Publications by authors named "Peter Schraml"

96 Publications

Cytoplasmic ADP-ribosylation levels correlate with markers of patient outcome in distinct human cancers.

Mod Pathol 2021 Mar 19. Epub 2021 Mar 19.

University of Zurich (UZH), Department of Molecular Mechanisms of Disease (DMMD), Zurich, Switzerland.

ADP-ribosylation (ADPR) is a posttranslational modification whose importance in oncology keeps increasing due to frequent use of PARP inhibitors (PARPi) to treat different tumor types. Due to the lack of suitable tools to analyze cellular ADPR levels, ADPR's significance for cancer progression and patient outcome is unclear. In this study, we assessed ADPR levels by immunohistochemistry using a newly developed anti-ADP-ribose (ADPr) antibody, which is able to detect both mono- and poly-ADPR. Tissue microarrays containing brain (n = 103), breast (n = 1108), colon (n = 236), lung (n = 138), ovarian (n = 142), and prostate (n = 328) cancers were used to correlate ADPR staining intensities to clinico-pathological data, including patient overall survival (OS), tumor grade, tumor stage (pT), lymph node status (pN), and the presence of distant metastasis (pM). While nuclear ADPR was detected only in a minority of the samples, cytoplasmic ADPR (cyADPR) staining was observed in most tumor types. Strong cyADPR intensities were significantly associated with better overall survival in invasive ductal breast cancer (p < 0.0001), invasive lobular breast cancer (p < 0.005), and high grade serous ovarian cancer patients (p < 0.01). Furthermore, stronger cytoplasmic ADPR levels significantly correlated with early tumor stage in colorectal and in invasive ductal breast adenocarcinoma (p < 0.0001 and p < 0.01, respectively) and with the absence of regional lymph node metastasis in colorectal adenocarcinoma (p < 0.05). No correlation to cyADPR was found for prostate and lung cancer or brain tumors. In conclusion, our new anti-ADP-ribose antibody revealed heterogeneous ADPR staining patterns with predominant cytoplasmic ADPR staining in most tumor types. Different cyADPR staining patterns could help to better understand variable response rates to PARP inhibitors in the future.
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http://dx.doi.org/10.1038/s41379-021-00788-9DOI Listing
March 2021

Dual functions of SPOP and ERG dictate androgen therapy responses in prostate cancer.

Nat Commun 2021 02 2;12(1):734. Epub 2021 Feb 2.

Institute of Oncology Research, Faculty of Biomedical Sciences, Università della Svizzera italiana, 6500, Bellinzona, TI, Switzerland.

Driver genes with a mutually exclusive mutation pattern across tumor genomes are thought to have overlapping roles in tumorigenesis. In contrast, we show here that mutually exclusive prostate cancer driver alterations involving the ERG transcription factor and the ubiquitin ligase adaptor SPOP are synthetic sick. At the molecular level, the incompatible cancer pathways are driven by opposing functions in SPOP. ERG upregulates wild type SPOP to dampen androgen receptor (AR) signaling and sustain ERG activity through degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG-function and enable oncogenic androgen receptor signaling. This dichotomy regulates the response to therapeutic interventions in the AR pathway. While mutant SPOP renders tumor cells susceptible to androgen deprivation therapies, ERG promotes sensitivity to high-dose androgen therapy and pharmacological inhibition of wild type SPOP. More generally, these results define a distinct class of antagonistic cancer drivers and a blueprint toward their therapeutic exploitation.
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http://dx.doi.org/10.1038/s41467-020-20820-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854732PMC
February 2021

Liquid Biopsies in Renal Cell Carcinoma-Recent Advances and Promising New Technologies for the Early Detection of Metastatic Disease.

Front Oncol 2020 28;10:582843. Epub 2020 Oct 28.

Department of Pathology and Molecular Pathology, University of Zurich and University Hospital Zurich, Zurich, Switzerland.

Clear cell renal cell carcinoma (ccRCC) displays a highly varying clinical progression, from slow growing localized tumors to very aggressive metastatic disease (mRCC). Almost a third of all patients with ccRCC show metastatic dissemination at presentation while another third develop metastasis during the course of the disease. Survival rates of mRCC patients remain low despite the development of novel targeted treatment regimens. Biomarkers indicating disease progression could help to define its aggressive potential and thus guide patient management. However, molecular markers that can reliably assess metastatic dissemination and disease recurrence in ccRCC have not been recommended for clinical practice to date. Liquid biopsies could provide an attractive and non-invasive method to determine the risk of recurrence or metastatic dissemination during follow-up and thus assist the search for surveillance biomarkers in ccRCC tumors. A wide spectrum of circulating molecules have already shown considerable potential for ccRCC diagnosis and prognostication. In this review, we outline state of the art of the key circulating analytes such as cfDNA, cfRNA, proteins, and exosomes that may serve as biomarkers for the longitudinal monitoring of ccRCC progression to metastasis. Moreover, we address some of the prevailing limitations in the past approaches and present promising adoptable technologies that could help to pursue the implementation of liquid biopsies as a prognostic tool for mRCC.
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http://dx.doi.org/10.3389/fonc.2020.582843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7656014PMC
October 2020

Frequent Germline and Somatic Single Nucleotide Variants in the Promoter Region of the Ribosomal RNA Gene in Japanese Lung Adenocarcinoma Patients.

Cells 2020 11 3;9(11). Epub 2020 Nov 3.

Histopathology Core Facility, Niigata University Faculty of Medicine, Niigata 951-8510, Japan.

