Katja Fischereder

Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia.

Mutations leading to activation of the RAF-mitogen-activated protein kinase/extracellular signal-regulated (ERK) kinase (MEK)-ERK pathway are key events in the pathogenesis of human malignancies. In a screen of 82 acute myeloid leukemia (AML) samples, 45 (55%) showed activated ERK and thus were further analyzed for mutations in B-RAF and C-RAF. Two C-RAF germ-line mutations, S427G and I448V, were identified in patients with therapy-related AML in the absence of alterations in RAS and FLT3. Both exchanges were located within the kinase domain of C-RAF. In vitro and in vivo kinase assays revealed significantly increased activity for (S427G)C-RAF but not for (I448V)C-RAF. The involvement of the S427G C-RAF mutation in constitutive activation of ERK was further confirmed through demonstration of activating phosphorylations on C-RAF, MEK, and ERK in neoplastic cells, but not in nonneoplastic cells. Transformation and survival assays showed oncogenic and antiapoptotic properties for both mutations. Screening healthy individuals revealed a <1/400 frequency of these mutations and, in the case of I448V, inheritance was observed over three generations with another mutation carrier suffering from cancer. Taken together, these data are the first to relate C-RAF mutations to human malignancies. As both mutations are of germ-line origin, they might constitute a novel tumor-predisposing factor.

Early results of a European multicentre experience with a new self-anchoring adjustable transobturator system for treatment of stress urinary incontinence in men.

Surgical treatment options for male stress urinary incontinence (SUI) include collagen injection, artificial urinary sphincter, or male sling placement. In recent years, various minimally invasive sling systems have been investigated as treatment options for post‐prostatectomy SUI. One of the drawbacks of using male slings is the lack of ability to make postoperative adjustments. To overcome the challenges associated with peri‐ and postoperative adjustment of male sling systems, the adjustable transobturator male system (ATOMS®) was introduced. Our initial European multicentre experience with this device treatment shows a significant improvement in the severity of incontinence and mean pad use as well as quality‐of‐life scores. Our data suggest that the ability at any time to make adjustments in male sling systems should be considered as a prerequisite when managing men with SUI.

Whole-genome plasma sequencing reveals focal amplifications as a driving force in metastatic prostate cancer

Genomic alterations in metastatic prostate cancer remain incompletely characterized. Here we analyse 493 prostate cancer cases from the TCGA database and perform whole-genome plasma sequencing on 95 plasma samples derived from 43 patients with metastatic prostate cancer. From these samples, we identify established driver aberrations in a cancer-related gene in nearly all cases (97.7%), including driver gene fusions (TMPRSS2:ERG), driver focal deletions (PTEN, RYBP and SHQ1) and driver amplifications (AR and MYC). In serial plasma analyses, we observe changes in focal amplifications in 40% of cases. The mean time interval between new amplifications was 26.4 weeks (range: 5–52 weeks), suggesting that they represent rapid adaptations to selection pressure. An increase in neuron-specific enolase is accompanied by clonal pattern changes in the tumour genome, most consistent with subclonal diversification of the tumour. Our findings suggest a high plasticity of prostate cancer genomes with newly occurring focal amplifications as a driving force in progression.

Are morphological criteria sufficient for the identification of circulating tumor cells in renal cancer

BackgroundSingle circulating tumor cells (CTCs) or circulating tumor microemboli (CTMs) are potential biomarkers of renal cell cancer (RCC), however studies of CTCs/CTMs in RCC are limited. In this pilot study we aimed to evaluate a novel blood filtration technique suited for cytomorphological classification, immunocytochemical and molecular characterization of filtered, so called circulating non-hematologic cells (CNHCs) - putative CTCs/CTMs - in patients with RCC.MethodsBlood of 40 patients with renal tumors was subjected to ScreenCell® filtration. CNHCs were classified according to cytomorphological criteria. Immunocytochemical analysis was performed with antibodies against CD45, CD31 and carbonic anhydrase IX (CAIX, a RCC marker). DNA of selected CNHCs and respective primary tumors was analysed by array-CGH.ResultsCNHC-clusters with malignant or uncertain malignant cytomorphological features - putative CTMs - were negative for CD45, positive for CD31, while only 6% were CAIX positive. Array-CGH revealed that 83% of malignant and uncertain malignant cells did represent with a balanced genome whereas 17% presented genomic DNA imbalances which did not match the aberrations of the primary tumors. Putative single CTCs were negative for CD45, 33% were positive for CD31 and 56% were positive for CAIX.ConclusionsThe majority of CNHC-clusters, putative CTMs, retrieved by ScreenCell® filtration may be of endothelial origin. Morphological criteria seem to be insufficient to distinguish malignant from non-malignant cells in renal cancer.

