Search

Objectives: TEL/AML1 (ETV6/RUNX1) fusion resulting from the translocation t(12;21)(p13;q22) constitutes the most common chimeric fusion gene in initial childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) (19–27%) and has been associated with good prognosis. Three secondary aberrations in TEL/AML1 positive ALL have been suspected to negatively influence outcome: deletion of the second TEL allele (T), gain of the second AML1 allele (A) and duplication of the derivative chromosome 21 (der(21), TA). Many studies have explored such aberrations in initial disease, while only few reports have investigated them in relapses. Methods: In this study, bone marrow samples from 38 children with relapsed TEL/AML1 RT-PCR positive and negative BCP-ALL were analyzed for these mutations by interphase fluorescence in situ hybridization and results were compared with published data. Results: In children with TEL/AML1 positive ALL relapse, additional (a) TEL loss, (b) combined AML1 and der(21) gain, (c) combined TEL loss and AML1 gain as well as (d) the occurrence of a subpopulation with the signal pattern 1T/3A/1TA appear to be related to higher peripheral blast counts (PBCs) at relapse diagnosis (a and d) or a tendency towards the occurrence of a subsequent relapse (b and c) (P-values

The histogenesis of Kaposi's 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. © 2006 Wiley-Liss, Inc.

Glucocorticoids are diabetogenic hormones because they decrease glucose uptake, increase hepatic glucose production, and inhibit insulin release. To study the long-term effects of increased glucocorticoid sensitivity in beta-cells, we studied transgenic mice overexpressing the rat glucocorticoid receptor targeted to the beta-cells using the rat insulin I promoter. Here we report that these mice developed hyperglycemia both in the fed and the overnight-fasted states at 12-15 months of age. Progression from impaired glucose tolerance, previously observed in the same colony at the age of 3 months, to manifest diabetes was not associated with morphological changes or increased apoptosis in the beta-cells. Instead, our current results suggest that the development of diabetes is due to augmented inhibition of insulin secretion through alpha(2)-adrenergic receptors (alpha(2)-ARs). Thus, we found a significantly higher density of alpha(2)-ARs in the islets of transgenic mice compared with controls, based on binding studies with the alpha(2)-AR agonist UK 14304. Furthermore, incubation of islets with benextramine, a selective antagonist of the alpha(2)-AR, restored insulin secretion in response to glucose in isolated islets from transgenic mice, whereas it had no effect on control islets. These results indicate that the chronic enhancement of glucocorticoid signaling in pancreatic beta-cells results in hyperglycemia and impaired glucose tolerance. This effect may involve signaling pathways that participate in the regulation of insulin secretion via the alpha(2)-AR.

Simian AIDS-related lymphomas (sARL) of cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied in relation to growth in severe combined immunodeficient (SCID) mice, karyotype abnormalities, and DNA sequence of the first noncoding region of the Bcl-6 gene. The tumors were diffuse large B cell lymphomas and expressed a simian homolog to Epstein-Barr virus (HVMF-1) in 12 of 13 primary tumors and corresponding cell lines. A tested cell line was tumorigenic in SCID mice. Tumors in the SCID mice showed cell growth features similar to those in the original lymphoma, suggesting that no subpopulation with growth advantage was selected for in the mice. Spectral karyotype analysis of sARL cell lines showed normal cytogenetic features except for a trisomy of monkey chromosome 2 (corresponding to human chromosomes 7 and 21) in two of five sARL lines, which was not recovered in SCID tumors established from the same cell line. Sequence analysis of a Bcl-6 gene fragment showed sequence variations indicative of population polymorphism(s) in 10 of 13 sARLs, and no evidence of Bcl-6 mutations. Thus Bcl-6 mutations in the first noncoding region are irrelevant for sARL development in cynomolgus monkeys and for tumorigenicity of sARL cell lines. We also demonstrate that no cytogenetic alterations are needed for the development of highly aggressive lymphomas in the SIV-immunosuppressed host.