Ribosomal RNA (rRNA), the most abundant non-coding RNA species, is a major component of the ribosome. Impaired ribosome biogenesis causes the dysfunction of protein synthesis and diseases called "ribosomopathies," including genetic disorders with cancer risk. However, the potential role of rRNA gene (rDNA) alterations in cancer is unknown. We investigated germline and somatic single-nucleotide variants (SNVs) in the rDNA promoter region (positions -248 to +100, relative to the transcription start site) in 82 lung adenocarcinomas (LUAC). Twenty-nine tumors (35.4%) carried germline SNVs, and eight tumors (9.8%) harbored somatic SNVs. Interestingly, the presence of germline SNVs between positions +1 and +100 ( = 12; 14.6%) was associated with significantly shorter recurrence-free survival (RFS) and overall survival (OS) by univariate analysis ( < 0.05, respectively), and was an independent prognostic factor for RFS and OS by multivariate analysis. LUAC cell line PC9, carrying rDNA promoter SNV at position +49, showed significantly higher ribosome biogenesis than H1650 cells without SNV. Upon nucleolar stress induced by actinomycin D, PC9 retained significantly higher ribosome biogenesis than H1650. These results highlight the possible functional role of SNVs at specific sites of the rDNA promoter region in ribosome biogenesis, the progression of LUAC, and their potential prognostic value.
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http://dx.doi.org/10.3390/cells9112409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692307PMC
November 2020

The DNA hypermethylation phenotype of colorectal cancer liver metastases resembles that of the primary colorectal cancers.

BMC Cancer 2020 Apr 6;20(1):290. Epub 2020 Apr 6.

Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.

Background: Identifying molecular differences between primary and metastatic colorectal cancers-now possible with the aid of omics technologies-can improve our understanding of the biological mechanisms of cancer progression and facilitate the discovery of novel treatments for late-stage cancer. We compared the DNA methylomes of primary colorectal cancers (CRCs) and CRC metastases to the liver. Laser microdissection was used to obtain epithelial tissue (10 to 25 × 10 μm) from sections of fresh-frozen samples of primary CRCs (n = 6), CRC liver metastases (n = 12), and normal colon mucosa (n = 3). DNA extracted from tissues was enriched for methylated sequences with a methylCpG binding domain (MBD) polypeptide-based protocol and subjected to deep sequencing. The performance of this protocol was compared with that of targeted enrichment for bisulfite sequencing used in a previous study of ours.

Results: MBD enrichment captured a total of 322,551 genomic regions (249.5 Mb or ~ 7.8% of the human genome), which included over seven million CpG sites. A few of these regions were differentially methylated at an expected false discovery rate (FDR) of 5% in neoplastic tissues (primaries: 0.67%, i.e., 2155 regions containing 279,441 CpG sites; liver metastases: 1%, i.e., 3223 regions containing 312,723 CpG sites) as compared with normal mucosa samples. Most of the differentially methylated regions (DMRs; 94% in primaries; 70% in metastases) were hypermethylated, and almost 80% of these (1882 of 2396) were present in both lesion types. At 5% FDR, no DMRs were detected in liver metastases vs. primary CRC. However, short regions of low-magnitude hypomethylation were frequent in metastases but rare in primaries. Hypermethylated DMRs were far more abundant in sequences classified as intragenic, gene-regulatory, or CpG shelves-shores-island segments, whereas hypomethylated DMRs were equally represented in extragenic (mainly, open-sea) and intragenic (mainly, gene bodies) sequences of the genome. Compared with targeted enrichment, MBD capture provided a better picture of the extension of CRC-associated DNA hypermethylation but was less powerful for identifying hypomethylation.

Conclusions: Our findings demonstrate that the hypermethylation phenotype in CRC liver metastases remains similar to that of the primary tumor, whereas CRC-associated DNA hypomethylation probably undergoes further progression after the cancer cells have migrated to the liver.
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http://dx.doi.org/10.1186/s12885-020-06777-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137338PMC
April 2020

Loss of CDKN1A mRNA and Protein Expression Are Independent Predictors of Poor Outcome in Chromophobe Renal Cell Carcinoma Patients.

Cancers (Basel) 2020 Feb 17;12(2). Epub 2020 Feb 17.

Department of Pathology and Molecular Pathology, University and University Hospital Zurich, Zurich CH-8091, Switzerland.

Chromophobe renal cell carcinoma (chRCC) patients have good prognosis. Only 5%-10% patients die of metastatic disease after tumorectomy, but tumor progression cannot be predicted by histopathological parameters alone. chRCC are characterized by losses of many chromosomes, whereas gene mutations are rare. In this study, we aim at identifying genes indicating chRCC progression. A bioinformatic approach was used to correlate chromosomal loss and mRNA expression from 15287 genes from The Cancer Genome Atlas (TCGA) database. All genes in TCGA chromophobe renal cancer dataset (KICH) for which a significant correlation between chromosomal loss and mRNA expression was shown, were identified and their associations with outcome was assessed. Genome-wide DNA copy-number alterations were analyzed by Affymetrix OncoScan CNV FFPE Microarrays in a second cohort of Swiss chRCC. In both cohorts, tumors with loss of chromosomes 2, 6, 10, 13, 17 and 21 had signs of tumor progression. There were 4654 genes located on these chromosomes, and 13 of these genes had reduced mRNA levels, which was associated with poor outcome in chRCC. Decreased CDKN1A expression at mRNA ( = 0.02) and protein levels ( = 0.02) were associated with short overall survival and were independent predictors of prognosis ( <0.01 and <0.05 respectively). CDKN1A expression status is a prognostic biomarker independent of tumor stage. CDKN1A immunohistochemistry may be used to identify chRCC patients at greater risk of disease progression.
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http://dx.doi.org/10.3390/cancers12020465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072616PMC
February 2020

Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity.

Nucleic Acids Res 2020 02;48(3):e17

IBM Research Zürich, Säumerstrasse 4, CH-8803 Rüschlikon, Switzerland.

Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.
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http://dx.doi.org/10.1093/nar/gkz1151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026647PMC
February 2020

Multi-institutional re-evaluation of prognostic factors in chromophobe renal cell carcinoma: proposal of a novel two-tiered grading scheme.

Virchows Arch 2020 Mar 23;476(3):409-418. Epub 2019 Nov 23.

Department of Pathology and Molecular Pathology, University and University Hospital Zurich, Schmelzbergstrasse 12, CH-8091, Zurich, Switzerland.

