Emergence of translocation t(9;11)-positive leukemia during treatment of childhood acute lymphoblastic leukemia

Therapy-related acute myeloid leukemia (t-AML) characterized by the t(9;11)(p22;q23) translocation is one of the most frequent secondary malignancies. The timing of the initiation of translocation and of development of the malignant t(9;11) clone during chemotherapy is presently unknown. In the present study, we backtracked bone marrow samples from three children during treatment for acute lymphoblastic leukemia (ALL). Two patients developed a t(9;11)-positive t-AML 19 and 30 months after therapy start, whereas the third patient, diagnosed with a rare t(9;11)-positive ALL, suffered from an ALL relapse 23 months after initial diagnosis. The genomic MLL-MLLT3 (MLL-AF9) fusion site was amplified by a multiplex, nested long-range PCR and used as a clonal marker for quantification of the MLL-MLLT3-positive cells during chemotherapy. The t(9;11)-positive clone was detectable 13 and 18 months after therapy start in both t-AML cases, which was 6-12 months before clinical diagnosis of the secondary malignancy. In the t(9;11)-positive ALL patient, the identical leukemic clone reoccurred during maintenance therapy after a short molecular remission, 8 months before clinically overt ALL relapse. The time course and characteristics of the genomic breakpoints in the present t-AML cases support the hypothesis of translocation formation as a result of defective breakage repair after topoisomerase II cleavage.     

Reactivation of codogenic endogenous retroviral (ERV) envelope genes in human endometrial carcinoma and prestages: Emergence of new molecular targets

Endometrial carcinoma (EnCa) is the most common invasive gynaecologic carcinoma. Over 85% of EnCa are classified as endometrioid, expressing steroid hormone receptors and mostly involving pathological prestages. Human endogenous retroviruses (ERV) are chromosomally integrated genes, account for about 8% of the human genome and are implicated in the etiology of carcinomas. The majority of ERV envelope (env) coding genes are either not present or not consistently represented between common gene expression microarrays. The aim of this study was to analyse the absolute gene expression of all known 21 ERV env genes including 19 codogenic and two env genes with premature stop codons in EnCa, endometrium as well as in hyperplasia and polyps. For EnCa seven env genes had high expression with >200 mol/ng cDNA (e.g. envH1-3, Syncytin-1, envT), two middle >50 mol/ng cDNA (envFc2, erv-3) and 12 low <50 mol/ng cDNA (e.g. Syncytin-2, envV2). Regarding tumor parameters, Syncytin-1 and Syncytin-2 were significantly over-expressed in advanced stage pT2 compared to pT1b. In less differentiated EnCa Syncytin-1, erv-3, envT and envFc2 were significantly over-expressed. Syncytin-1, Syncytin-2 and erv-3 were specific to glandular epithelial cells of polyps, hyperplasia and EnCa using immunohistochemistry. An analysis of 10 patient-matched EnCa with endometrium revealed that the ERV-W 5'' long terminal repeat regulating Syncytin-1 was hypomethylated, including the ERE and CRE overlapping MeCP2 sites. Functional analyses showed that 10 env genes were regulated by methylation in EnCa using the RL95-2 cell line. In conclusion, over-expressed env genes could serve as indicators for pathological pre-stages and EnCa.     

Angiogenic growth factors in maternal and fetal serum in pregnancies complicated by intrauterine growth restriction

The present study was performed to compare serum concentrations of maternal and fetal angiogenic growth factors in IUGR (intrauterine growth restriction) and normal pregnancy at the time of delivery. VEGF (vascular endothelial growth factor), PlGF (placental growth factor), sFlt-1 (soluble fms-like tyrosine kinase 1), sKDR (soluble kinase domain receptor) and bFGF (basic fibroblast growth factor) were measured by ELISA in serum from a maternal peripheral vein, the umbilical vein and the umbilical arteries in 15 women with pregnancies complicated by IUGR and 16 controls (women with normal pregnancies). In IUGR, sFlt-1 was increased, and PlGF and sKDR were decreased, in both maternal serum and serum from the umbilical vein. Additionally, bFGF was increased in serum from the umbilical vein of women with pregnancies complicated by IUGR. No significant differences in growth factor concentrations between the groups were found in serum from the umbilical artery. In both groups, levels of VEGF were higher and levels of sFlt-1 were lower in serum from the umbilical vein and umbilical artery compared with maternal serum. PlGF levels were found to be lower in serum from the umbilical vein compared with maternal serum in both groups, whereas PlGF levels in serum from the umbilical artery were significantly lower only in the control group. These findings suggest an imbalance of angiogenic and anti-angiogenic factors in IUGR, with formation of an anti-angiogenic state in maternal and, to a lesser extent, umbilical vein blood. The placenta appears to play a central role in the release of sFlt-1 into maternal and umbilical blood. Umbilical artery blood was unaffected in IUGR, indicating that the fetus does not contribute to changes in angiogenic growth factor concentrations.     