Aims: To determine the sensitivity and specificity of flow‐ and image‐cytometry for the detection of DNA‐aneuploidy as a marker for malignant cells in effusions.Methods: 200 effusions (80 tumor cell‐positive, 74 negative and 46 cytologically equivocal) were stained with DAPI‐SR for DNA‐flow‐ and with Feulgen‐Pararosaniline for ‐image‐cytometry. They were measured using a PAS‐flow‐cytometer and an AutoCyte‐QUIC‐DNA‐workstation according to the ESACP consensus reports for DNA‐flow‐ and ‐image‐cytometry, respectively [7,23,29,49].Results: Sensitivity of DNA‐aneuploidy for the identification of malignant cells was 32.1% for DNA‐flow‐ and 75.0% for ‐image‐cytometry, specificity of ‐euploidy in benign cells was 100.0% for both methods. Positive predictive value of DNA‐aneuploidy for the identification of malignant cells was 100.0% for both techniques, negative predictive value of DNA‐euploidy was 48.6% for DNA‐flow‐ and 72.0% for ‐image‐cytometry.Conclusions: Searching for DNA‐aneuploidy as a diagnostic marker for neoplastic cells in serous effusions image‐cytometry revealed superior sensitivity as compared with monoparametric flow cytometry.

Kaposi's sarcoma (KS) is a multifocal lesion that occurs predominantly in the skin, most frequently in people infected with HIV-1, and that evolves through early stages (patch and plaque) to a tumor-like late stage (nodular). Both, endemic African (EKS) and AIDS-associated (AKS) KS expressed human herpesvirus 8 (HHV-8) as shown by PCR. By immunohistochemistry the expression of cellular Bcl-2 and c-myc was confined in early stages of both EKS and AKS to relatively few endothelial cells (EC) whereas in nodular KS most of spindle cells (SC) strongly expressed both genes. CD40 was usually strongly expressed in SC at all KS stages as well as in EC of non-involved tissue whereas CD40L (CD154) was not demonstrable. Fas (CD95) was moderately to weakly expressed by SC whereas p53 and Waf-1 were found in less than 5% of the SC. In both AKS and EKS at nodular stage almost no apoptotic SC were detected. In most AKS and EKS low levels of cell proliferation were seen but AKS showed consistently higher values compared to EKS. All clinical types and stages of KS showed a diploid cellular DNA content by flow cytometric analysis of microselected lesions. Thus, we conclude that KS during evolution represents diploid, probably reactive, cell proliferation, which progressively increases the expression of strong cellular and also viral (HHV-8) antiapoptotic factors.

Lymphomas in 10 cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied with regard to proliferative activity and apoptosis-related gene expression. All were diffuse large-cell lymphomas, showed mono or oligoclonality and a 9/10 diploid cellular DNA content. Expression of a simian homologue to Epstein-Barr virus (HVMF-1) was shown in nine cases. The lymphomas showed moderate to high proliferative activity by Ki67 immunostaining and DNA flow cytometry, and a low number of apoptotic cells detected by TdT-mediated dUTP nick-end labeling (TUNEL). Immunohistochemistry showed abundant tumor infiltrating TIA-1+ cytotoxic lymphocytes (CTL) and macrophages. Bcl-2, Mcl-1, and also Bax and Bak, but not p53 were demonstrable in the tumor cells by immunostaining. Our findings suggest a causal relationship between HVMF-1 infection and a low apoptotic index of the lymphomas due to the expression of Bcl-2. The apparent inefficient function of tumor-infiltrating CTL could be due to inactivation of CTL and/or resistance of the lymphoma cells to CTL effects. The tumors showed immunoreactivity for CD18, CD29, and CD49d, but not for CD11a, mimicking the phenotype of human Epstein-Barr virus (EBV)–related lymphomas. In summary, our observations indicate a high similarity between this simian model of acquired immunodeficiency syndrome (AIDS)-related lymphomas (ARL) and human ARL and other immunosuppression-related lymphomas.

Background Malignant lymphomas (ML) are increasingly important causes of morbidity and mortality in sub-Saharan Africa including Tanzania, possibly due to HIV and AIDS. However, the biological characterization ML including their HIV and Epstein-Barr virus (EBV) association as well as DLBCL subtypes in Tanzania is still sketchy. This prevents diagnostic/prognostic comparison as well as application of established therapeutic protocols.

Background In 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. Methods Archival, 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). Results A 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. Conclusions According 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.