A histological grading system of chromophobe renal cell carcinoma (chRCC) is highly desirable to identify approximately 5-10% of tumors at risk for progression. Validation studies failed to demonstrate a correlation between the four-tiered WHO/ISUP grade and outcome. Previous proposals with three-tiered chromophobe grading systems could not be validated. In this study, the presence of sarcomatoid differentiation, necrosis, and mitosis was analyzed in a Swiss cohort (n = 42), an Italian cohort (n = 103), a German cohort (n = 54), a Japanese cohort (n = 119), and The Cancer Genome Atlas cohort (n = 64). All 3 histological parameters were significantly associated with shorter time to tumor progression and overall survival in univariate analysis. Interobserver variability for identification of these parameters was measured by Krippendorff's alpha coefficient and showed high concordance for the identification of sarcomatoid differentiation and tumor necrosis, but only low to medium concordance for the identification of mitosis. Therefore, we tested a two-tiered tumor grading system (low versus high grade) based only on the presence of sarcomatoid differentiation and/or necrosis finding in the combined cohorts (n = 382). pT stage, patient's age (> 65 vs ≤ 65), lymph node and/or distant metastasis, and the two-tiered grading system (low versus high grade) were significantly associated with overall survival and were independent prognostic parameters in multivariate analysis (Cox proportional hazard). This multi-institutional evaluation of prognostic parameters suggests tumor necrosis and sarcomatoid differentiation as reproducible components of a two-tiered chromophobe tumor grading system.
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http://dx.doi.org/10.1007/s00428-019-02710-wDOI Listing
March 2020

Cancer Sample Biobanking at the Next Level: Combining Tissue With Living Cell Repositories to Promote Precision Medicine.

Front Cell Dev Biol 2019 22;7:246. Epub 2019 Oct 22.

Department of Pathology and Molecular Pathology, University Hospital of Zürich, Zurich, Switzerland.

Biorespositories of formalin-fixed and paraffin-embedded (FFPE) or fresh frozen human tissues from malignant diseases generated as integral part of the diagnostic workup in many pathology departments have been pivotal resources for translational cancer studies. However, such tissue biobanks have traditionally contained only non-viable specimens and thus cannot enable functional assays for the discovery and validation of therapeutic targets or the assessment of drug responses and resistance to treatment. To overcome these limitations, we have developed a next-generation comprehensive biobanking platform that includes the generation of patient-derived cell models from colorectal, pancreatic and kidney cancers among others. As such patient-derived cell (PDC) models retain important features of the original human tumors, they have emerged as relevant tools for more dynamic clinical and experimental analyses of cancer. Here, we describe details of the complex processes of acquisition and processing of patient-derived samples, propagation, annotation, characterization and distribution of resulting cell models and emphasize the requirements of quality assurance, organizational considerations and investment into resources. Taken together, we show how clinical tissue collections can be taken to the next level thus promising major new opportunities for understanding and treating cancer in the context of precision medicine.
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http://dx.doi.org/10.3389/fcell.2019.00246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817465PMC
October 2019

Classic Chromophobe Renal Cell Carcinoma Incur a Larger Number of Chromosomal Losses Than Seen in the Eosinophilic Subtype.

Cancers (Basel) 2019 Oct 3;11(10). Epub 2019 Oct 3.

Department of Pathology and Molecular Pathology, University and University Hospital Zurich, CH-8091 Zurich, Switzerland.

Chromophobe renal cell carcinoma (chRCC) is a renal tumor subtype with a good prognosis, characterized by multiple chromosomal copy number variations (CNV). The World Health Organization (WHO) chRCC classification guidelines define a classic and an eosinophilic variant. Large cells with reticular cytoplasm and prominent cell membranes (pale cells) are characteristic for classic chRCC. Classic and eosinophilic variants were defined in 42 Swiss chRCCs, 119 Japanese chRCCs and in whole-slide digital images of 66 chRCCs from the Cancer Genome Atlas (TCGA) kidney chromophobe (KICH) dataset. 32 of 42 (76.2%) Swiss chRCCs, 90 of 119 (75.6%) Japanese chRCCs and 53 of 66 (80.3%) TCGA-KICH were classic chRCCs. There was no survival difference between eosinophilic and classic chRCC in all three cohorts. To identify a genotype/phenotype correlation, we performed a genome-wide CNV analysis using Affymetrix OncoScan CNV Assay (Affymetrix/Thermo Fisher Scientific, Waltham, MA, USA) in 33 Swiss chRCCs. TCGA-KICH subtypes were compared with TCGA CNV data. In the combined Swiss and TCGA-KICH cohorts, losses of chromosome 1, 2, 6, 10, 13, and 17 were significantly more frequent in classic chRCC ( < 0.05, each), suggesting that classic chRCC are characterized by higher chromosomal instability. This molecular difference justifies the definition of two chRCC variants. Absence of pale cells could be used as main histological criterion to define the eosinophilic variant of chRCC.
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http://dx.doi.org/10.3390/cancers11101492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826417PMC
October 2019

Tracing Clonal Dynamics Reveals that Two- and Three-dimensional Patient-derived Cell Models Capture Tumor Heterogeneity of Clear Cell Renal Cell Carcinoma.

Eur Urol Focus 2021 Jan 29;7(1):152-162. Epub 2019 Jun 29.

Department of Pathology and Molecular Pathology, University Hospital and University of Zurich, Zurich, Switzerland.

Background: Extensive DNA sequencing has led to an unprecedented view of the diversity of individual genomes and their evolution among patients with clear cell renal cell carcinoma (ccRCC).

Objective: To understand subclonal architecture and dynamics of patient-derived two-dimensional (2D) and three-dimensional (3D) ccRCC models in vitro, in order to determine whether they mirror ccRCC inter- and intratumor heterogeneity.

Design, Setting, And Participants: We have established a comprehensive platform of living renal cancer cell models from ccRCC surgical specimens.

Outcome Measurements And Statistical Analysis: We confirmed the concordance of 2D and 3D patient-derived cell (PDC) models with the original tumor tissue in terms of histology, biomarker expression, cancer driver mutations, and copy number alterations. We addressed inter- and intrapatient heterogeneity by analyzing clonal dynamics during serial passaging.

Results And Limitations: In-depth genetic characterization verified the presence of heterogeneous cell populations, and revealed a high degree of similarity between subclonal compositions of monolayer and organoid cell cultures and the corresponding parental ccRCCs. Clonal dynamics were evident during serial passaging of cells in vitro, suggesting that PDC cultures can offer insights into evolutionary potential and treatment susceptibility of ccRCC subclones in vivo. Proof-of-concept drug profiling using selected ccRCC-targeted therapy agents highlighted patient-specific vulnerabilities in PDC models that could not be anticipated by interrogating commercially available cell lines.

