Fatemeh Pak

Lymphatic and vascular origin of Kaposi's sarcoma spindle cells during tumor development.

The histogenesis of Kaposis sarcoma (KS) tumor spindle cells (SC) remains controversial but several immunohistochemical studies favor a lymphatic origin. Twenty KS surgical biopsies were analyzed for the coexpression of LANA, CD34, LYVE‐1, D2‐40, VEGFR‐2, VEGFR3 by using double or triple immunostaining. Most of the SC in both early and late KS expressed the lymphatic markers LYVE‐1, D2‐40 and VEGFR‐3 and the blood vascular endothelial/endothelial precursor cell markers CD34 and endothelial stem cell marker VEGFR‐2. All the LANA+ SC in early and late KS were LYVE‐1+, but only 75% of these LANA+ cells were CD34+. The CD34+/LANA+ cells increased from early‐ (68.8%) to late‐stage KS (82.2%). However, approximately 18% of the LANA+ SC in early KS were CD34− but were LYVE‐1+, suggesting that resident lymphatic endothelial cells (LEC) are targeted for primary infection by human herpesvirus‐8. This LANA+/LYVE‐1+/CD34− (resident LEC) cell population clearly decreased during the development of KS from early (18.7%) to late KS (2.9%). Thus, in late stages of KS, most SC were LANA+/CD34+/LYVE‐1+. However, in both early‐ and late‐stage KS, approximately 18% of the SC were CD34+/LANA‐/LYVE‐1− and could represent newly recruited endothelial precursor cells, which become infected in the lesion and eventually undergo a phenotype switch expressing LEC markers. Our study apparently indicates that KS represents a unique variant of tumor growth with continues recruitment of tumor precursor cells as well as proliferation and decreased apoptosis of SC.

HHV‐8/KSHV during the development of Kaposi's sarcoma: evaluation by polymerase chain reaction and immunohistochemistry

The human γ‐herpes virus‐8 (HHV‐8) was first described in AIDS‐related Kaposis sarcoma (KS) tumour samples. In this study, we report comparative studies on paraffin‐embedded biopsies of AIDS‐related KS (AKS) and endemic KS (EKS) with regard to HHV‐8 content as evaluated using polymerase chain reaction (PCR) and immunohistochemistry. DNA was extracted either using Chelex‐100 or using Qia‐gene kit and was evaluated with the help of a semiquantitative PCR assay. The PCR detection of HHV‐8 was more sensitive to the Chelex method than to Qia‐gene. The threshold for PCR test sensitivity with the help of serial dilution of DNA was at the level of five plasmid ORF‐26 regions, and DNA from 25 body cavity‐based lymphoma‐1 cells. The results expressed as virus load/actin unit showed progressively higher HHV‐8 levels in late (nodular) cases, compared to those in early (patch/plaque) stages. Evaluation of HHV‐8 DNA levels in tumour tissues, thus, indicates a correlation between virus load and KS stage. Double immunostaining of spindle cells (SC) in KS biopsies for CD34 and HHV‐8/latency‐associated nuclear antigen (LANA) showed an increase in double‐positive SC in the lesions of nodular AKS and EKS cases, compared to that in plaque and patch stages. However, 10–15% of CD34+/LANA– SC cells were observed during the development from patch to nodular cases of AKS and EKS. Our results indicate that PCR analysis is a simple and sensitive diagnostic method for HHV‐8 evaluation in KS tissues, processed for conventional histopathology.

Tanzanian malignant lymphomas: WHO classification, presentation, ploidy, proliferation and HIV/EBV association.