B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell–null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell–null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.     

Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop.

The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.     

Oncocytic adrenal cortical tumor with cytoplasmic inclusions and hyaline globules

Adrenal cortical tumors, particularly oncocytic tumors, have been reported to contain a variety of intracytoplasmic and intramitochondrial inclusions. Oncocytic cortical tumors can also morphologically mimic pheochromocytomas. We report an unusual, partially oncocytic cortical neoplasm with nesting architecture, intranuclear inclusions, and hyaline globules reminiscent of pheochromocytoma, together with numerous, small, brightly eosinophilic, periodic acid-Schiff-positive cytoplasmic inclusions and typical cytoplasmic lipid droplets. Ultrastructural study revealed oncocytes containing numerous mitochondria with intramitochondrial crystals and lipid droplets. Immunohistochemistry and immunoblots were utilized to further characterize the tumor. Immunohistochemistry demonstrated immunoreactivity of both the eosinophilic inclusions and the hyaline globules for adipose differentiation-related protein (ADRP), which is one of a group of proteins associated with storage of neutral lipids in many cell types. Immunoblots confirmed the presence of ADRP and demonstrated an imbalance between ADRP and perilipin, another neutral lipid-associated protein, in tumor tissue compared to normal adrenal cortex. The findings suggest that mitochondrial dysfunction in oncocytic cortical tumors may lead to abnormal processing of proteins related to the lipid-storing functions of the adrenal cortex, resulting in unusual cytoplasmic inclusions and extracellular globules resembling the globules in pheochromocytomas. The finding of ADRP as a constituent of inclusions in adrenal cortical tumors has not been previously reported.     

Single nucleotide polymorphisms of the aromatase gene (CYP19A1), HER2/neu status, and prognosis in breast cancer patients

Purpose Estrogen exposure is involved in both breast cancer susceptibility and the prognosis in patients with breast cancer. Aromatase is involved in the production of estrogens, and altered expression of it might be associated with the prognosis. The aim of this study was to examine the effect of single nucleotide polymorphisms (SNPs) in the aromatase gene, CYP19A1, on the prognosis, and in relation to tumor and patient characteristics in a cohort of breast cancer patients. Patients and methods The cohort analyzed in this study consisted of 1,257 patients with invasive primary breast cancer. Polymorphisms rs10046, rs4646 and rs700519 were genotyped within this group. Results The variant genotypes of rs10046 and rs4646 were associated with a lower percentage of HER2-positive tumors. There was no association of rs700519 and rs4646 with disease-free survival (DFS) or overall survival (OS). The variant genotype of rs10046 was significantly associated with a better 5-year DFS (hazards ratio 0.51; 95% CI, 0.32 to 0.81; P = 0.004) adjusted for age, nodal status, tumor size grading, and hormone receptor status. This effect appeared to be determined in the subgroup of premenopausal patients. Conclusion SNPs rs10046 and rs4646 may influence the HER2 status of breast cancer tumors, and rs10046 genotypes are associated with an altered DFS. Genotypes of aromatase polymorphisms may influence the prognosis for breast cancer patients not only by affecting the extent of estrogen exposure but also through an alteration in tumor characteristics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia

Chromosomal translocations involving the MLL gene occur in about 80% of infant leukemia. In the search for possible agents inducing infant leukemia, we identified bioflavonoids, natural substances in food as well as in dietary supplements, that cause site-specific DNA cleavage in the MLL breakpoint cluster region (BCR) in vivo. The MLL BCR DNA cleavage was shown in primary progenitor hematopoietic cells from healthy newborns and adults as well as in cell lines; it colocalized with the MLL BCR cleavage site induced by chemotherapeutic agents, such as etoposide (VP16) and doxorubicin (Dox). Both in vivo and additional in vitro experiments demonstrated topoisomerase II (topo II) as the target of bioflavonoids similar to VP16 and Dox. Based on 20 bioflavonoids tested, we identified a common structure essential for topo II-induced DNA cleavage. Reversibility experiments demonstrated a religation of the bioflavonoid as well as the VP16-induced MLL cleavage site. Our observations support a two-stage model of cellular processing of topo II inhibitors: The first and reversible stage of topo II-induced DNA cleavage results in DNA repair, but also rarely in chromosome translocations; whereas the second, nonreversible stage leads to cell death because of an accumulation of DNA damage. These results suggest that maternal ingestion of bioflavonoids may induce MLL breaks and potentially translocations in utero leading to infant and early childhood leukemia.