Conclusions: We demonstrate that PDC models mirror inter- and intratumor heterogeneity of ccRCC in vitro. Based on our findings, we envision that the use of these models will advance our understanding of the trajectories that cause genetic diversity and their consequences for treatment on an individual level.

Patient Summary: In this study, we developed two- and three-dimensional patient-derived models from clear cell renal cell carcinoma (ccRCC) as "mini-tumors in a dish." We show that these cell models retain important features of the human ccRCCs such as the profound tumor heterogeneity, thus highlighting their importance for cancer research and precision medicine.
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http://dx.doi.org/10.1016/j.euf.2019.06.009DOI Listing
January 2021

Allele Loss and Reduced Expression of CYCLOPS Genes is a Characteristic Feature of Chromophobe Renal Cell Carcinoma.

Transl Oncol 2019 Sep 11;12(9):1131-1137. Epub 2019 Jun 11.

Department of Pathology and Molecular Pathology, Schmelzbergstrasse 12, University and University Hospital Zurich, CH-8091 Zurich, Switzerland. Electronic address:

Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS (CYCLOPS) genes have been recently identified as the most enriched class of copy-number associated gene dependencies in human cancer. These genes are cell essential and render tumor cells highly sensitive to the expression of the remaining copy. Chromophobe renal cell carcinoma (chRCC) is characterized by frequent chromosomal deletions, but the relevance of CYCLOPS genes in this tumor subtype is unclear. We found 39 (31%) of 124 recently published candidate CYCLOPS genes (B. Paolella et al., eLife 2017;6:e23268) located on 7 autosomes that are frequently lost in chRCC. GISTIC and RNA-seq data obtained from the TCGA-KICH database showed that 62% of these CYCLOPS genes had significantly lower expression levels in samples with deletion of the respective gene. As copy number (CN) loss of the CYCLOPS gene SF3B1 (Splicing factor 3B subunit 1) has been recently reported in 71% chRCC, we explored the relevance of SF3B1 CN alteration and SF3B1 expression in a set of chRCC and additional oncocytic renal neoplasms. The frequency of SF3B1 CN loss (65%) was similar to that obtained from the TCGA-KICH database and correlated significantly with both lower SF3B1 mRNA (P < .05) and protein expression (P < .001). Other tumor subtypes with oncocytic cytoplasm had normal SF3B1 CN and displayed strong SF3B1 protein expression. These results suggest that CN loss of CYCLOPS genes is a characteristic feature in chRCC. Since many CYCLOPS genes code for components of proteasomes and transcriptional regulation, their alteration could make chRCC vulnerable to targeted drugs.
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http://dx.doi.org/10.1016/j.tranon.2019.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563336PMC
September 2019

Quantitative microimmunohistochemistry for the grading of immunostains on tumour tissues.

Nat Biomed Eng 2019 06 8;3(6):478-490. Epub 2019 Apr 8.

IBM Research-Zurich, Rüschlikon, Switzerland.

Immunohistochemistry is the gold-standard method for cancer-biomarker identification and patient stratification. Yet, owing to signal saturation, its use as a quantitative assay is limited as it cannot distinguish tumours with similar biomarker-expression levels. Here, we introduce a quantitative microimmunochemistry assay that enables the acquisition of dynamic information, via a metric of the evolution of the immunohistochemistry signal during tissue staining, for the quantification of relative antigen density on tissue surfaces. We used the assay to stratify 30 patient-derived breast-cancer samples into conventional classes and to determine the proximity of each sample to the other classes. We also show that the assay enables the quantification of multiple biomarkers (human epidermal growth factor receptor, oestrogen receptor and progesterone receptor) in a standard breast-cancer panel. The integration of quantitative microimmunohistochemistry into current pathology workflows may lead to improvements in the precision of biomarker quantification.
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http://dx.doi.org/10.1038/s41551-019-0386-3DOI Listing
June 2019

Identification and Validation of a Biomarker Signature in Patients With Resectable Pancreatic Cancer via Genome-Wide Screening for Functional Genetic Variants.

JAMA Surg 2019 06 19;154(6):e190484. Epub 2019 Jun 19.

Institute for Regenerative Medicine, University of Zurich, Zurich, Switzerland.

Importance: Surgery currently offers the only chance for a cure in pancreatic ductal adenocarcinoma (PDAC), but it carries a significant morbidity and mortality risk and results in varying oncologic outcomes. At present, to our knowledge, there are no tests available before surgical resection to identify tumors with an aggressive biological phenotype that could guide personalized treatment strategies.

Objective: Identification of noninvasive genetic biomarkers that could direct therapy in patients whose cases are amenable to pancreatic cancer resection.

Design, Setting, And Participants: This multicenter study combined a prospective European cohort of patients with PDAC who underwent pancreatic resection (from University Hospital of Zurich, Zurich, Switzerland; Cantonal Hospital of Winterthur, Winterthur, Switzerland; and University Clinic of Ulm, Ulm, Germany) with data from the Cancer Genome Atlas database in the United States, which includes prospectively registered patients with PDAC. A genome-wide screening for functional single-nucleotide polymorphisms (SNPs) that affect PDAC survival was conducted using the European cohort for identification and the Cancer Genome Atlas cohort for validation. We used Cox proportional hazards models to screen for high-frequency polymorphic variants that are associated with allelic differences in tumor-associated survival and either result in an altered protein structure and function or reside in known regulatory noncoding genomic regions. The false-discovery rate method was applied for multiple hypothesis-testing corrections. Data analysis occurred from November 2017 to May 2018.

Exposures: Pancreatic resection.

Main Outcomes And Measures: Tumor-associated survival.

