The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) gui... more The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) guidelines are used for human epidermal growth factor receptor 2 (HER2) reporting in breast carcinoma. Cases that demonstrate weak to moderate complete membrane immunohistochemical staining in >10% of the tumor are scored as 2+ (equivocal). This study aimed to determine what proportion of HER2 immunohistochemistry (IHC) score = 2+ breast carcinomas were confirmed to be positive by HER2 fluorescent in situ hybridization (FISH). There were 241 HER2 IHC score = 2+ breast carcinomas included. Most (74.3%) carcinomas were estrogen and progesterone receptor-positive. Invasive breast carcinoma of no special type (89.2%) was the commonest histologic subtype. Most tumors were grade 2 (64.3%). As per the FISH report, at the time of diagnosis, 27 cases (11.2%) were HER2 FISH positive. All HER2 FISH equivocal cases and one FISH positive case assessed using the 2013 ASCO/CAP HER2 criteria were reclassified to HER2 FISH negative when the 2018 criteria were applied. There was a high level of agreement (κ = 0.979) between HER2 FISH results obtained using the 2013 and the 2018 criteria. This study provides insight into the frequency of HER2 FISH positivity (11.2%) among HER2 IHC score = 2+ breast carcinomas and the impact of modifications to the ASCO/CAP HER2 guidelines. Elimination of the HER2 FISH equivocal category by the 2018 guidelines has reduced the need for repeat testing and simplified clinical management. Reclassification of previous HER2 FISH positive to negative has resulted in some patients being ineligible for costly anti-HER therapy.
Human Immunodeficiency Virus (HIV) infection is known to be strongly associated with an increased... more Human Immunodeficiency Virus (HIV) infection is known to be strongly associated with an increased risk of high grade B-Cell Lymphoma (such as Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma). As South Africa has more than 5 million people living with HIV infection, this region sees a sharp increase in the diagnosis of high-grade lymphomas. HIV infection may associate with a number of atypical morphological features making it difficult to differentiate between classically described DLBCL and BL. As these two diseases have different prognosis and treatment, it is essential to accurately differentiate the type of disease and/or to diagnose the intermediate category of B-Cell Lymphoma, unclassifiable, with features intermediate between DLBCL and BL newly introduced in the 2008 WHO classification.
High-grade non-Hodgkin's lymphomas with plasmablastic differentiation are frequently encountered ... more 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 CD20negative, 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.
AimsPlasmablastic lymphoma (PBL) occurs mainly in immunocompromised individuals, usually secondar... more AimsPlasmablastic lymphoma (PBL) occurs mainly in immunocompromised individuals, usually secondary to human immunodeficiency virus (HIV) infection. It classically occurs intraorally, but has been described in extraoral locations. The aim of this study was to define the immunophenotype and Epstein–Barr virus (EBV) status in a large single‐centre cohort of extraoral PBL (EPBL) in South Africa, a high‐prevalence HIV setting.Methods and resultsThis retrospective study of 45 EPBLs included patients' age, gender, race, HIV status, and site. Cases were reviewed histologically, and classified morphologically as pure plasmablastic or plasmablastic with plasmacytic differentiation, and assessed immunohistochemically with antibodies against CD45, CD20, CD79a, PAX5, CD138, MUM1/IRF4, BLIMP1, VS38c, Ki67, bcl‐6, CD10, cyclin D1, and human herpesvirus‐8, by the use of standard automated procedures. EBV was assessed by the use of chromogenic in‐situ hybridisation. Tumours were assessed with a fluorescence in‐situ hybridisation (FISH) MYC break‐apart probe. Twenty‐seven PBLs showed pure plasmablastic morphology, and 18 showed plasmacytic differentiation. The male/female ratio was 1.5:1. The anus was the favoured extraoral site (31.1%), followed by lymph nodes (15.6%). All 29 patients with known HIV status were HIV‐positive. The immunohistochemical profile recapitulated that reported for oral PBLs and EPBLs in HIV‐positive and HIV‐negative patients. EBV was positive in 92.5% of PBLs. FISH analysis showed MYC rearrangement in 48% of cases.ConclusionThis study showed a strong association of EPBLs with HIV and EBV infection, similarly to the previously described oral PBL. The strong EBV association together with other clinicopathological parameters and an immunohistochemical profile that includes CD45, CD20, MUM1/IRF4, CD138 and Ki67 may be used in distinguishing PBL from diffuse large B‐cell lymphoma and plasma cell myeloma.
