Georg Schaefer

Targeted high throughput sequencing in clinical cancer Settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity

BackgroundMassively parallel sequencing technologies have brought an enormous increase in sequencing throughput. However, these technologies need to be further improved with regard to reproducibility and applicability to clinical samples and settings.MethodsUsing identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV) detection in samples of formalin-fixed paraffin embedded (FFPE) tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity.ResultsWe show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance.Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations.ConclusionsThe application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - in the long term - result in a more reliable prediction of individual cancer therapies.

Prostate cancer diagnosis: value of real-time elastography.

It is well known that prostate cancer (PCa) has a higher cell density than the surrounding normal tissue. This increased cell density leads to an alteration in tissue elasticity, which can be measured and displayed by sonographic-based elastography under real-time conditions. Real-time sonoelastography (RTE) has been proven capable to visualize PCa areas as “hard” lesions and therefore can be used for PCa detection and for targeted ultrasound-guided biopsy. Further applications such as the assessment of local extent of PCa should be considered. This overview describes the capabilities, advantages, and limitations of this new ultrasound technique for PCa diagnosis.

Distinct ERG rearrangement prevalence in prostate cancer: higher frequency in young age and in low PSA prostate cancer.

Background:The TMPRSS2-ERG gene fusion resulting in ERG overexpression has been found in around 50% of prostate cancers (PCa) and is a very early event in tumorigenesis. Most studies have reported on selected surgical cohorts with inconsistent results. We hypothesized that ERG gene rearrangements impact tumor development and investigated the frequency of ERG overexpression in the context of clinicopathological tumor characteristics.Methods:ERG overexpression (ERG+ or ERG-) was determined by immunohistochemistry (IHC) in 1039 radical prostatectomy (RP) tumors and association with PSA, D’Amico risk score, histopathology, biochemical recurrence, body mass index and age of PCa cases was analyzed.Results:ERG+ was associated with younger age at diagnosis (P<0.0001), lower serum PSA (P=0.002) and lower prostate volume (PV) (P=0.001). It was most frequent in the youngest age quartile (⩽55 years, 63.9% ERG+) and decreased constantly with increasing age to 40.8% in the oldest age quartile (⩾67 years, P<0.0001). In the PSA range <4 ng ml−1 the frequency of ERG positivity was 60.2% compared with 47.5 and 49.1% in the PSA ranges 4–10 and ⩾10 ng ml−1, respectively. In the first age quartile, ERG+ patients had lower median serum PSA and fPSA% and smaller PV. In the highest age quartile tumor volume (TV) was increased. Similar differences were observed in the low PSA range. Multivariate analysis identified the first age quartile as a predictor for ERG status (odds ratios (OR) 2.05, P=0.007). No association was found with the D’Amico progression risk score and with biochemical tumor recurrence.Conclusions:ERG+ tumors manifest clinically at lower PSA levels and their prevalence is age dependent. This suggests acceleration of tumor development by ERG overexpression that results in earlier tumor detection in young patients. Long-term results are warranted to determine the impact of ERG overexpression on disease outcome.

Identification of functionally active, low frequency copy number variants at 15q21.3 and 12q21.31 associated with prostate cancer risk

Copy number variants (CNVs) are a recently recognized class of human germ line polymorphisms and are associated with a variety of human diseases, including cancer. Because of the strong genetic influence on prostate cancer, we sought to identify functionally active CNVs associated with susceptibility of this cancer type. We queried low-frequency biallelic CNVs from 1,903 men of Caucasian origin enrolled in the Tyrol Prostate Specific Antigen Screening Cohort and discovered two CNVs strongly associated with prostate cancer risk. The first risk locus (P = 7.7 × 10−4, odds ratio = 2.78) maps to 15q21.3 and overlaps a noncoding enhancer element that contains multiple activator protein 1 (AP-1) transcription factor binding sites. Chromosome conformation capture (Hi-C) data suggested direct cis-interactions with distant genes. The second risk locus (P = 2.6 × 10−3, odds ratio = 4.8) maps to the α-1,3-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase C (MGAT4C) gene on 12q21.31. In vitro cell-line assays found this gene to significantly modulate cell proliferation and migration in both benign and cancer prostate cells. Furthermore, MGAT4C was significantly overexpressed in metastatic versus localized prostate cancer. These two risk associations were replicated in an independent PSA-screened cohort of 800 men (15q21.3, combined P = 0.006; 12q21.31, combined P = 0.026). These findings establish noncoding and coding germ line CNVs as significant risk factors for prostate cancer susceptibility and implicate their role in disease development and progression.