BackgroundIn Tanzania, the International Working Formulation [WF] rather than the WHO Classification is still being used in diagnosing malignant lymphomas (ML) and the biological characterization including the HIV/EBV association is sketchy, thus restraining comparison, prognostication and application of established therapeutic protocols.MethodsArchival, diagnostic ML biopsies (N = 336), available sera (N = 35) screened by ELISA for HIV antibodies and corresponding clinical/histological reports at Muhimbili National Hospital (MNH) in Tanzania between 1996 and 2006 were retrieved and evaluated. A fraction (N = 174) were analyzed by histopathology and immunohistochemistry (IHC). Selected biopsies were characterized by flow-cytometry (FC) for DNA ploidy (N = 60) and some by in-situ hybridization (ISH) for EBV-encoded RNA (EBER, N = 37).ResultsA third (38.8%, 109/281) of the ML patients with available clinical information had extranodal disease presentation. A total of 158 out of 174 biopsies selected for immunophenotyping were confirmed to be ML which were mostly (84. 8%, 134/158) non-Hodgkin lymphoma (NHL). Most (83.6%, 112/134) of NHL were B-cell lymphomas (BCL) (CD20+), of which 50.9%, (57/112) were diffuse large B-cell (DLBCL). Out of the 158 confirmed MLs, 22 (13.9%) were T-cell [CD3+] lymphomas (TCL) and 24 (15.2%) were Hodgkin lymphomas (HL) [CD30+]. Furthermore, out of the 60 FC analyzed ML cases, 27 (M:F ratio 2:1) were DLBCL, a slight majority (55.6%, 15/27) with activated B-cell like (ABC) and 45% (12/27) with germinal center B-cell like (GCB) immunophenotype. Overall, 40% (24/60) ML were aneuploid mostly (63.0%, 17/27) the DLBCL and TCL (54.5%, 6/11). DNA index (DI) of FC-analyzed ML ranged from 1.103-2.407 (median = 1.51) and most (75.0%) aneuploid cases showed high (>40%) cell proliferation by Ki-67 reactivity. The majority (51.4%, 19/37) of EBER ISH analyzed lymphoma biopsies were positive. Of the serologically tested MLs, 40.0% (14/35) were HIV positive, mostly with high (≥40.0%) Ki-67 reactivity.ConclusionsAccording to the 2001 WHO Classification, most subtypes are represented in Tanzanian ML. Extranodal presentation was common among MNH lymphoma patients who also showed high aneuploidy, tumor proliferation (KI-67) and EBER positivity. DLBCL was frequent and phenotype heterogeneity appeared similar to observations in Western countries suggesting applicability of established intervention approaches. HIV was apparently associated with high ML cell proliferation but extended studies are needed to clarify this.

Human herpesvirus-8 (HHV-8) sero-detection and HIV association in Kaposi's sarcoma (KS), non-KS tumors and non-neoplastic conditions

BackgroundThe association of the human herpesvirus-8/Kaposis sarcoma (KS)-associated herpesvirus (HHV-8/KSHV) serology with various malignancies in Tanzania is not currently well established while previous studies were based on either PCR or immunofluorescence assays [IFA] but not with a sensitive enzyme-linked immunosorbent assay (ELISA). Selected archival diagnostic biopsies (n = 184) and sera from indigenous patients with KS (n = 120), non-KS tumors (n = 24) and non-neoplastic lesions (n = 40) at Muhimbili National Hospital (MNH), Tanzania, were evaluated by diagnostic histopathology, immunohistology [anti-HHV-8 latency-associated nuclear antigen (LANA)] and serology for HIV (ELISA) and HHV-8 (IFA and ELISA).ResultsAbout 66.3% (n = 122) cases including AIDS-associated Kaposis sarcoma (AKS) (n = 93), reactive conditions (n = 28) and only one non-KS tumour were HIV positive. Endemic KS (EKS) patients were mostly males (96.3%, 26/27) who were less (69.9%, 65/93) predominant in AIDS-associated (AKS). A high (89%) percentage of patients with anti-HHV-8 antibodies was found in the cohort including the HIV positive (92%) cases, males (81.2%), KS patients (93%), non-KS tumors (92%), and reactive conditions (75%). All HHV-8 seronegative KS cases were nodular stage whereas both sera and corresponding biopsies from early stage KS were HHV-8+. Assay sensitivity, positive predictive value (PPV) and specificity were 98.6%, 93.5% and 16.7% for IFA and 93.5%, 98.6% and 50.0% for ELISA respectively.ConclusionHHV-8 seroprevalence at MNH appears high as expected among AKS cases and males but also in non-KS patients. ELISA showed a combination of high HHV-8 sensitivity as well as higher PPV and specificity than IFA which however, showed higher sensitivity. The apparent stage-dependent, inverted serum HHV-8 immunoreactivity supports a notion of viral immune-segregation during KS development. Routine HHV-8 screening should be considered particularly in patients at risk of KS and for selection of blood/organ donations.