Abstract We report on the finding of at (1; 17) in two primary neuroblastomas. Subsequent fluores... more Abstract We report on the finding of at (1; 17) in two primary neuroblastomas. Subsequent fluorescence in situ hybridization (FISH) analysis revealed the presence of 1; 17 transfocations in four out of nine neuroblastoma cell lines. The chromosome 1 short arm ...
Background: Fragile sites are regions of the genome sensitive to replication stress and to exposu... more Background: Fragile 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. Methods: Multiplex 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. Results: Both 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. Conclusion: This 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.
Granulocytic sarcomas (GS) have been associated with t(8;21). The prognosis of patients with GS i... more Granulocytic sarcomas (GS) have been associated with t(8;21). The prognosis of patients with GS is generally regarded as being less favorable than of patients with acute myeloblastic leukemia (AML). GS occurs relatively commonly in Africa and has been reported to affect 10-25% of black children presenting with AML. We sought to establish the incidence of GS in our pediatric population, to determine whether an association with t(8;21) existed, and to report on the outcome of these cases in a single series. The records of consecutive pediatric patients treated for de novo AML in Johannesburg between January 1985-December 1995 were reviewed. Fifteen cases of GS among a total of 88 cases of AML presented to the Paediatric Haematology/Oncology Clinics of the Johannesburg and Baragwanath Hospitals. Fourteen (93%) of these patients were black male children. All 9 cases of orbital GS (60%) and almost all cases with concurrent AML M2 had t(8;21). This translocation was present in only 4 n(8.5%) of the remaining 47 AML cases without GS for which cytogenetic data were available. One case presented with a complex chromosomal translocation not previously associated with GS. The median disease-free survival of the GS patients, using conventional chemotherapy treatment protocols, was significantly better than for the patients with AML and no GS (P = 0.0004). Our data support a strong association between orbital GS, t(8;21), and AML M2 in the pediatric population. This entity occurred virtually exclusively in black male children at presentation. One third of these children who presented with AML had a GS. The favorable prognosis noted in our GS patients on standard induction and intensification therapy without local irradiation conflicts with some previous reports but is consistent with the favorable outcome documented in AML with t(8;21).
Background: Plasma cell neoplasms (PCNs) encompass a spectrum of disorders including monoclonal g... more Background: Plasma cell neoplasms (PCNs) encompass a spectrum of disorders including monoclonal gammopathy of undetermined significance, smoldering myeloma, plasma cell myeloma, and plasma cell leukemia. Molecular subtypes have been defined by recurrent cytogenetic abnormalities and somatic mutations that are prognostic and predictive. Karyotype and fluorescence in situ hybridization (FISH) have historically been used to guide management; however, new technologies and markers raise the need to reassess current testing algorithms. Methods: We convened a panel of representatives from international clinical laboratories to capture current state-of-the-art testing from published reports and to put forward recommendations for cytogenomic testing of plasma cell neoplasms. We reviewed 65 papers applying FISH, chromosomal microarray (CMA), next-generation sequencing, and gene expression profiling for plasma cell neoplasm diagnosis and prognosis. We also performed a survey of our peers to capture current laboratory practice employed outside our working group. Results: Plasma cell enrichment is widely used prior to FISH testing, most commonly by magnetic bead selection. A variety of strategies for direct, short-and long-term cell culture are employed to ensure clonal representation for karyotyping. Testing of clinically-informative 1p/1q, del(13q) and del(17p) are common using karyotype, FISH and, increasingly, CMA testing. FISH for a variety of clinically-informative balanced IGH rearrangements is prevalent. Literature review found that CMA analysis can detect abnormalities in 85-100% of patients with PCNs; more specifically, in 5-53% (median 14%) of cases otherwise normal by FISH and cytogenetics. CMA results in plasma cell neoplasms are usually complex, with alteration counts ranging from 1 to 74 (median 10-20), pr imar ily affecting loci not covered by FISH testing. Emerging biomarkers