Results: A total of 195 patients in the European cohort were included, as well as 136 patients in the Cancer Genome Atlas cohort (overall median [range] age, 66 [19-87] years; 156 [47.1%] were women, and 175 [52.9%] were men). Two SNPs in noncoding, functional regions of genes that regulate cancer progression, invasion, and metastasis were identified (CHI3L2 SNP rs684559 and CD44 SNP rs353630). These were associated with survival after PDAC resection; patients who carry the risk alleles at 1 of both SNP loci had a 2.63-fold increased risk for tumor-associated death compared with those with protective genotypes (hazard ratio for survival, 0.38 [95% CI, 0.27-0.53]; P = 1.0 × 10-8).

Conclusions And Relevance: The identified polymorphisms may serve as a noninvasive biomarker signature of prospective survival after pancreatic resection that is readily available at the time of PDAC diagnosis. This signature can be used to identify a subset of high-risk patients with PDAC with very low survival probability who might be eligible for inclusion in clinical trials of new therapeutic strategies, including neoadjuvant chemotherapy protocols. In addition, the biological knowledge about these SNPs could help guide the development of individualized genomic strategies for PDAC therapies.
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http://dx.doi.org/10.1001/jamasurg.2019.0484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6583393PMC
June 2019

IL-8 and CXCR1 expression is associated with cancer stem cell-like properties of clear cell renal cancer.

J Pathol 2019 07 11;248(3):377-389. Epub 2019 Apr 11.

Department of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland.

Recent studies suggest that clear cell renal cell carcinoma (ccRCC) possesses a rare population of cancer stem cells (CSCs) that might contribute to tumor heterogeneity, metastasis and therapeutic resistance. Nevertheless, their relevance for renal cancer is still unclear. In this study, we successfully isolated CSCs from established human ccRCC cell lines. CSCs displayed high expression of the chemokine IL-8 and its receptor CXCR1. While recombinant IL-8 significantly increased CSC number and properties in vitro, CXCR1 inhibition using an anti-CXCR1 antibody or repertaxin significantly reduced these features. After injection into immune-deficient mice, CSCs formed primary tumors that metastasized to the lung and liver. All xenografted tumors in mice expressed high levels of IL-8 and CXCR1. Furthermore, IL-8/CXCR1 expression significantly correlated with decreased overall survival in ccRCC patients. These results suggest that the IL-8/CXCR1 phenotype is associated with CSC-like properties in renal cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618115PMC
July 2019

Specific immune cell and lymphatic vessel signatures identified by image analysis in renal cancer.

Mod Pathol 2019 07 8;32(7):1042-1052. Epub 2019 Feb 8.

Department of Pathology and Molecular Pathology, University Hospital Zurich and University Zurich, Schmelzbergstrasse 12, CH-8091, Zurich, Switzerland.

Anti-angiogenic therapy and immune checkpoint inhibition are novel treatment strategies for patients with renal cell carcinoma. Various components and structures of the tumor microenvironment are potential predictive biomarkers and also attractive treatment targets. Macrophages, tumor infiltrating lymphocytes, vascular and lymphatic vessels represent an important part of the tumor immune environment, but their functional phenotypes and relevance for clinical outcome are yet ill defined. We applied Tissue Phenomics methods including image analysis for the standardized quantification of specific components and structures within the tumor microenvironment to profile tissue sections from 56 clear cell renal cell carcinoma patients. A characteristic composition and unique spatial relationship of CD68+ macrophages and tumor infiltrating lymphocytes correlated with overall survival. An inverse relationship was found between vascular (CD34) and lymphatic vessel (LYVE1) density. In addition, outcome was significantly better in patients with high blood vessel density in the tumors, whereas increased lymphatic vessel density in the tumors was associated with worse outcome. The Tissue Phenomics imaging analysis approach allowed visualization and simultaneous quantification of immune environment components, adding novel contextual information, and biological insights with potential applications in treatment response prediction.
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http://dx.doi.org/10.1038/s41379-019-0214-zDOI Listing
July 2019

Expression and Mutation Patterns of PBRM1, BAP1 and SETD2 Mirror Specific Evolutionary Subtypes in Clear Cell Renal Cell Carcinoma.

Neoplasia 2019 02 16;21(2):247-256. Epub 2019 Jan 16.

Department of Pathology and Molecular Pathology, University Hospital Zurich and University Zurich, Zurich, Switzerland. Electronic address:

Bi-allelic inactivation of the VHL gene on chromosome 3p is the characteristic feature in most clear cell renal cell carcinomas (ccRCC). Frequent gene alterations were also identified in SETD2, BAP1 and PBRM1, all of which are situated on chromosome 3p and encode histone/chromatin regulators. The relationship between gene mutation, loss of protein expression and the correlations with clinicopathological parameters is important for the understanding of renal cancer progression. We analyzed PBRM1 and BAP1 protein expression as well as the tri-methylation state of H3K36 as a surrogate marker for SETD2 activity in more than 700 RCC samples. In ccRCC loss of nuclear PBRM1 (68%), BAP1 (40%) and H3K36me3 (47%) expression was significantly correlated with each other, advanced tumor stage, poor tumor differentiation (P < .0001 each), and necrosis (P < .005) Targeted next generation sequencing of 83 ccRCC samples demonstrated a significant association of genetic mutations in PBRM1, BAP1, and SETD2 with absence of PBRM1, BAP1, and HEK36me3 protein expression (P < .05, each). By assigning the protein expression patterns to evolutionary subtypes, we revealed similar clinical phenotypes as suggested by TRACERx Renal. Given their important contribution to tumor suppression, we conclude that combined functional inactivation of PBRM1, BAP1, SETD2 and pVHL is critical for ccRCC progression.
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http://dx.doi.org/10.1016/j.neo.2018.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355619PMC
February 2019

Clear cell renal cell carcinoma with wild-type von Hippel-Lindau gene: a non-existent or new tumour entity?

Histopathology 2019 Jan;74(1):60-67

Department of Pathology and Molecular Pathology, University and University Hospital Zurich, Zurich, Switzerland.

