Mutations in the CCND1 and CCND2 genes are frequent events in adult patients with t(8;21)(q22;q22) acute myeloid leukemia.

Core-binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a 'second hit' to initiate leukemogenesis. Mutation assessment of 177 adults with CBF-AML, including 68 with t(8;21) and 109 with inv(16)/t(16;16), identified not only mutations well known in CBF-AML but also mutations in the CCND1 and CCND2 genes, which represent novel frequent molecular alterations in AML with t(8;21). Altogether, CCND1 (n=2) and CCND2 (n=8) mutations were detected in 10 (15%) patients with t(8;21) in our cohort. A single CCND2 mutation was also found in 1 (0.9%) patient with inv(16). In contrast, CCND1 and CCND2 mutations were detected in only 11 (0.77%) of 1426 non-CBF-AML patients. All CCND2 mutations cluster around the highly conserved amino-acid residue threonine 280 (Thr280). We show that Thr280Ala-mutated CCND2 leads to increased phosphorylation of the retinoblastoma protein, thereby causing significant cell cycle changes and increased proliferation of AML cell lines. The identification of CCND1 and CCND2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease.

Prognostic and biologic significance of DNMT3B expression in older patients with cytogenetically normal primary acute myeloid leukemia.

DNMT3B encodes a DNA methyltransferase implicated in aberrant epigenetic changes contributing to leukemogenesis. We tested whether DNMT3B expression, measured by NanoString nCounter assay, associates with outcome, gene and microRNA expression and DNA methylation profiles in 210 older (⩾60 years) adults with primary, cytogenetically normal acute myeloid leukemia (CN-AML). Patients were dichotomized into high versus low expressers using median cut. Outcomes were assessed in the context of known CN-AML prognosticators. Gene and microRNA expression, and DNA methylation profiles were analyzed using microarrays and MethylCap-sequencing, respectively. High DNMT3B expressers had fewer complete remissions (CR; P=0.002) and shorter disease-free (DFS; P=0.02) and overall (OS; P<0.001) survival. In multivariable analyses, high DNMT3B expression remained an independent predictor of lower CR rates (P=0.04) and shorter DFS (P=0.04) and OS (P=0.001). High DNMT3B expression associated with a gene expression profile comprising 363 genes involved in differentiation, proliferation and survival pathways, but with only four differentially expressed microRNAs (miR-133b, miR-148a, miR-122, miR-409-3p) and no differential DNA methylation regions. We conclude that high DNMT3B expression independently associates with adverse outcome in older CN-AML patients. Gene expression analyses suggest that DNMT3B is involved in the modulation of several genes, although the regulatory mechanisms remain to be investigated to devise therapeutic approaches specific for these patients.

A stem cell-like gene expression signature associates with inferior outcomes and a distinct microRNA expression profile in adults with primary cytogenetically normal acute myeloid leukemia.

Acute myeloid leukemia (AML) is hypothesized to be sustained by self-renewing leukemia stem cells (LSCs). Recently, gene expression signatures (GES) from functionally defined AML LSC populations were reported, and expression of a 'core enriched' (CE) GES, representing 44 genes activated in LCSs, conferred shorter survival in cytogenetically normal (CN) AML. The prognostic impact of the CE GES in the context of other molecular markers, including gene mutations and microRNA (miR) expression alterations, is unknown and its clinical utility is unclear. We studied associations of the CE GES with known molecular prognosticators, miR expression profiles, and outcomes in 364 well-characterized CN-AML patients. A high CE score (CE(high)) associated with FLT3-internal tandem duplication, WT1 and RUNX1 mutations, wild-type CEBPA and TET2, and high ERG, BAALC and miR-155 expression. CE(high) patients had a lower complete remission (CR) rate (P=0.003) and shorter disease-free (DFS, P<0.001) and overall survival (OS, P<0.001) than CE(low) patients. These associations persisted in multivariable analyses adjusting for other prognosticators (CR, P=0.02; DFS, P<0.001; and OS, P<0.001). CE(high) status was accompanied by a characteristic miR expression signature. Fifteen miRs were upregulated in both younger and older CE(high) patients, including miRs relevant for stem cell function. Our results support the clinical relevance of LSCs and improve risk stratification in AML.

Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: a novel epigenetic-targeting approach in acute myeloid leukemia.

Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously, we demonstrated that AML patients with higher miR (microRNA)-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here, we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (that is, SP1, DNMT1, DNMT3A and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represent a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDACIs.

UVA (UVA-1) therapy for the treatment of acute GVHD of the skin.

Long-wavelength UVA (340-400 nm UVA-1) phototherapy has been reported to be effective in atopic dermatitis, localized scleroderma and T-cell-derived skin diseases. We retrospectively investigated 70 patients with acute cutaneous GVHD after allogeneic haematopoietic cell transplantation or donor lymphocyte infusion. Complete and partial responses with a median duration of 10 months were achieved in 49 (70%) and 17 (24.3%) patients, respectively. Overall, 47 (67.1%) patients were not treated with systemic steroids. Furthermore, immunosuppression could be tapered in 24 (34.3%) patients while they were receiving UVA-1 treatment. Responses were seen irrespective of age or type of conditioning. Treatment was very well tolerated. After a median follow-up of 18 (range 10-60) months, three patients developed epithelial skin neoplasia. We conclude that UVA-1 therapy is feasible, well tolerated and can be an effective treatment for acute GVHD of the skin, thereby avoiding the use of systemic steroids and/or allowing a more rapid tapering of systemic immunosuppression in a substantial number of patients. The results of this retrospective analysis warrant larger, prospective studies and the effectiveness of UVA-1 therapy should be compared with other established treatment modalities.

Identification of SOX2 as a novel glioma-associated antigen and potential target for T cell-based immunotherapy.

Prognosis for patients suffering from malignant glioma has not substantially improved. Specific immunotherapy as a novel treatment concept critically depends on target antigens, which are highly overexpressed in the majority of gliomas, but the number of such antigens is still very limited. SOX2 was identified by screening an expression database for transcripts that are overexpressed in malignant glioma, but display minimal expression in normal tissues. Expression of SOX2 mRNA was further investigated in tumour and normal tissues by real-time PCR. Compared to cDNA from pooled normal brain, SOX2 was overexpressed in almost all (9 out of 10) malignant glioma samples, whereas expression in other, non-malignant tissues was almost negligible. SOX2 protein expression in glioma cell lines and tumour tissues was verified by Western blot and immunofluorescence. Immunohistochemistry demonstrated SOX2 protein expression in all malignant glioma tissues investigated ranging from 6 to 66% stained tumour cells. Human leucocyte antigen-A(*)0201-restricted SOX2-derived peptides were tested for the activation of glioma-reactive CD8+ cytotoxic T lymphocytes (CTLs). Specific CTLs were raised against the peptide TLMKKDKYTL and were capable of lysing glioma cells. The abundant and glioma-restricted overexpression of SOX2 and the generation of SOX2-specific and tumour-reactive CTLs may recommend this antigen as target for T-cell-based immunotherapy of glioma.

Elevated des-AngI-angiotensinogen levels by nephrectomy and adrenalectomy.

This study investigates the time course of plasma levels of angiotensinogen (Aogen) and of the Aogen metabolite des-AngI-angiotensiongen (des-AngI-Aogen) in nephrectomized rats with and without adrenals for 24 h. After nephrectomy the plasma Aogen levels increased 5-fold over the following 24 h. The increase is significantly lower after sham nephrectomy (3.7-fold, P < 0.05) and if the kidneys are withdrawn without decapsulization (2.4-fold, P < 0.05). A small and transient increase arise after nephrectomy plus adrenalectomy (1.6-fold after 8 h, P < 0.005). After adrenalectomy alone Aogen levels continuously shrink to 38% of control values after 24 h. Plasma des-AngI-Aogen levels increase 2.1- to 3.7-fold 24 h after the different nephrectomy procedures. In connection with recent findings these data support the notion that the increase in Aogen plasma levels after bilateral nephrectomy is triggered by renin, released during surgery. High plasma levels of des-AngI-Aogen after nephrectomy indicate that AngI is generated by tissue renin, e.g., in the adrenals. This suggests that after nephrectomy the plasma des-AngI-Aogen levels should be a valuable proof for the evaluation of the amount of generated angiotensin.