MALDI-MS tissue imaging identification of biliverdin reductase B overexpression in prostate cancer

UNLABELLED New biomarkers are needed to improve the specificity of prostate cancer detection and characterisation of individual tumors. In a proteomics profiling approach using MALDI-MS tissue imaging on frozen tissue sections, we identified discriminating masses. Imaging analysis of cancer, non-malignant benign epithelium and stromal areas of 15 prostatectomy specimens in a test and 10 in a validation set identified characteristic m/z peaks for each tissue type, e.g. m/z 10775 for benign epithelial, m/z 6284 and m/z 6657.5 for cancer and m/z 4965 for stromal tissue. A 10-fold cross-validation analysis showed highest discriminatory ability to separate tissue types for m/z 6284 and m/z 6657.5, both overexpressed in cancer, and a multicomponent mass peak cluster at m/z 10775-10797.4 overexpressed in benign epithelial tissue. ROC AUC values for these three masses ranged from 0.85 to 0.95 in the discrimination of malignant and non-malignant tissue. To identify the underlying proteins, prostate whole tissue extract was separated by nano-HPLC and subjected to MALDI TOF/TOF analysis. Proteins in fractions containing discriminatory m/z masses were identified by MS/MS analysis and candidate marker proteins subsequently validated by immunohistochemistry (IHC). Biliverdin reductase B (BLVRB) turned out to be overexpressed in PCa tissue. BIOLOGICAL SIGNIFICANCE In this study on cryosections of radical prostatectomies of prostate cancer patients, we performed a MALDI-MS tissue imaging analysis and a consecutive protein identification of significant m/z masses by nano-HPLC, MALDI TOF/TOF and MS/MS analysis. We identified BLVRB as a potential biomarker in the discrimination of PCa and benign tissue, also suggesting BVR as a feasible therapeutic target.

Characterization of normal and malignant prostate tissue by Fourier transform infrared microspectroscopy

Prostate cancer has become one of the most common malignancies worldwide. Morphological and histomorphological evaluation of this disease is a well established technique for the cancer classification and has remained relatively unchanged since several decades, although it remains a time consuming and subjective technique, with unsatisfactory levels of inter- and intra-observer discrepancy. Novel approaches for histological recognition are necessary to identify and to investigate cancer in detail. Fourier transform infrared (FTIR) spectroscopic imaging has become an essential tool for the detection, identification and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of cancer progression. Major advantage of this new technique is the acquisition of local molecular expression profiles while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification and separation steps. By using this method it is possible to investigate the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids and small molecules within biological systems by in situ analysis of tissue sections. We applied this technique on prostate cancer patients radical prostatectomy specimens in order to develop new tools for histomorphological analysis and the characterization of snap frozen prostate cancer tissues. As a first step, an optimization of sample preparation, tissue section thickness and IR slide material was performed. Special preparation methods for FTIR imaging are the essential requirements to maintain the spatial arrangement of compounds and avoid delocalization and degradation of the analytes. Subsequently, selected cancer samples were characterized with the prior optimized parameters and analyzed by univariate and cluster analysis. For the interpretation and calibration of the system we correlated the FTIR-images with the histopathological information. With this method it is possible to distinguish between cancer and noncancer areas within a prostate cancer tissue with a resolution of 6.25 μm × 6.25 μm on frozen sections.