In 49 pairs of contiguous sections from paraffin-embedded prostatic cancer tissue, the DNA indices (DIs) were determined by flow cytometry (FCM) at 2 different laboratories. In 3 of 45 pairs of evaluable nuclear suspensions, DIs of 1.1 (DNA aneuploid) were found at Laboratory 1, whereas all 3 tumours were classified as DNA diploid at Laboratory 2. In the remaining 42 specimens, the correlation between the DIs was excellent, though the application of strictly defined DNA ploidy ranges led to different DNA ploidy allocation in 3 cases. It is concluded that in 85-90% of the cases, reliable DIs can be obtained by FCM done in paraffin-embedded material at different laboratories. Slight technical variations and interpretation differences may lead to different ploidy allocation in 10-15% of the cases.

Acute lymphoblastic leukemia (ALL), a clinically and biologically heterogeneous disease, is the most common type of childhood cancer. Approximately 25% of B-cell precursor ALL (BCP-ALL) carry the cryptic chromosomal translocation t(12;21)(p13;q22), the most frequent genetic aberration in pediatric ALL. This translocation combines two transcription factors and essential regulators of normal hematopoiesis, ETV6 and RUNX1, generating the fusion oncogene ETV6/RUNX1 (synonym TEL/AML1). Recent studies in various animal models have strengthened the view that ETV6/RUNX1 positive cells give rise to preleukemic clones with a differentiation block in the pro/pre-B stage of B cell development that, after acquisition of additional mutations, may transform into full malignancy. However, the effects triggered by the expression of ETV6/RUNX1 and the associated additional mutated genes are not well understood. We compared global gene expression data from ETV6/RUNX1 positive ALL and normal hematopoietic progenitor cells to identify expression signatures underlying the malignant phenotype. Our meta-analysis comprised Affymetrix HG-U133A und HG-U133 Plus 2.0 data from a total of 321 patients with ETV6/RUNX1 positive ALL, different ETV6/RUNX1 positive cell models, leukemias with 11q23 rearrangements and hyperdiploid ALL, as well as 251 samples from various normal controls including normal bone marrow, hematopoietic stem cells (HSCs) and normal B-(progenitor) cells (multipotent progenitor, pro-, pre-, immature-, naive-, centrocyte-, centroblast-, memory-, plasmablast-B cells). To eliminate potential lab batch effects, we performed RMA normalization of all microarray CEL files and next applied the batch effect removal algorithm ComBat. For determination of differentially expressed genes and pathway analyses, the R/Bioconductor packages limma and SPIA were used, respectively. We generated a gene expression landscape of the normal B-cell development and were able to show that pre-B cells are the closest normal counterpart of ETV6/RUNX1 positive ALL. Furthermore, in comparisons with normal HSCs the p53-, mTOR-, PI3K-AKT-, Apoptosis-, and JAK-STAT signaling pathways were found to be deregulated in those ALL. The combination of multiple global gene expression data sets from ETV6/RUNX1 positive ALL offers the possibility of an integrated large-scale meta-analysis and reveals novel insights into deregulated gene networks in this type of leukemia. Disclosures No relevant conflicts of interest to declare.

Background: It is still unclear if Kaposi's sarcoma (KS) is a monoclonal cell proliferation or a polyclonal, hyperplastic, reactive process. Reports on KS cytogenetics are few and restricted to late stage disease and cell lines. Method: We analysed 27 KS, early and late, AIDS related (AKS) and endemic (EKS) by laser microdissection, global DNA amplification and comparative genomic hybridization (CGH). Result: Loss of Y chromosome was detected in 20/23 male KS, which was the only recurrent chromosomal aberration in all nine male early (patch) KS. Only one patch EKS showed in addition to the Y loss a loss of Xq. Late (nodular) AKS and EKS showed recurrent copy number changes in chromosomes 16, 17, 21, X and Y, as well as other random changes. The loss of chromosome 16, 17 and Y was confirmed by interphase fluorescence in situ hybridization (FISH) on paraffin sections. EKS showed a higher number of chromosomal abnormalities than AKS, indicating that rapid growth of AKS is less dependent on genetic changes than is EKS, possibly because of the immunosuppressed host environment in AKS. Conclusion: Clonal loss of chromosome Y was detected in all early male KS, while additional chromosomal aberrations appeared during development to late KS. This increase in chromosomal abnormalities during tumour growth indicates genetic instability and the selection of survival cell clones establishing late, aggressive sarcoma growth. Our data support the view that KS (in males) develops into a clonal tumour yet initially is a hyperplastic reactive cell proliferation.