Preventing HHV-8 transmission and Kaposi’s sarcoma (KS) risk prediction and prognostication in resource-poor countries

(p<0.001, highly statistically significant, Chi 2 Test) and as expected the majority (68.3%, 112/164) were KS. HHV-8 serology tests by IFA and ELISA were mostly (92.4%, 73/79) concordant. Sensitivity, positive predictive value (PPV), and specificity were 98.6%, 93.5%, and 16.7% for IFA and 93.5%, 98.6%, and 50.0% for ELISA, respectively. All patients with early-stage KS were HHV8 seropositive but two late-stage cases were seronegative despite LANA expression in their corresponding biopsies.

Kaposi (KS) pathogenesis: cell origin, HHV-8 permissiveness, proliferation, genomic instability

Results LANA+ cells varied with frequency of CD34+ cells at all stages of KS. However about 25% of CD34+ cells were LANAand LYVE-1-. All LANA+ SC were LYVE-1+, but only 75% were CD34+. About 18% of LANA+ SC in early KS were CD34but LYVE-1+. This LANA+/LYVE-1+/ CD34(resident LEC) cells decreased from early to late KS. Ki67+ cells were moderate (4.5–11.5%) at all KS stages, but usually higher in early KS. CGH showed Y chrom. deletion in most KS and was the only aberration in early KS, whereas late KS also showed deletions of chrom. 16 and 17, confirmed by FISH. Chk2 which is upregulated at early stage of DNA damage was more frequent in early than late KS.

Autologous T cells expressing the oncogenic transcription factor KLF6-SV1 prevent apoptosis of chronic lymphocytic leukemia cells

Crosstalk between leukemic cells and the tumor microenvironment is of importance in chronic lymphocytic leukemia (CLL). T cells seem to sustain the survival of CLL cells by various mechanisms. The Krüppel-like family of transcription factors (KLFs) are identified as regulators of proliferation and cell death. In the present study, we analyzed the expression of the wild type (WT) gene KLF6 and the oncogenic splice variant 1 (KLF6–SV1) at the mRNA level in subsets of T cells from CLL patients (n = 29), multiple myeloma patients (n = 6) and normal donors (n = 10). RNA Silencing was used for wtKLF6 and KLF6-SV1. Tumor cell apoptosis was measured. A significant overexpression of wtKLF6 and KLF6-SV1 in T cells of CLL patients compared to normal donors and myeloma patients was noted (p<0.002). Western blot showed that both wtKLF6 and KLF6–SV1 were expressed in purified T cells from CLL patients. KLF6-SV1 siRNA transfection induced a significant down-regulation of KLF6-SV1 in CLL T cells, which lost the capability to sustain the growth of leukemic cells. However, no such a significant effect was seen after wtKLF6 transfection of the autologous T cells. The results suggest that KLF6-SV1 may play a role in the regulation of survival CLL cells.

Virus (KSHV/HHV8) Infection and Genomic Aberrations in Developing AIDS Kaposi's Sarcoma (KS)

Material and Methods Biopsies of AKS and EKS were compared by triple immunoflourscence for possible stage related phenotypic differences in HHV8 (LANA) infected tumor spindle cells (CD34+SC) and proliferating (Ki67+) cells. Histological tumor areas were alsolaser microdissected and the DNA analyzed by CGH and interphase FISH for cytogenetic changes in early and late stages of tumor development.