first reported to our knowledge.