Daniel Steinbach

Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate.

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotests 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol®). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.

Prenatal origin of childhood acute lymphoblastic leukemia, association with birth weight and hyperdiploidy

Recent studies with very small numbers of patients showed that in some cases of childhood acute lymphoblastic leukemia (ALL), preleukemic cells are detectable on Guthrie cards that were used for newborn screening. We present here the largest series of ALL patients (n=32) in whom Guthrie cards were analyzed for the presence of preleukemic cells. Rearranged immunoglobulin heavy-chain genes were used as a marker for leukemic clones. We combined our set of patients with 17 previously published cases. Preleukemic cells were detected in 31 of all 49 patients (63%). Positive screening cards were not associated with patients age at diagnosis but were almost always found in patients with hyperdiploidy (10/11; 91%; P=0.04). High birth weight is an established risk factor for childhood ALL. Positive screening cards were strongly associated with low birth weight (P=0.01). In conclusion, the majority of childhood B-precursor ALL arise prior to birth. In the search for causes of childhood leukemia we should concentrate on prenatal factors as well as postnatal factors. Our results suggest that autologous cord bloods could be a poor choice as the source of stem cells for transplantation in leukemia, which may contain preleukemic cells. Pending the development of suitable methods, childhood leukemia is a potentially screenable disease.

Polymorphism of interleukin-23 receptor gene but not of NOD2/CARD15 is associated with graft-versus-host disease after hematopoietic stem cell transplantation in children.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). The selection of a suitable donor is the most critical issue in preventing severe GVHD. Recent data suggest that the risk of GVHD does not only depend on human leukocyte antigens (HLA) but also on polymorphisms of genes that influence immune responses. We analyzed the 1142 G>A single-nucleotide polymorphism (SNP) in the interleukin-23 receptor gene (IL23R) and 3 SNPs in the NOD2/CARD15 gene in a cohort of 231 children who underwent allogeneic stem cell transplantation and/or their respective donors. No association was observed between any of the NOD2/CARD15 polymorphisms and GVHD in either donor or recipient. Likewise, the IL23R polymorphism in the recipient was not significantly associated with GVHD. We found a significantly reduced incidence of acute GVHD (aGVHD) grade II-IV in patients who were transplanted from a donor with the IL23R polymorphism (5.0% versus 33.3%; P=.009). There was no case of aGVHD grade III-IV if this polymorphism occurred in the donor. These findings could be particularly relevant for children with inborn metabolic or immunologic disorders who do not benefit from a graft-versus-tumor effect, and therefore, selection of a donor with the IL23R polymorphism might be beneficial.

Clinical implications of c-Kit mutations in acute myelogenous leukemia

Abstractc-Kit is a receptor tyrosine kinase (RTK) with a pivotal role in melanogenesis, gametogenesis, and hematopoiesis. Aberrantly activated RTK and related downstream signaling partners were identified as key elements in the molecular pathogenesis of several malignancies. This finding culminated in a two-class model integrating constitutive activating and maturation arrest-inducing mutations as key elements for the pathogenesis of acute myelogenous leukemia (AML). c-Kit is expressed by myeloblasts in about 60% to 80% of patients, and the most frequently observed activating RTK mutations in AML (next to FLT3) are mutations or internal tandem duplications in c-Kit, with an overall incidence of 17%. The identification of small-molecule tyrosine kinase inhibitors capable of blocking key kinase switches introduced a paradigm change in the treatment of diseases like gastrointestinal stromal tumors and chronic myelogenous leukemia. Despite encouraging preclinical data, it appears that a complex clonal disease like AML will probably benefit from a synergistic approach of targeted drugs used (at least for now) in combination with conventional chemotherapy.

A promising DNA methylation signature for the triage of high-risk human papillomavirus DNA-positive women.

High-risk human papillomavirus (hrHPV)-DNA testing is frequently performed parallel to cytology for the detection of high-grade dysplasia and cervical cancer particularly in women above 30 years of age. Although highly sensitive, hrHPV testing cannot distinguish between HPV-positive women with or without clinically relevant lesions. However, in principle discrimination is possible on the basis of DNA methylation markers. In order to identify novel DNA regions which allow an effective triage of hrHPV-positive cases, hypermethylated DNA enriched from cervical cancers was compared with that from cervical scrapes of HPV16-positive cases with no evidence for disease by CpG island microarray hybridization. The most promising marker regions were validated by quantitative methylation-specific PCR (qMSP) using DNA from archived cervical tissues and cervical scrapes. The performance of these markers was then determined in an independent set of 217 hrHPV-positive cervical scrapes from outpatients with histopathological verification. A methylation signature comprising the 5′ regions of the genes DLX1, ITGA4, RXFP3, SOX17 and ZNF671 specific for CIN3 and cervical cancer (termed CIN3+) was identified and validated. A high detection rate of CIN3+ was obtained if at least 2 of the 5 markers were methylated. In the subsequent cross-sectional study all cervical carcinomas (n = 19) and 56% (13/23) of CIN3 were identified by this algorithm. Only 10% (11/105) of hrHPV-positive women without histological evidence of cervical disease were scored positive by the methylation assay. Of note is that the detection rate of CIN3 differed between age groups. Eight of nine CIN3 were detected among women ≥30 years of age but only five of fourteen among <30 year old group (p = 0.03). The specificity for CIN3+ in the older age group was 76.6% (95% CI 65.6–85.5%). Clinical validation studies are required to determine the usefulness of these novel markers for triage after primary hrHPV testing in a cervical cancer screening setting.