The current World Health Organisation (WHO) classification of renal tumours is based on characteristic histological features or specific molecular alterations. von Hippel-Lindau (VHL) alteration is the hallmark of clear cell renal cell carcinoma (RCC). After identification of the MiT translocation family of tumours, clear cell papillary renal cancer and others, the group of ccRCC with wild-type VHL is small. TCEB1 mutation combined with chromosome 8q loss is an emerging tumour entity with wild-type VHL. Inactivation of TCEB1 increases HIF stabilisation via the same mechanism as VHL inactivation. Importantly, recent molecular analyses suggest the existence of another 'VHL wild-type' evolutionary subtype of clear cell RCC in addition to TCEB1 mutated RCC and clear cell papillary renal cancer. These tumours are characterised by an aggressive behaviour, high tumour cell proliferation rate, elevated chromosomal instability and frequent presence of sarcomatoid differentiation. Future clinicopathological studies will have to provide data to determine whether TCEB1 tumours and clear cell RCC with wild-type VHL are separate tumour entities or represent variants of a clear cell RCC tumour family.
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http://dx.doi.org/10.1111/his.13749DOI Listing
January 2019

Scavenger receptor BI promotes cytoplasmic accumulation of lipoproteins in clear-cell renal cell carcinoma.

J Lipid Res 2018 11 1;59(11):2188-2201. Epub 2018 Sep 1.

Institute of Clinical Chemistry University of Zurich and University Hospital of Zurich, Zurich, Switzerland

Clear-cell renal cell carcinomas (ccRCCs) are characterized by inactivation of the von Hippel-Lindau (VHL) gene and intracellular lipid accumulation by unknown pathomechanisms. The immunochemical analysis of 356 RCCs revealed high abundance of apoA-I and apoB, as well as scavenger receptor BI (SR-BI) in the ccRCC subtype. Given the characteristic loss of VHL function in ccRCC, we used VHL-defective and VHL-proficient cells to study the potential influence of VHL on lipoprotein uptake. VHL-defective patient-derived ccRCC cells and cell lines (786O and RCC4) showed enhanced uptake as well as less resecretion and degradation of radio-iodinated HDL and LDL (I-HDL and I-LDL, respectively) compared with the VHL-proficient cells. The ccRCC cells showed enhanced vascular endothelial growth factor (VEGF) and SR-BI expression compared with normal kidney epithelial cells. Uptake of I-HDL and I-LDL by patient-derived normal kidney epithelial cells as well as the VHL-reexpressing ccRCC cell lines, 786-O-VHL and RCC4-O-VHL cells, was strongly enhanced by VEGF treatment. The knockdown of the VEGF coreceptor, neuropilin-1 (NRP1), as well as blocking of SR-BI significantly reduced the uptake of lipoproteins into ccRCC cells in vitro. LDL stimulated proliferation of 786-O cells more potently than 786-O-VHL cells in a NRP1- and SR-BI-dependent manner. In conclusion, enhanced lipoprotein uptake due to increased activities of VEGF/NRP1 and SR-BI promotes lipid accumulation and proliferation of VHL-defective ccRCC cells.
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http://dx.doi.org/10.1194/jlr.M083311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210910PMC
November 2018

Fluorocholine Transport Mediated by the Organic Cation Transporter 2 (OCT2, SLC22A2): Implication for Imaging of Kidney Tumors.

Drug Metab Dispos 2018 08 24;46(8):1129-1136. Epub 2018 May 24.

Departments of Clinical Pharmacology and Toxicology (M.V., A.T., Z.G., S.H., C.L., C.H., G.A.K.-U.) and Pathology and Molecular Pathology (P.H.S., H.M.), University Hospital Zurich, University of Zurich, Zurich, Switzerland; and Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China (C.L.).

[F]fluorocholine is the fluorinated analog of [C]choline and is used in positron emission tomography to monitor tumor metabolic activity. Although important to optimize its use and expand the clinical indications, the molecular determinants of fluorocholine cellular uptake are poorly characterized. In this work, we described the influx kinetics of fluorocholine mediated by the organic cation transporter 2 (OCT2, SLC22A2) and compared with that of choline. Then we characterized the expression pattern of OCT2 in renal cell carcinoma (RCC). In HEK293 cells stably transfected with OCT2 fluorocholine influx, kinetics was biphasic, suggesting two independent binding sites: a high-affinity (K = 14 ± 8 M, V = 1.3 ± 0.5 nmol mg min) and a low-affinity component (K = 1.8 ± 0.3 mM, V = 104 ± 4.5 nmol mg min). Notably, choline was found to be transported with sigmoidal kinetics typical of homotropic positive cooperativity (h = 1.2, 95% confidence interval 1.1-1.3). OCT2 mRNA expression level was found significantly decreased in primary but not in metastatic RCC. Tissue microarray immunostaining of 216 RCC biopsies confirmed that the OCT2 protein level was consistent with that of the mRNA. The kinetic properties described in this work suggest that OCT2 is likely to play a dominant role in [F]fluorocholine uptake in vivo. OCT2-altered expression in primary and metastatic cancer cells, as compared with the surrounding tissues, could be exploited in RCC imaging, especially to increase the detection sensitivity for small metastatic lesions, a major clinical challenge during the initial staging of RCC.
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http://dx.doi.org/10.1124/dmd.118.081091DOI Listing
August 2018

Maturation of tertiary lymphoid structures and recurrence of stage II and III colorectal cancer.

Oncoimmunology 2018;7(2):e1378844. Epub 2017 Dec 18.

Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT), Carinagasse 47, Feldkirch, Austria.

Tertiary lymphoid structures (TLS) are associated with favorable outcome in non-metastatic colorectal carcinoma (nmCRC), but the dynamics of TLS maturation and its association with effective anti-tumor immune surveillance in nmCRC are unclear. Here, we hypothesized that not only the number of TLS but also their composition harbors information on recurrence risk in nmCRC. In a comprehensive molecular, tissue, laboratory, and clinical analysis of 109 patients with stage II/III nmCRC, we assessed TLS numbers and degree of maturation in surgical specimens by multi-parameter immunofluorescence of follicular dendritic cell (FDC) and germinal center (GC) markers. TLS formed in most tumors and were significantly more prevalent in highly-microsatellite-instable (MSI-H) and/or BRAF-mutant nmCRC. We could distinguish three sequential TLS maturation stages which were characterized by increasing prevalence of FDCs and mature B-cells: [1] Early TLS, composed of dense lymphocytic aggregates without FDCs, [2] Primary follicle-like TLS, having FDCs but no GC reaction, and [3] Secondary follicle-like TLS, having an active GC reaction. A simple integrated TLS immunoscore reflecting these parameters identified a large subgroup of nmCRC patients with a very low risk of recurrence independently of clinical co-variables such as ECOG performance status, age, stage, and adjuvant chemotherapy. We conclude that (1) mismatch repair and BRAF mutation status are associated with the formation of TLS in nmCRC, (2) TLS formation in nmCRC follows sequential maturation steps, culminating in germinal center formation, and (3) this maturation process harbors important prognostic information on the risk of disease recurrence.
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http://dx.doi.org/10.1080/2162402X.2017.1378844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798199PMC
December 2017

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

Nat Med 2017 09 14;23(9):1046-1054. Epub 2017 Aug 14.