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.

The aim of the study was to evaluate proton magnetic resonance spectroscopy ((1)H MRS) for noninvasive biological characterisation of neuroblastoma xenografts in vivo. For designing the experiments, human neuroblastoma xenografts growing subcutaneously in nude rats were analysed in vivo with (1)H MRS and magnetic resonance imaging at 4.7 T. The effects of spontaneous tumour growth and antiangiogenesis treatment, respectively, on spectral characteristics were evaluated. The spectroscopic findings were compared to tumour morphology, proliferation and viable tumour tissue fraction. The results showed that signals from choline (Cho)-containing compounds and mobile lipids (MLs) dominated the spectra. The individual ML/Cho ratios for both treated and untreated tumours were positively correlated with tumour volume (P0.05). There was an inverse correlation between the ML/Cho ratio and the viable tumour fraction (r=-0.86, P0.001). Higher ML/Cho ratios concomitant with pronounced histological changes were seen in spectra from tumours treated with the antiangiogenic drug TNP-470, compared to untreated control tumours (P0.05). In conclusion, the ML/Cho ratio obtained in vivo by (1)H MRS enabled accurate assessment of the viable tumour fraction in a human neuroblastoma xenograft model. (1)H MRS also revealed early metabolic effects of antiangiogenesis treatment. (1)H MRS could prove useful as a tool to monitor experimental therapy in preclinical models of neuroblastoma, and possibly also in children.

Using flow cytometric DNA measurement and sorting combined with morphometric light microscopy, different groups of cells were studied in a human melanoma pleural effusion, a human melanoma lymph node metastasis and a mouse tumor, as well as in normal reference tissues. Beside cells of the predominant tumor cell population, three types of rare tumor cells were studied after enrichment by sorting: a) giant cells from the greater than 8c region, comprising about 5% of the tumor cells, b) binucleated and multinucleated cells with unequal nuclear sizes within the same cell, found at frequencies of about 1.5%, and c) less than 2c cells which were derived from the so-called "debris"-region of the DNA histogram, found at frequencies of about 1 to 6%. All these rare cells were found only in the malignant tumors and not in the benign reference tissues. Morphometry showed that the increase in the cellular DNA content in the different fractions of tumor cells was combined with an increase in the cellular and nuclear sizes. However, the n/c-ratio was constant in the whole range of tumor cell fractions, including the fractions from the the less than 2c and the greater than 8c regions. The n/c-ratio of the less than 2c cells and giant cells differed from that of corresponding normal cells underlining their origin from the predominant tumor cell population. The possible linkage between the occurrence of the three rare cell types and genetic instability of tumors related to faulty nucleus and cell division is discussed.

In this study, we have investigated the model DNA values and the expression of estramustine-binding protein (EMBP) in formalin-fixed and paraffin-embedded TUR specimens from 76 untreated patients with prostatic cancer. In addition, specimens from 13 patients were analyzed for tumour EMBP expression only. Ploidy was measured as diploid, tetraploid and non-tetraploid aneuploid or aneuploid in the near-diploid region. All patients had been referred during 1978-1981, and were subjected to TUR due to urinary obstruction. Survival data were obtained for all patients through March 1988. Statistical analyses were performed using a Cox's regression model with respect to survival and cause specific survival and correlated to the DNA pattern and the expression of EMBP. The existence of a near-diploid aneuploid cell population as well as poor differentiation grade were both statistically significantly correlated with poor survival. Near-diploid aneuploid cell lines were seen in 9/76 (12%) of the patients and were also seen in well differentiated cancers (4/17). The expression of EMBP was most abundant in the moderately differentiated cancers. However, all prostatic cancer specimens investigated were positive for the antigen. Patients with poorly differentiated carcinomas and high EMBP expression showed a tendency towards better prognosis than those with poorly differentiated carcinomas and low EMBP expression. The present patient material was, however, too small to show a statistically significant correlation between EMBP and survival.