Plasmablastic lymphoma (PBL) is an aggressive B-cell non-Hodgkin lymphoma associated with immunod... more Plasmablastic lymphoma (PBL) is an aggressive B-cell non-Hodgkin lymphoma associated with immunodeficiency in the context of human immunodeficiency virus (HIV) infection or iatrogenic immunosuppression. While a rare disease in general, the incidence is dramatically increased in regions of the world with high HIV prevalence. The molecular pathogenesis of this disease is poorly characterized. Here, we defined the genomic features of PBL in a cohort of 110 patients from South Africa (15 by whole-exome sequencing and 95 by deep targeted sequencing). We identified recurrent mutations in genes of the JAK-STAT signaling pathway, including STAT3 (42%), JAK1 (14%), and SOCS1 (10%), leading to its constitutive activation. Moreover, 24% of cases harbored gain-of-function mutations in RAS family members (NRAS and KRAS). Comparative analysis with other B-cell malignancies uncovered PBL-specific somatic mutations and transcriptional programs. We also found recurrent copy number gains encompassing the CD44 gene (37%), which encodes for a cell surface receptor involved in lymphocyte activation and homing, and was found expressed at high levels in all tested cases, independent of genetic alterations. These findings have implications for the understanding of the pathogenesis of this disease and the development of personalized medicine approaches. SIGNIFICANCE: Plasmablastic lymphoma is a poorly studied and extremely aggressive tumor. Here we define the genomic landscape of this lymphoma in HIV-positive individuals from South Africa and identify pervasive mutations in JAK-STAT3 and RAS-MAPK signaling pathways. These data offer a genomic framework for the design of improved treatment strategies targeting these circuits.
Setting: The Johannesburg state sector. Methods: All cases of ALL diagnosed by flow cytometry in ... more Setting: The Johannesburg state sector. Methods: All cases of ALL diagnosed by flow cytometry in the state-sector hospitals of Johannesburg over 42 months between 2016 and 2019 were identified and pertinent data recorded from the laboratory information system. Results: One hundred and eighty-one cases of ALL were identified, of which 59 (32.6%) were of T-cell lineage. The proportion of adult and paediatric ALL made up by TALL was similar (19/54 [35.2%] vs 40/127 [31.5%] respectively). Crude survival rates were very poor, with 80.0% having demised at the time of data collection. The mortality rate was overall significantly poorer amongst patients with TALL (80.0%) as compared to those with BALL (53.8%; p = 0.005) but was similarly poor in adults with BALL (83.3%) vs TALL (86.7%) (p = 0.53). The mortality rate did not differ between those with low-risk versus high-risk clinical features (77.8% vs 80.6%; p = 1.00). Conclusion: T-cell lymphoblastic leukaemia makes up a larger proportion of ALL in Johannesburg than is reported elsewhere, and it is a high-risk disease that is not well stratified by conventional risk factors.
Journal of Oral Pathology & Medicine, Dec 31, 2021
AIMS Originally described exclusively orally in HIV-infected patients, plasmablastic lymphoma (PB... more AIMS Originally described exclusively orally in HIV-infected patients, plasmablastic lymphoma (PBL) is increasingly described extra-orally and in non-HIV infected persons. The study comparatively analysed the clinico-pathologic features of oral PBLs (n=55) to previously published extra-oral PBLs (n=45 +1) diagnosed over a seven-year period at the same institution in an HIV prevalent setting in South Africa in order to clarify any distinction between oral and extra-oral PBLs. METHODS AND RESULTS Tumours were assessed histologically and immunohistochemically with CD45 (LCA), CD3, CD20, CD79a, PAX5, CD138, MUM1, BLIMP1, VS38c, Ki-67, BCL6 and CD10 using standard protocols. Age ranged from 22-76 years (oral) and 9-59 years (extra-oral). Most PBL patients were HIV positive [oral (84%); extra-oral (65%)]. Male:female ratio was 2.7:1 for oral and 1.4:1 for extra-oral PBLs. Favoured oral and extra-oral sites were the maxilla and anus. PBLs displayed an indistinguishable immunohistochemical profile with unusually high CD45 expression (oral: 98%, extra-oral: 84%). EBV assessed by chromogenic in-situ hybridisation (ISH) showed positivity in all oral PBLs and 95% extra-oral PBLs. MYC rearrangements (fluorescence ISH MYC break-apart probe) were similar in all the PBLs. CONCLUSIONS Extra-oral PBL is identical to its oral counterpart in gender and age distribution, HIV status, morphological appearances, immunophenotypic profile and EBV association. PBL should be regarded as the same tumour irrespective of oral or extra-oral site of origin.