Rapid Identification of Plasma DNA Samples with Increased ctDNA Levels by a Modified FAST-SeqS Approach

BACKGROUND Recent progress in the analysis of cell-free DNA fragments [cell-free circulating tumor DNA (ctDNA)] now allows monitoring of tumor genomes by noninvasive means. However, previous studies with plasma DNA from patients with cancer demonstrated highly variable allele frequencies of ctDNA. The comprehensive analysis of tumor genomes is greatly facilitated when plasma DNA has increased amounts of ctDNA. Therefore, a fast and cost-effective prescreening method to identify such plasma samples without previous knowledge about alterations in the respective tumor genome could assist in the selection of samples suitable for further extensive qualitative analysis. METHODS We adapted the recently described Fast Aneuploidy Screening Test-Sequencing System (FAST-SeqS) method, which was originally established as a simple, effective, noninvasive screening method for fetal aneuploidy from maternal blood. RESULTS We show that our modified FAST-SeqS method (mFAST-SeqS) can be used as a prescreening tool for an estimation of ctDNA percentage. With a combined evaluation of genome-wide and chromosome arm-specific z-scores from dilution series with cell line DNA and by comparisons of plasma-Seq profiles with data from mFAST-SeqS, we established a detection limit of ≥10% mutant alleles. Plasma samples with an mFAST-SeqS z-score >5 showed results that were highly concordant with those of copy number profiles obtained from our previously described plasma-Seq approach. CONCLUSIONS Advantages of this approach include the speed and cost-effectiveness of the assay and that no prior knowledge about the genetic composition of tumor samples is necessary to identify plasma DNA samples with >10% ctDNA content.

Behandlung der Belastungsinkontinenz nach radikaler Prostatektomie

BACKGROUND The adjustable transobturator male system (ATOMS®) is a new method for the treatment of male stress urinary incontinence. This article presents the results of a prospective multicenter observational study with this system. PATIENTS AND METHODS Between March 2009 and March 2011 a total of 124 patients with persistent stress urinary incontinence after radical prostatectomy received the ATOMS system. Postoperative adjustments via the implanted port chamber were performed after 6 weeks and thereafter when necessary. Postoperative evaluation consisted of medical history, mictionary protocol, 24-h pad tests, 24-h pad counts and sonography. RESULTS The mean age of the patients was 71.2 ± 5.5 years (range 58-85 years). Previous incontinence surgery had been carried out in 36.3% of patients while 34.5% of patients had a previous history of radiation treatment. The mean operation time was 48.3 ± 11.2 min (range 36-116 min) and the mean hospital stay was 3.8 ± 1.2 days (range 2-6 days). No intraoperative urethral or bladder injuries occurred. After removal of the transurethral catheter on the first postoperative day, temporary urinary retention occurred in 3 patients who were conservatively treated. Transient perineal/scrotal pain or dysesthesia was observed in 75 patients (60.5%) and resolved after 3-4 weeks of non-opioid analgesics. There were no perineal infections; however, infections at the port site occurred in 3 patients (2.4%) leading to explantation of the system in all cases. The average number of adjustments to achieve the desired result was 4.3 ± 1.8 (range 2-7). After a mean follow-up of 19.1 ± 2.2 months (range 12-36 months), there was a significant reduction in the mean number of pads/24 h from 8.8 to 1.8 (p<0.001). The overall success rate was 93.8% with 61.6% of the patients being dry and 32.2% of the patients showing improvement. CONCLUSIONS The results of the study demonstrate the safety and efficacy to date of the ATOMS system for treatment of stress urinary incontinence after radical prostatectomy.

Sampling of the anterior apical region results in increased cancer detection and upgrading in transrectal repeat saturation biopsy of the prostate.

To evaluate whether biopsy cores taken via a transrectal approach from the anterior apical region of the prostate in a repeat‐biopsy population can result in an increased overall cancer detection rate and in more accurate assessment of the Gleason score.

Cell phone usage and erectile function.