Institute of Oncology Research, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.
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http://dx.doi.org/10.1038/nm.4372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592092PMC
September 2017

Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections.

PLoS One 2017 11;12(5):e0176691. Epub 2017 May 11.

IBM Research - Zurich, Rüschlikon, Switzerland.

We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and β-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol-dewaxing, rehydration and staining-with the same microfluidic probe head. Tissue lithography brings a new level of versatility and flexibility in sample processing and budgeting in biobanks, which may alleviate current sample limitations for retrospective studies in biomarker discovery and drug screening.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176691PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426611PMC
September 2017

An international reproducibility study validating quantitative determination of ERBB2, ESR1, PGR, and MKI67 mRNA in breast cancer using MammaTyper®.

Breast Cancer Res 2017 05 11;19(1):55. Epub 2017 May 11.

European Institute of Oncology, University of Milan, Milan, Italy.

Background: Accurate determination of the predictive markers human epidermal growth factor receptor 2 (HER2/ERBB2), estrogen receptor (ER/ESR1), progesterone receptor (PgR/PGR), and marker of proliferation Ki67 (MKI67) is indispensable for therapeutic decision making in early breast cancer. In this multicenter prospective study, we addressed the issue of inter- and intrasite reproducibility using the recently developed reverse transcription-quantitative real-time polymerase chain reaction-based MammaTyper® test.

Methods: Ten international pathology institutions participated in this study and determined messenger RNA expression levels of ERBB2, ESR1, PGR, and MKI67 in both centrally and locally extracted RNA from formalin-fixed, paraffin-embedded breast cancer specimens with the MammaTyper® test. Samples were measured repeatedly on different days within the local laboratories, and reproducibility was assessed by means of variance component analysis, Fleiss' kappa statistics, and interclass correlation coefficients (ICCs).

Results: Total variations in measurements of centrally and locally prepared RNA extracts were comparable; therefore, statistical analyses were performed on the complete dataset. Intersite reproducibility showed total SDs between 0.21 and 0.44 for the quantitative single-marker assessments, resulting in ICC values of 0.980-0.998, demonstrating excellent agreement of quantitative measurements. Also, the reproducibility of binary single-marker results (positive/negative), as well as the molecular subtype agreement, was almost perfect with kappa values ranging from 0.90 to 1.00.

Conclusions: On the basis of these data, the MammaTyper® has the potential to substantially improve the current standards of breast cancer diagnostics by providing a highly precise and reproducible quantitative assessment of the established breast cancer biomarkers and molecular subtypes in a decentralized workup.
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http://dx.doi.org/10.1186/s13058-017-0848-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426065PMC
May 2017

VHL missense mutations in the p53 binding domain show different effects on p53 signaling and HIFα degradation in clear cell renal cell carcinoma.

Oncotarget 2017 Feb;8(6):10199-10212

Department of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland.

Clear cell Renal Cell Carcinoma (ccRCC) formation is connected to functional loss of the von Hippel-Lindau (VHL) gene. Recent data identified its gene product, pVHL, as a multifunctional adaptor protein which interacts with HIFα subunits but also with the tumor suppressor p53. p53 is hardly expressed and rarely mutated in most ccRCC. We showed that low and absent p53 expression correlated with the severity of VHL mutations in 262 analyzed ccRCC tissues. In contrast to nonsense and frameshift mutations which abrogate virtually all pVHL functions, missense mutations may rather influence one or few functions. Therefore, we focused on four VHL missense mutations, which affect the overlapping pVHL binding sites of p53 and Elongin C, by investigating their impact on HIFα degradation, p53 expression and signaling, as well as on cellular behavior using ccRCC cell lines and tissues. TP53 mRNA and its effector targets p21, Bax and Noxa, were altered both in engineered cell lines and in tumor tissues which carried the same missense mutations. Two of these mutations were not able to degrade HIFα whereas the remaining two mutations led to HIFα downregulation, suggesting the latter are p53 binding site-specific. The selected VHL missense mutations further enhanced tumor cell survival, but had no effects on cell proliferation. Whereas Sunitinib was able to efficiently reduce cell proliferation, Camptothecin was additionally able to increase apoptotic activity of the tumor cells. It is concluded that systematic characterization of the VHL mutation status may help optimizing targeted therapy for patients with metastatic ccRCC.
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http://dx.doi.org/10.18632/oncotarget.14372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354652PMC
February 2017

Detailed simulation of cancer exome sequencing data reveals differences and common limitations of variant callers.

BMC Bioinformatics 2017 Jan 3;18(1). Epub 2017 Jan 3.

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstr, Basel, 26, 4058, Switzerland.

Background: Next-generation sequencing of matched tumor and normal biopsy pairs has become a technology of paramount importance for precision cancer treatment. Sequencing costs have dropped tremendously, allowing the sequencing of the whole exome of tumors for just a fraction of the total treatment costs. However, clinicians and scientists cannot take full advantage of the generated data because the accuracy of analysis pipelines is limited. This particularly concerns the reliable identification of subclonal mutations in a cancer tissue sample with very low frequencies, which may be clinically relevant.