We document the clinical, morphologic, and cytogenetic findings in a 2 1/2-year-old patient with ... more 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.
ABSTRACT INTRODUCTION As many people in South Africa are infected with HIV, it is has become impo... more ABSTRACT INTRODUCTION As many people in South Africa are infected with HIV, it is has become important to investigate possible changes in cellular radiosensitivity of such individuals to ionizing radiation. This study was prompted in part by observations in radiotherapy clinics that the normal tissue tolerance of HIV positive patients are compro- mised when treated with MV photons. MATERIALS AND METHODS Blood samples were collected from a cohort of HIV infected donors as well as from a group of HIV negative individuals. Lymphocytes were exposed in vitro to 6 MV X-rays to doses ranging between 0 to 4 Gy. Following the culture of cells over 3 days to yield binucleated lymphoblasts (T-type), micronuclei were numerated using fluores- cence microscopy. Micronuclei frequencies are a measure of chro- mosomal damage. At least 500 cells were analysed in each irradiated as well as in control samples for each donor. RESULTS AND CONCLUSION Micronuclei frequencies were consistently higher in HIV positive individuals compared to that observed in blood cells from HIV negative donors. The dose response curves show a clear linear- quadratic shape for both sets of donors indicating that residual radiation damage has been successfully quantified in all samples. As the micronuclei frequencies is higher in HIV donors with statistically significance, a clear dose modifying factor can be stated for such patients that may need radiation therapy.
ABSTRACT Rearrangements of the short arm of chromosome 12 (12p) are a common finding in hematolog... more ABSTRACT Rearrangements of the short arm of chromosome 12 (12p) are a common finding in hematologic malignancies. There has recently been considerable interest in chromosome 12 abnormalities in view of the mapping of the TEL gene to 12p13 and frequent 12p interstitial deletions. Overrepresentation of 12p sequences is, on the other hand, a consistent finding in testicular germ cell tumor (TGCT), and the 12p11.2-p12.1 subregion has been found to be specifically involved. We have studied a secondary leukemic patient whose cells contained 12p rearrangements with a view to clarifying the underlying molecular events. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) have revealed the presence of 12p11 breakpoints on both 12 homologs as well as amplification of 12p11-p12-derived sequences. Six YACs and a cosmid probe have been used in an attempt to map the amplification unit on 12p. The two YACs contigs WC-1468 and WC-985 were not amplified, and our results suggested a small amplicon localized in the 12p11.2-p12 subregion. We speculate that this region harbors gene(s) which are critical in tumor formation and could be involved in both TGCT and our patient. Whether the same gene(s) are involved in both amplification and translocation is unknown.
Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the digestive... more Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the digestive tract. Pathogenesis is linked to activating mutations identified in two proto-oncogenes, v-kit Hardy/Zuckerman 4 feline sarcoma viral oncogene homologue KIT (KIT) and the platelet-derived growth factor α (PDGFRα). In addition, these mutations affect response to treatment with tyrosine kinase inhibitors. In the present study, we report on the molecular characterisation of GISTs in the South African population. Tumour DNA was extracted from 46 GIST samples, followed by cycle sequencing of KIT exons 11, 13 and 17 and PDGFRα exons 12, 14 and 18. Fragment length analysis was used to detect a 6-bp duplication in KIT exon 9. Wild-type duplications were analysed further by PCR and sequencing of additional KIT and PDGFRα exons was performed. Overall, 78.3% of the samples had a mutation in KIT or PDGFRα. Of these, mutations were detected in KIT exon 11 (88.9%), PDGFRα exon 18 (8.3%) and KIT exon 9 (2.8%). Mutations varied from simple substitutions and duplications to large deletions (some with nucleotide insertions) resulting in missense mutations. In addition, seven single nucleotide polymorphisms were detected in 17 patients, one of which appears novel. The incidence of mutations in KIT exon 11 and PDGFRα exon 18 is consistent with the literature, however, the low incidence of KIT exon 9 mutations detected was unexpected. In contrast to previous western and Asian studies, this mutation appears to be rare in the South African population. The present study contributes to the molecular understanding of GISTs in the South African population.