Value of magnetic resonance imaging in prostate cancer diagnosis

MRI has shown its potential in prostate cancer (PCa) imaging. MRI is able to demonstrate zonal anatomy with excellent contrast resolution. Furthermore it can detect PCa dependent not only on tumor-size, histological grading, PSA levels, but also on technical equipment and reader’s experience. Non-palpable PCas in the inner and outer gland can be detected by this technique. Another potential is that MRI is helpful for tumor staging and treatment planning as well as response evaluation. Besides the morphological information, MRI can give functional information based on metabolic evaluation with proton magnetic resonance spectroscopy and of tumor angiogenesis based on dynamic contrast-material enhanced MRI and diffusion-weighted imaging. In addition MRI can be used for targeted prostate biopsies; however, the clinical practicability is questionable. Furthermore many data about the value of MRI for PCa diagnosis are based on transrectal ultrasound (TRUS) biopsy findings. Since there is lack of accuracy in fusing MRI images with TRUS images these limit the results of MRI for cancer diagnosis. However, in the future MRI may play an additional role in planning and monitoring minimally invasive PCa therapies. Although, MRI of the prostate seems to be useful, nevertheless this method remains expensive and lacks availability regarding the oncoming requirements.

Tubulin tyrosine ligase like 12 links to prostate cancer through tubulin posttranslational modification and chromosome ploidy.

Prostate cancer is a common cause of death, and an important goal is to establish the pathways and functions of causative genes. We isolated RNAs that are differentially expressed in macrodissected prostate cancer samples. This study focused on 1 identified gene, TTLL12, which was predicted to modify tubulins, an established target for tumor therapy. TTLL12 is the most poorly characterized member of a recently discovered 14‐member family of proteins that catalyze posttranslational modification of tubulins. We show that human TTLL12 is expressed in the proliferating layer of benign prostate. Expression increases during cancer progression to metastasis. It is highly expressed in many metastatic prostate cancer cell lines. It partially colocalizes with vimentin intermediate filaments and cellular structures containing tubulin, including midbodies, centrosomes, intercellular bridges and the mitotic spindle. Downregulation of TTLL12 affects several posttranslational modifications of tubulin (detyrosination and subsequent deglutamylation and polyglutamylation). Overexpression alters chromosomal ploidy. These results raise the possibility that TTLL12 could contribute to tumorigenesis through effects on the cytoskeleton, tubulin modification and chromosome number stability. This study contributes a step toward developing more selective agents targeting microtubules, an already successful target for tumor therapy.

The influence of prostate volume on prostate cancer detection using a combined approach of contrast-enhanced ultrasonography-targeted and systematic grey-scale biopsy

To evaluate the influence of prostate volume (PV) on the detection of prostate cancer using a combined approach of contrast‐enhanced ultrasonography (CEUS) and grey‐scale US‐guided systematic biopsy (SB).

Upgrading of Gleason score 6 prostate cancers on biopsy after prostatectomy in the low and intermediate tPSA range

When offering watchful waiting or active monitoring protocols to prostate cancer (PCa) patients, differentiation between Gleason scores (GS) 6 and 7 at biopsy is important. However, upgrading after prostatectomy is common. We investigated the impact of different PSA levels on misclassification in the PSA range of 2–3.9 and 4–10 ng ml−1. A total of 448 patients with GS 6 PCa on prostate biopsy were evaluated by comparing biopsy and prostatectomy GS. Possible over diagnosis was defined as GS <7, pathological stage pT2a and negative surgical margins, and possible under diagnosis was defined as pT3a or greater, or positive surgical margins; the percentage of over- or under diagnosis was determined for correctly and upgraded tumors after prostatectomy. A match between biopsy and prostatectomy GS was found in 210 patients (46.9%). Patients in the PSA range of 2.0–3.9 and 4.0–10.0 ng ml−1 were upgraded in 32.6 and 44.0%, respectively. Over diagnosis was more common than under diagnosis (23.2% vs 15.6%). When upgraded there was a significant increase in under diagnosis. As almost 40% of GS 6 tumors on biopsy are GS 7 or higher after surgery with a significant rise in under diagnosis there is a risk of misclassification and subsequent delayed or even insufficient treatment, when relying on favorable biopsy GS.