Angiotensin II is the mediator of the increase in hepatic angiotensinogen synthesis after bilateral nephrectomy.

The present study investigated the hypothesis that the increase in plasma angiotensinogen after nephrectomy is mediated by endogenous renin and angiotensin (ANG) II. Rats were divided into control, nephrectomy, or nephrectomy plus adrenalectomy groups. In addition, similar cohorts were divided as just mentioned and then given either atenolol (selective beta 1-adrenoceptor inhibitor that prevents renin release) or Dup-753 [ANG II (AT1) receptor antagonist]. The plasma angiotensinogen levels increase approximately fivefold after 24 h in nephrectomized rats. Pretreatment with atenolol blunted this increase. A significant reduction was observed 4 h (P < 0.05) and 8 h (P < 0.005) after surgery. Dup-753 nearly abolished the increase in angiotensinogen plasma levels. After pretreatment with Dup-753, significantly higher angiotensinogen levels (P < 0.005) were found only after 24 h. Nephrectomy plus adrenalectomy also blunted the rise in plasma angiotensinogen. A significant increase in angiotensinogen plasma levels could only be observed after 8 h (P < 0.005) and 12 h (P < 0.005) but not after 24 h. Atenolol further reduced this increase. After atenolol pretreatment, significantly higher angiotensinogen levels could only be observed after 12 h (P < 0.05). Dup-753 completely abolished the increase of plasma angiotensinogen after nephrectomy plus adrenalectomy. In anesthetized control rats at time 0 the plasma ANG I levels were 0.7 nM. Pretreatment with atenolol decreased the ANG I values by 30%, whereas Dup-753 caused a sixfold increase in plasma ANG I levels in the control rats at time 0.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the renin-angiotensin system after nephrectomy and adrenalectomy.

The study investigates the change in angiotensinogen (Aogen), angiotensin I (AngI) and renin plasma concentration after nephrectomy and adrenalectomy. The aim of the study was to elucidate the mechanisms that are involved in the up regulation of the Aogen plasma levels after nephrectomy and the contribution of the adrenals. Rats were treated with the beta 1-selective adrenoceptor blocker, atenolol, and with the angiotensin antagonist, DuP 753 in order to inhibit renal renin release and to check whether the increase in plasma Aogen after nephrectomy is mediated by angiotensin (AngII), respectively. The plasma Aogen levels increase approx. 5-fold 24 h after nephrectomy. This increase is significantly reduced in the presence of atenolol. After nephrectomy plus adrenalectomy there is a maximal increase of 60% in plasma Aogen levels 8 h after surgery and a subsequent decline. In the presence of atenolol this increase is even smaller. In contrast after adrenalectomy the plasma Aogen levels continuously declined. In the presence of atenolol the plasma Aogen levels were approx. 20% higher at time 0 but declined with the same slope as after adrenalectomy without atenolol treatment. Treatment with DuP 753 caused an almost complete inhibition of the increase in Aogen plasma levels after nephrectomy. Significantly higher Aogen levels were found only after 24 h. At time 0, immediately after nephrectomy the plasma AngI levels were increased compared to the respective control rats. Significantly higher AngI values (P < 0.05) could also be observed in nephrectomized rats and in nephrectomized plus adrenalectomized rats at time 0 in the presence and absence of atenolol and DuP 753, respectively. In contrast after adrenalectomy alone the AngI levels at time 0, were not different from those of the controls. Subsequently the AngI levels increased at a similar rate as after adrenalectomy in the presence of atenolol. These findings suggest that the increase in plasma Aogen after nephrectomy is essentially mediated by AngII via an adrenal mechanism. It seems likely that this process is triggered by renin released during surgery. The increased renin release after adrenalectomy that is responsible for the increased degradation of Aogen seems not to be mediated by a sympathetic stimulation of the renal beta 1-adrenoceptors.

[Toxic shock syndrome following cesarean section]. / Toxic Shock Syndrom nach Sectio Caesarea.

The typical onset, development and therapy of the Staphylococcus aureus exotoxin (TSST-1)-induced Toxic Shock Syndrome is reported in a case of a 32 years old I. Para/I. Gravida following caesarean section. The outcome of this rare but often lethal disease depends on early diagnosis and treatment.