The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) gui... more The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) guidelines are used for human epidermal growth factor receptor 2 (HER2) reporting in breast carcinoma. Cases that demonstrate weak to moderate complete membrane immunohistochemical staining in >10% of the tumor are scored as 2+ (equivocal). This study aimed to determine what proportion of HER2 immunohistochemistry (IHC) score = 2+ breast carcinomas were confirmed to be positive by HER2 fluorescent in situ hybridization (FISH). There were 241 HER2 IHC score = 2+ breast carcinomas included. Most (74.3%) carcinomas were estrogen and progesterone receptor-positive. Invasive breast carcinoma of no special type (89.2%) was the commonest histologic subtype. Most tumors were grade 2 (64.3%). As per the FISH report, at the time of diagnosis, 27 cases (11.2%) were HER2 FISH positive. All HER2 FISH equivocal cases and one FISH positive case assessed using the 2013 ASCO/CAP HER2 criteria were reclassified to HER2 FISH negative when the 2018 criteria were applied. There was a high level of agreement (κ = 0.979) between HER2 FISH results obtained using the 2013 and the 2018 criteria. This study provides insight into the frequency of HER2 FISH positivity (11.2%) among HER2 IHC score = 2+ breast carcinomas and the impact of modifications to the ASCO/CAP HER2 guidelines. Elimination of the HER2 FISH equivocal category by the 2018 guidelines has reduced the need for repeat testing and simplified clinical management. Reclassification of previous HER2 FISH positive to negative has resulted in some patients being ineligible for costly anti-HER therapy.
Human Immunodeficiency Virus (HIV) infection is known to be strongly associated with an increased... more Human Immunodeficiency Virus (HIV) infection is known to be strongly associated with an increased risk of high grade B-Cell Lymphoma (such as Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma). As South Africa has more than 5 million people living with HIV infection, this region sees a sharp increase in the diagnosis of high-grade lymphomas. HIV infection may associate with a number of atypical morphological features making it difficult to differentiate between classically described DLBCL and BL. As these two diseases have different prognosis and treatment, it is essential to accurately differentiate the type of disease and/or to diagnose the intermediate category of B-Cell Lymphoma, unclassifiable, with features intermediate between DLBCL and BL newly introduced in the 2008 WHO classification.
High-grade non-Hodgkin's lymphomas with plasmablastic differentiation are frequently encountered ... more 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 CD20negative, 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.
AimsPlasmablastic lymphoma (PBL) occurs mainly in immunocompromised individuals, usually secondar... more AimsPlasmablastic lymphoma (PBL) occurs mainly in immunocompromised individuals, usually secondary to human immunodeficiency virus (HIV) infection. It classically occurs intraorally, but has been described in extraoral locations. The aim of this study was to define the immunophenotype and Epstein–Barr virus (EBV) status in a large single‐centre cohort of extraoral PBL (EPBL) in South Africa, a high‐prevalence HIV setting.Methods and resultsThis retrospective study of 45 EPBLs included patients' age, gender, race, HIV status, and site. Cases were reviewed histologically, and classified morphologically as pure plasmablastic or plasmablastic with plasmacytic differentiation, and assessed immunohistochemically with antibodies against CD45, CD20, CD79a, PAX5, CD138, MUM1/IRF4, BLIMP1, VS38c, Ki67, bcl‐6, CD10, cyclin D1, and human herpesvirus‐8, by the use of standard automated procedures. EBV was assessed by the use of chromogenic in‐situ hybridisation. Tumours were assessed with a fluorescence in‐situ hybridisation (FISH) MYC break‐apart probe. Twenty‐seven PBLs showed pure plasmablastic morphology, and 18 showed plasmacytic differentiation. The male/female ratio was 1.5:1. The anus was the favoured extraoral site (31.1%), followed by lymph nodes (15.6%). All 29 patients with known HIV status were HIV‐positive. The immunohistochemical profile recapitulated that reported for oral PBLs and EPBLs in HIV‐positive and HIV‐negative patients. EBV was positive in 92.5% of PBLs. FISH analysis showed MYC rearrangement in 48% of cases.ConclusionThis study showed a strong association of EPBLs with HIV and EBV infection, similarly to the previously described oral PBL. The strong EBV association together with other clinicopathological parameters and an immunohistochemical profile that includes CD45, CD20, MUM1/IRF4, CD138 and Ki67 may be used in distinguishing PBL from diffuse large B‐cell lymphoma and plasma cell myeloma.