Toll-Like 4 Receptor Variant, Asp299Gly, and Reduced Risk of Hemorrhagic Cystitis after Hematopoietic Stem Cell Transplantation

Hemorrhagic cystitis (HC) is a major cause of morbidity after hematopoietic stem cell transplantation. Toll-like receptor 4 (TLR4) is a pattern recognition receptor of the innate immune system and induces inflammation. Individuals with the single nucleotide polymorphisms Thr399Ile (rs4986791) or Asp299Gly (rs4986790) of TLR4 show diminished inflammatory responsiveness to endotoxins. The genotype of TLR4 was determined in 166 children who underwent allogeneic hematopoietic stem cell transplantation and in their donors. Asp299Gly was present in 21 patients (13%) and 24 donors (14%). Thr399Ile was found in 22 patients (13%) and 25 donors (15%). The incidence of HC was significantly lower in patients with Asp299Gly (0% vs 23%; P = .009) and in patients who underwent transplantation from a donor with Asp299Gly (4% vs 23%; P = .05). The trend was the same for Thr399Ile-donor positive (8% vs 22%; P = .17), recipient positive (9% vs 22%; P = .25), donor or recipient positive (8% vs 23%; P = .04). Multivariate analysis revealed age, conditioning with busulfan, and absence of Asp299Gly as independent risk factors for HC. In conclusion, the TLR4 Asp299Gly variant seems to confer protection against hemorrhagic cystitis. This study provides the first indication that the innate immune system through TLR4 signaling pathway plays a role in the pathogenesis of HC after hematopoietic stem cell transplantation.

Evaluation of Plasmatic Kisspetin-10 as a Biomarker for Malignancy and Subtype Differentiation in Small Renal Tumours.

Objective: To evaluate Kisspeptin-10 (Kiss-10) in patients with small renal tumours (SRTs) and controls. Material and Methods: Kiss-10 was measured in preoperative plasma samples in a cohort of 143 patients with unilateral renal tumours smaller than or equal to 4 cm and 40 age-matched controls by a competitive ELISA test kit. The cohort of patients included 56 patients with clear cell renal cell carcinoma (ccRCC), 43 with papillary RCC (pRCC), 12 with chromophobe RCC (chRCC) and 32 with oncocytomas. Results: Kiss-10 was detected in all patients and controls. SRT patients revealed significantly higher Kiss-10 levels than controls (mean value 10.04 vs. 6.37 pmol/l, p < 0.001). In SRT patients, Kiss-10 was detected at significantly different concentrations between the subgroups (p = 0.021). The highest concentration was observed in those with oncocytomas (11.50 pmol/) followed by chRCC, pRCC and ccRCC patients (9.89, 10.01 and 9.25 pmol/l, respectively). Receiver operating characteristic curve analyses revealed an area under the curve (AUC) of 0.82 for the comparison of all tumours vs. controls (p < 0.001) and an AUC of 0.671 for all malignant tumours vs. oncoytomas (p = 0.003). Conclusion: This study shows that Kiss-10 levels are significantly altered by malignancy and tumour subtypes even in patients with SRTs. Kiss-10 therefore deserves further attention as a plasmatic biomarker for renal tumours.

MP15-15 URINE SAMPLE DERIVED CK20- AND IGF2-EXPRESSION AS BIOMARKER FOR THE DETECTION OF BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Non muscle invasive bladder cancer is a recurrent and progressive disease; currently we are unable to forecast recurrence in the individual patient. Recently we developed a mathematical model that found NLR as a good prognostic tool. The model was tested retrospectively in an additional study and found accurate too. The aim of the current study is to assess its accuracy to forecast recurrence prospectively in patients with NMIBC METHODS: All patients admitted to bladder tumor resection (TURBT) and agreed to participate in the study had blood drawn for blood count 24 hours prior to surgery. Patients with non-muscle invasive tumor were recruited and prospectively followed. Patients had urine cytology and cystoscopy every 3 months for 2 years following resection. Time to recurrence and recurrence free of tumor were recorded. Statistical analysis was done with X2 test for categorical parameters and T test for serial parameters. Logistic regression was performed to forecast prognosis. RESULTS: 123 patients were recruited, mean age was 71 years, all patients had at least 1 year follow up. Twenty nine patients (23.6%) experienced biopsy proven tumor recurrence. The mean time for recurrence was 7.38 months.Neutrophil to Lymphocyte rate > 2 showed direct statistically significant correlation with tumor recurrence (p1⁄40.038), tumor stage showed the same correlation (p1⁄40.048). The specificity of our recurrence forecasting model was 96.8%. EORTC score did not demonstrate significance between the recurrent and nonrecurrent groups. CONCLUSIONS: Our mathematical model that found NLR as a prognostic tool in patients with NMIBC was tested for the first time prospectively. The model demonstrated its ability to forecast recurrence more accurately then tumor stage grade and EORT score in the individual patient with NMIBC.The main limitation of this work is the relatively low number of patients.