Results: Using simulations based on kidney tumor data, we compared the performance of nine state-of-the-art variant callers, namely deepSNV, GATK HaplotypeCaller, GATK UnifiedGenotyper, JointSNVMix2, MuTect, SAMtools, SiNVICT, SomaticSniper, and VarScan2. The comparison was done as a function of variant allele frequencies and coverage. Our analysis revealed that deepSNV and JointSNVMix2 perform very well, especially in the low-frequency range. We attributed false positive and false negative calls of the nine tools to specific error sources and assigned them to processing steps of the pipeline. All of these errors can be expected to occur in real data sets. We found that modifying certain steps of the pipeline or parameters of the tools can lead to substantial improvements in performance. Furthermore, a novel integration strategy that combines the ranks of the variants yielded the best performance. More precisely, the rank-combination of deepSNV, JointSNVMix2, MuTect, SiNVICT and VarScan2 reached a sensitivity of 78% when fixing the precision at 90%, and outperformed all individual tools, where the maximum sensitivity was 71% with the same precision.

Conclusions: The choice of well-performing tools for alignment and variant calling is crucial for the correct interpretation of exome sequencing data obtained from mixed samples, and common pipelines are suboptimal. We were able to relate observed substantial differences in performance to the underlying statistical models of the tools, and to pinpoint the error sources of false positive and false negative calls. These findings might inspire new software developments that improve exome sequencing pipelines and further the field of precision cancer treatment.
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http://dx.doi.org/10.1186/s12859-016-1417-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209852PMC
January 2017

MiR-99b-5p expression and response to tyrosine kinase inhibitor treatment in clear cell renal cell carcinoma patients.

Oncotarget 2016 Nov;7(48):78433-78447

Institute of Surgical Pathology, University Hospital Zürich, Zurich, Switzerland.

A number of treatments targeting VEGF or mTOR pathways have been approved for metastatic clear cell Renal Cell Carcinoma (ccRCC), but the majority of patients show disease progression after first line therapy with a very low rate of complete or long-term responders. It has been shown that miRs may play a role in prediction of treatment response in various cancer types. The aim of our study was to identify a miR signature predictive for RCC patients' response to antiangiogenic tyrosine kinase inhibitor (TKI) treatment in the first line therapy. Sequencing of 40 paired normal/tumor formalin fixed and paraffin embedded ccRCC tissues revealed separate clustering via unsupervised dendrograms. With supervised analysis, the strongest differential expression was obtained with miR-99b-5p, which was significantly lower in patients with short progression free survival (<8 months) and TKI non-responders (progressive disease patients according to RECIST) (p<0.0001, each). Validation using RTqPCR and a second patient cohort compiled from three different hospitals (n=65) showed higher expression of miR-99b-5p in complete responders, but this trend did not reach statistical significance. It is concluded that low miR-99b-5p expression analyzed with sequencing methodology may correlate with tumor progression in TKI-treated ccRCC patients.
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http://dx.doi.org/10.18632/oncotarget.12618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346651PMC
November 2016

Discretization of Gene Expression Data Unmasks Molecular Subgroups Recurring in Different Human Cancer Types.

PLoS One 2016 18;11(8):e0161514. Epub 2016 Aug 18.

Institute of Surgical Pathology, University Hospital Zurich, Schmelzbergstrasse 12, 8091 Zurich, Switzerland.

Despite the individually different molecular alterations in tumors, the malignancy associated biological traits are strikingly similar. Results of a previous study using renal cell carcinoma (RCC) as a model pointed towards cancer-related features, which could be visualized as three groups by microarray based gene expression analysis. In this study, we used a mathematic model to verify the presence of these groups in RCC as well as in other cancer types. We developed an algorithm for gene-expression deviation profiling for analyzing gene expression data of a total of 8397 patients with 13 different cancer types and normal tissues. We revealed three common Cancer Transcriptomic Profiles (CTPs) which recurred in all investigated tumors. Additionally, CTPs remained robust regardless of the functions or numbers of genes analyzed. CTPs may represent common genetic fingerprints, which potentially reflect the closely related biological traits of human cancers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161514PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990327PMC
July 2017

Characterization of VHL missense mutations in sporadic clear cell renal cell carcinoma: hotspots, affected binding domains, functional impact on pVHL and therapeutic relevance.

BMC Cancer 2016 08 17;16:638. Epub 2016 Aug 17.

Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.

Background: The VHL protein (pVHL) is a multiadaptor protein that interacts with more than 30 different binding partners involved in many oncogenic processes. About 70 % of clear cell renal cell carcinoma (ccRCC) have VHL mutations with varying impact on pVHL function. Loss of pVHL function leads to the accumulation of Hypoxia Inducible Factor (HIF), which is targeted by current targeted treatments. In contrast to nonsense and frameshift mutations that highly likely nullify pVHL multipurpose functions, missense mutations may rather specifically influence the binding capability of pVHL to its partners. The affected pathways may offer predictive clues to therapy and response to treatment. In this study we focused on the VHL missense mutation pattern in ccRCC, and studied their potential effects on pVHL protein stability and binding partners and discussed treatment options.

Methods: We sequenced VHL in 360 sporadic ccRCC FFPE samples and compared observed and expected frequency of missense mutations in 32 different binding domains. The prediction of the impact of those mutations on protein stability and function was assessed in silico. The response to HIF-related, anti-angiogenic treatment of 30 patients with known VHL mutation status was also investigated.

Results: We identified 254 VHL mutations (68.3 % of the cases) including 89 missense mutations (35 %). Codons Ser65, Asn78, Ser80, Trp117 and Leu184 represented hotspots and missense mutations in Trp117 and Leu 184 were predicted to highly destabilize pVHL. About 40 % of VHL missense mutations were predicted to cause severe protein malfunction. The pVHL binding domains for HIF1AN, BCL2L11, HIF1/2α, RPB1, PRKCZ, aPKC-λ/ι, EEF1A1, CCT-ζ-2, and Cullin2 were preferentially affected. These binding partners are mainly acting in transcriptional regulation, apoptosis and ubiquitin ligation. There was no correlation between VHL mutation status and response to treatment.

Conclusions: VHL missense mutations may exert mild, moderate or strong impact on pVHL stability. Besides the HIF binding domain, other pVHL binding sites seem to be non-randomly altered by missense mutations. In contrast to LOF mutations that affect all the different pathways normally controlled by pVHL, missense mutations may be rather appropriate for designing tailor-made treatment strategies for ccRCC.
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http://dx.doi.org/10.1186/s12885-016-2688-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987997PMC
August 2016