Abstract We report on the finding of at (1; 17) in two primary neuroblastomas. Subsequent fluores... more Abstract We report on the finding of at (1; 17) in two primary neuroblastomas. Subsequent fluorescence in situ hybridization (FISH) analysis revealed the presence of 1; 17 transfocations in four out of nine neuroblastoma cell lines. The chromosome 1 short arm ...
Background: Fragile sites are regions of the genome sensitive to replication stress and to exposu... more Background: Fragile 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. Methods: Multiplex 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. Results: Both 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. Conclusion: This 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.
Granulocytic sarcomas (GS) have been associated with t(8;21). The prognosis of patients with GS i... more Granulocytic sarcomas (GS) have been associated with t(8;21). The prognosis of patients with GS is generally regarded as being less favorable than of patients with acute myeloblastic leukemia (AML). GS occurs relatively commonly in Africa and has been reported to affect 10-25% of black children presenting with AML. We sought to establish the incidence of GS in our pediatric population, to determine whether an association with t(8;21) existed, and to report on the outcome of these cases in a single series. The records of consecutive pediatric patients treated for de novo AML in Johannesburg between January 1985-December 1995 were reviewed. Fifteen cases of GS among a total of 88 cases of AML presented to the Paediatric Haematology/Oncology Clinics of the Johannesburg and Baragwanath Hospitals. Fourteen (93%) of these patients were black male children. All 9 cases of orbital GS (60%) and almost all cases with concurrent AML M2 had t(8;21). This translocation was present in only 4 n(8.5%) of the remaining 47 AML cases without GS for which cytogenetic data were available. One case presented with a complex chromosomal translocation not previously associated with GS. The median disease-free survival of the GS patients, using conventional chemotherapy treatment protocols, was significantly better than for the patients with AML and no GS (P = 0.0004). Our data support a strong association between orbital GS, t(8;21), and AML M2 in the pediatric population. This entity occurred virtually exclusively in black male children at presentation. One third of these children who presented with AML had a GS. The favorable prognosis noted in our GS patients on standard induction and intensification therapy without local irradiation conflicts with some previous reports but is consistent with the favorable outcome documented in AML with t(8;21).
Background: Plasma cell neoplasms (PCNs) encompass a spectrum of disorders including monoclonal g... more Background: Plasma cell neoplasms (PCNs) encompass a spectrum of disorders including monoclonal gammopathy of undetermined significance, smoldering myeloma, plasma cell myeloma, and plasma cell leukemia. Molecular subtypes have been defined by recurrent cytogenetic abnormalities and somatic mutations that are prognostic and predictive. Karyotype and fluorescence in situ hybridization (FISH) have historically been used to guide management; however, new technologies and markers raise the need to reassess current testing algorithms. Methods: We convened a panel of representatives from international clinical laboratories to capture current state-of-the-art testing from published reports and to put forward recommendations for cytogenomic testing of plasma cell neoplasms. We reviewed 65 papers applying FISH, chromosomal microarray (CMA), next-generation sequencing, and gene expression profiling for plasma cell neoplasm diagnosis and prognosis. We also performed a survey of our peers to capture current laboratory practice employed outside our working group. Results: Plasma cell enrichment is widely used prior to FISH testing, most commonly by magnetic bead selection. A variety of strategies for direct, short-and long-term cell culture are employed to ensure clonal representation for karyotyping. Testing of clinically-informative 1p/1q, del(13q) and del(17p) are common using karyotype, FISH and, increasingly, CMA testing. FISH for a variety of clinically-informative balanced IGH rearrangements is prevalent. Literature review found that CMA analysis can detect abnormalities in 85-100% of patients with PCNs; more specifically, in 5-53% (median 14%) of cases otherwise normal by FISH and cytogenetics. CMA results in plasma cell neoplasms are usually complex, with alteration counts ranging from 1 to 74 (median 10-20), pr imar ily affecting loci not covered by FISH testing. Emerging biomarkers