Introduction The objective of this pilot study was to report our experience concerning the effects of cell phone usage on erectile function (EF) in men. Material and Methods We recruited 20 consecutive men complaining of erectile dysfunction (ED) for at least six months (Group A), and another group of 10 healthy men with no complaints of ED (Group B). Anamnesis, basic laboratory investigations, and clinical examinations were performed. All men completed the German version of the Sexual Health Inventory for Men (SHIM) for evaluation of the International Index of Erectile Function (IIEF), as well as another questionnaire designed by our clinicians that assessed cell phone usage habits. Results There was no significant difference between both groups regarding age, weight, height, and total testosterone (Table 1). The SHIM scores of Group A were significantly lower than that of Group B, 11.2 ±5 and 24.2 ±2.3, respectively. Total time spent talking on the cell phone per week was not significantly higher in Group A over B, 17.6 ±11.1 vs. 12.5 ±7 hours. Men with ED were found to carry their ‘switched on’ cell phones for a significantly longer time than those without ED, 4.4 ±3.6 vs. 1.8 ±1 hours per day. Conclusions We found a potential correlation with cell phone usage and a negative impact on EF. Further large–scale studies confirming our initial data and exploring the mechanisms involved in this phenomenon are recommended.

mFast-SeqS as a Monitoring and Pre-screening Tool for Tumor-Specific Aneuploidy in Plasma DNA

Recent progress in the analysis of cell-free DNA fragments (cell-free circulating tumor DNA, ctDNA) now allows monitoring of tumor genomes by non-invasive means. However, previous studies with plasma DNA from patients with cancer demonstrated highly variable allele frequencies of ctDNA. Comprehensive genome-wide analysis of tumor genomes is greatly facilitated when plasma DNA has increased amounts of ctDNA. In order to develop a fast and cost-effective pre-screening method for the identification of plasma samples suitable for further extensive qualitative analysis, we adapted the recently described FAST-SeqS method. We show that our modified FAST-SeqS method (mFAST-SeqS) can be used as a pre-screening tool for an estimation of the ctDNA percentage. Moreover, since the genome-wide mFAST-SeqS z-scores correlate with the actual tumor content in plasma samples, changes in ctDNA levels associated with response to treatment can be easily monitored without prior knowledge of the genetic composition of tumor samples.

Abstract PR05: Analysis of ctDNA using the mFastSeqS and plasma–Seq methods for screening and therapy monitoring in prostate cancer patients

Background: Prostate cancer is the most common malignancy in males. Prostate cancer progression can be inhibited by androgen-deprivation therapy, but nearly all patients eventually develop castration-resistance prostate cancer (CRPC). In the management of prostate cancer treatment, there are still unsolved questions as to how patients can best be matched to targeted therapies in accordance with the characteristics of their tumor genome. Recent progress in the analysis of cell-free circulating tumor DNA (ctDNA) now allows for the monitoring of tumor genomes by non-invasive means. Therefore, there is an unmet need to develop methods which will allow cost-effective pre-screening and therapy monitoring. Methods: We analyzed a total of 71 metastatic prostate cancer patients with our recently published plasma-Seq method in order to establish genome-wide copy number aberrations (CNAs). Furthermore, we used a targeted resequencing approach to screen for mutations in a set of 58 cancer-associated genes and for the presence of TMPRSS-ERG fusions. In order to assess the amount of ctDNA in plasma, we used a modified FAST-Seq (mFAST-Seq) approach in a subset of samples. Follow-up samples were available from a total of 30 patients, which enabled us to monitor the constantly changing tumor genomes. Results: Using plasma-Seq, we observed a variety of copy number changes characteristic of prostate cancer, i.e. 8q gains as well as losses at 3p, 8p, 10q, 13q, 17p and 21q in 67% of patients. AR gene amplifications were found in 70% of metastatic castration-resistant patients. Furthermore, when analyzing follow-up samples, we observed the occurrence of novel copy number aberrations and clonal shifts in one-third of the patients. In one extreme case we observed the loss of an AR focal amplification, which was accompanied by shifts in mutant allele frequencies for TP53 and EP300 mutations from 76.9% to 94.8% and from 14.4% to undetectable levels, respectively, after switching to cytotoxic chemotherapy. In a subset of samples (n=61), we performed mFAST-SeqS in order to estimate the ctDNA content and to identify samples with a ctDNA content of 10% or more. For samples that did not show any CNAs after plasma-Seq, a low amount of ctDNA ( 10%), we observed highly consistent results (r=0.667) for plasma-Seq and mFastSeqS copy number profiles. Conclusion: Plasma-Seq serves as a non-invasive method for monitoring a patient9s response to therapy and may identify the occurrence of novel changes associated with resistance to a given therapy. The mFastSeqS approach has the advantage that no prior knowledge about the genetic composition of tumor samples is needed for the identification of plasma DNA samples with more than 10% of ctDNA content. Plasma DNA analyses may evolve to become a novel tool for the monitoring of patients with cancer and for the development of personalized medicine. This abstract is also presented as Poster 28. Citation Format: Jelena Belic, Ellen Heitzer, Peter Ulz, Martina Auer, Katja Fischereder, Thomas Bauernhofer, Jochen B. Geigl, Michael R. Speicher. Analysis of ctDNA using the mFastSeqS and plasma–Seq methods for screening and therapy monitoring in prostate cancer patients. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr PR05.