Plasmablastic lymphoma (PBL) is an aggressive B-cell non-Hodgkin lymphoma associated with immunod... more Plasmablastic lymphoma (PBL) is an aggressive B-cell non-Hodgkin lymphoma associated with immunodeficiency in the context of human immunodeficiency virus (HIV) infection or iatrogenic immunosuppression. While a rare disease in general, the incidence is dramatically increased in regions of the world with high HIV prevalence. The molecular pathogenesis of this disease is poorly characterized. Here, we defined the genomic features of PBL in a cohort of 110 patients from South Africa (15 by whole-exome sequencing and 95 by deep targeted sequencing). We identified recurrent mutations in genes of the JAK-STAT signaling pathway, including STAT3 (42%), JAK1 (14%), and SOCS1 (10%), leading to its constitutive activation. Moreover, 24% of cases harbored gain-of-function mutations in RAS family members (NRAS and KRAS). Comparative analysis with other B-cell malignancies uncovered PBL-specific somatic mutations and transcriptional programs. We also found recurrent copy number gains encompassing the CD44 gene (37%), which encodes for a cell surface receptor involved in lymphocyte activation and homing, and was found expressed at high levels in all tested cases, independent of genetic alterations. These findings have implications for the understanding of the pathogenesis of this disease and the development of personalized medicine approaches. SIGNIFICANCE: Plasmablastic lymphoma is a poorly studied and extremely aggressive tumor. Here we define the genomic landscape of this lymphoma in HIV-positive individuals from South Africa and identify pervasive mutations in JAK-STAT3 and RAS-MAPK signaling pathways. These data offer a genomic framework for the design of improved treatment strategies targeting these circuits.
Setting: The Johannesburg state sector. Methods: All cases of ALL diagnosed by flow cytometry in ... more Setting: The Johannesburg state sector. Methods: All cases of ALL diagnosed by flow cytometry in the state-sector hospitals of Johannesburg over 42 months between 2016 and 2019 were identified and pertinent data recorded from the laboratory information system. Results: One hundred and eighty-one cases of ALL were identified, of which 59 (32.6%) were of T-cell lineage. The proportion of adult and paediatric ALL made up by TALL was similar (19/54 [35.2%] vs 40/127 [31.5%] respectively). Crude survival rates were very poor, with 80.0% having demised at the time of data collection. The mortality rate was overall significantly poorer amongst patients with TALL (80.0%) as compared to those with BALL (53.8%; p = 0.005) but was similarly poor in adults with BALL (83.3%) vs TALL (86.7%) (p = 0.53). The mortality rate did not differ between those with low-risk versus high-risk clinical features (77.8% vs 80.6%; p = 1.00). Conclusion: T-cell lymphoblastic leukaemia makes up a larger proportion of ALL in Johannesburg than is reported elsewhere, and it is a high-risk disease that is not well stratified by conventional risk factors.
Journal of Oral Pathology & Medicine, Dec 31, 2021
AIMS Originally described exclusively orally in HIV-infected patients, plasmablastic lymphoma (PB... more AIMS Originally described exclusively orally in HIV-infected patients, plasmablastic lymphoma (PBL) is increasingly described extra-orally and in non-HIV infected persons. The study comparatively analysed the clinico-pathologic features of oral PBLs (n=55) to previously published extra-oral PBLs (n=45 +1) diagnosed over a seven-year period at the same institution in an HIV prevalent setting in South Africa in order to clarify any distinction between oral and extra-oral PBLs. METHODS AND RESULTS Tumours were assessed histologically and immunohistochemically with CD45 (LCA), CD3, CD20, CD79a, PAX5, CD138, MUM1, BLIMP1, VS38c, Ki-67, BCL6 and CD10 using standard protocols. Age ranged from 22-76 years (oral) and 9-59 years (extra-oral). Most PBL patients were HIV positive [oral (84%); extra-oral (65%)]. Male:female ratio was 2.7:1 for oral and 1.4:1 for extra-oral PBLs. Favoured oral and extra-oral sites were the maxilla and anus. PBLs displayed an indistinguishable immunohistochemical profile with unusually high CD45 expression (oral: 98%, extra-oral: 84%). EBV assessed by chromogenic in-situ hybridisation (ISH) showed positivity in all oral PBLs and 95% extra-oral PBLs. MYC rearrangements (fluorescence ISH MYC break-apart probe) were similar in all the PBLs. CONCLUSIONS Extra-oral PBL is identical to its oral counterpart in gender and age distribution, HIV status, morphological appearances, immunophenotypic profile and EBV association. PBL should be regarded as the same tumour irrespective of oral or extra-oral site of origin.
We document the clinical, morphologic, and cytogenetic findings in a 2 1/2-year-old patient with ... more 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.
ABSTRACT INTRODUCTION As many people in South Africa are infected with HIV, it is has become impo... more ABSTRACT INTRODUCTION As many people in South Africa are infected with HIV, it is has become important to investigate possible changes in cellular radiosensitivity of such individuals to ionizing radiation. This study was prompted in part by observations in radiotherapy clinics that the normal tissue tolerance of HIV positive patients are compro- mised when treated with MV photons. MATERIALS AND METHODS Blood samples were collected from a cohort of HIV infected donors as well as from a group of HIV negative individuals. Lymphocytes were exposed in vitro to 6 MV X-rays to doses ranging between 0 to 4 Gy. Following the culture of cells over 3 days to yield binucleated lymphoblasts (T-type), micronuclei were numerated using fluores- cence microscopy. Micronuclei frequencies are a measure of chro- mosomal damage. At least 500 cells were analysed in each irradiated as well as in control samples for each donor. RESULTS AND CONCLUSION Micronuclei frequencies were consistently higher in HIV positive individuals compared to that observed in blood cells from HIV negative donors. The dose response curves show a clear linear- quadratic shape for both sets of donors indicating that residual radiation damage has been successfully quantified in all samples. As the micronuclei frequencies is higher in HIV donors with statistically significance, a clear dose modifying factor can be stated for such patients that may need radiation therapy.
ABSTRACT Rearrangements of the short arm of chromosome 12 (12p) are a common finding in hematolog... more ABSTRACT Rearrangements of the short arm of chromosome 12 (12p) are a common finding in hematologic malignancies. There has recently been considerable interest in chromosome 12 abnormalities in view of the mapping of the TEL gene to 12p13 and frequent 12p interstitial deletions. Overrepresentation of 12p sequences is, on the other hand, a consistent finding in testicular germ cell tumor (TGCT), and the 12p11.2-p12.1 subregion has been found to be specifically involved. We have studied a secondary leukemic patient whose cells contained 12p rearrangements with a view to clarifying the underlying molecular events. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) have revealed the presence of 12p11 breakpoints on both 12 homologs as well as amplification of 12p11-p12-derived sequences. Six YACs and a cosmid probe have been used in an attempt to map the amplification unit on 12p. The two YACs contigs WC-1468 and WC-985 were not amplified, and our results suggested a small amplicon localized in the 12p11.2-p12 subregion. We speculate that this region harbors gene(s) which are critical in tumor formation and could be involved in both TGCT and our patient. Whether the same gene(s) are involved in both amplification and translocation is unknown.
Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the digestive... more Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the digestive tract. Pathogenesis is linked to activating mutations identified in two proto-oncogenes, v-kit Hardy/Zuckerman 4 feline sarcoma viral oncogene homologue KIT (KIT) and the platelet-derived growth factor α (PDGFRα). In addition, these mutations affect response to treatment with tyrosine kinase inhibitors. In the present study, we report on the molecular characterisation of GISTs in the South African population. Tumour DNA was extracted from 46 GIST samples, followed by cycle sequencing of KIT exons 11, 13 and 17 and PDGFRα exons 12, 14 and 18. Fragment length analysis was used to detect a 6-bp duplication in KIT exon 9. Wild-type duplications were analysed further by PCR and sequencing of additional KIT and PDGFRα exons was performed. Overall, 78.3% of the samples had a mutation in KIT or PDGFRα. Of these, mutations were detected in KIT exon 11 (88.9%), PDGFRα exon 18 (8.3%) and KIT exon 9 (2.8%). Mutations varied from simple substitutions and duplications to large deletions (some with nucleotide insertions) resulting in missense mutations. In addition, seven single nucleotide polymorphisms were detected in 17 patients, one of which appears novel. The incidence of mutations in KIT exon 11 and PDGFRα exon 18 is consistent with the literature, however, the low incidence of KIT exon 9 mutations detected was unexpected. In contrast to previous western and Asian studies, this mutation appears to be rare in the South African population. The present study contributes to the molecular understanding of GISTs in the South African population.
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Papers by Pascale Willem