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Figure S1. Fluorescence in situ hybridization (FISH) analysis of FGFR1 gene copy number status in mesothelioma cell lines. Figure S2. Bioluminescence imaging of orthotopically implanted H226 tumors treated with or without ponatinib. Supplementary Tables S1 through S3.

Purpose: FGFR1 copy-number gain (CNG) occurs in head and neck squamous cell cancers (HNSCC) and is used for patient selection in FGFR-specific inhibitor clinical trials. This study explores FGFR1 mRNA and protein levels in HNSCC cell lines, primary tumors, and patient-derived xenografts (PDX) as predictors of sensitivity to the FGFR inhibitor, NVP-BGJ398.Experimental Design: FGFR1 status, expression levels, and BGJ398 sensitive growth were measured in 12 HNSCC cell lines. Primary HNSCCs (n = 353) were assessed for FGFR1 CNG and mRNA levels, and HNSCC TCGA data were interrogated as an independent sample set. HNSCC PDXs (n = 39) were submitted to FGFR1 copy-number detection and mRNA assays to identify putative FGFR1-dependent tumors.Results: Cell line sensitivity to BGJ398 is associated with FGFR1 mRNA and protein levels, not FGFR1 CNG. Thirty-one percent of primary HNSCC tumors expressed FGFR1 mRNA, 18% exhibited FGFR1 CNG, 35% of amplified tumors were also positive for FGFR1 mRNA. This relationship was confirmed with the TCGA dataset. Using high FGFR1 mRNA for selection, 2 HNSCC PDXs were identified, one of which also exhibited FGFR1 CNG. The nonamplified tumor with high mRNA levels exhibited in vivo sensitivity to BGJ398.Conclusions: FGFR1 expression associates with BGJ398 sensitivity in HNSCC cell lines and predicts tyrosine kinase inhibitor sensitivity in PDXs. Our results support FGFR1 mRNA or protein expression, rather than FGFR1 CNG as a predictive biomarker for the response to FGFR inhibitors in a subset of patients suffering from HNSCC. Clin Cancer Res; 21(19); 4356–64. ©2015 AACR.

Figure S6. Correlation of HPV-expression and FGFR1 mRNA expression.

Figure S5. In situ hybridization (ISH) assay for FGFR1 mRNA in primary HNSCC tissue.

Table S1. The panel of HNSCC PDXs was submitted to FGFR1 gene copy number (GCN) by CISH and mRNA levels by in situ hybridization (ISH) assay.

Figure S4. FGFR1 dependency in 584-A2 and CCL30 cells assessed by RNAi-mediated FGFR1 silencing.

Figure S1. Representative images of FGFR1 GCN assayed by FISH in HNSCC cell lines.

Figure S3. Correlation of FGFR1 mRNA and protein expression with BGJ398 sensitivity.

Figure S2. Effect of BGJ398 on Ki67 staining, cleaved caspase 3 levels and phospho-ERK levels in HNSCC cell lines.

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking. We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

Purpose: FGFR1 copy-number gain (CNG) occurs in head and neck squamous cell cancers (HNSCC) and is used for patient selection in FGFR-specific inhibitor clinical trials. This study explores FGFR1 mRNA and protein levels in HNSCC cell lines, primary tumors, and patient-derived xenografts (PDX) as predictors of sensitivity to the FGFR inhibitor, NVP-BGJ398. Experimental Design: FGFR1 status, expression levels, and BGJ398 sensitive growth were measured in 12 HNSCC cell lines. Primary HNSCCs (n = 353) were assessed for FGFR1 CNG and mRNA levels, and HNSCC TCGA data were interrogated as an independent sample set. HNSCC PDXs (n = 39) were submitted to FGFR1 copy-number detection and mRNA assays to identify putative FGFR1-dependent tumors. Results: Cell line sensitivity to BGJ398 is associated with FGFR1 mRNA and protein levels, not FGFR1 CNG. Thirty-one percent of primary HNSCC tumors expressed FGFR1 mRNA, 18% exhibited FGFR1 CNG, 35% of amplified tumors were also positive for FGFR1 mRNA. This relationship was confirmed with the TCGA dataset. Using high FGFR1 mRNA for selection, 2 HNSCC PDXs were identified, one of which also exhibited FGFR1 CNG. The nonamplified tumor with high mRNA levels exhibited in vivo sensitivity to BGJ398. Conclusions: FGFR1 expression associates with BGJ398 sensitivity in HNSCC cell lines and predicts tyrosine kinase inhibitor sensitivity in PDXs. Our results support FGFR1 mRNA or protein expression, rather than FGFR1 CNG as a predictive biomarker for the response to FGFR inhibitors in a subset of patients suffering from HNSCC. Clin Cancer Res; 21(19); 4356–64. ©2015 AACR.

Malignant pleural mesothelioma (MPM) is associated with asbestos exposure and is a cancer that has not been significantly affected by small molecule-based targeted therapeutics. Previously, we demonstrated the existence of functional subsets of lung cancer and head and neck squamous cell carcinoma (HNSCC) cell lines in which fibroblast growth factor receptor (FGFR) autocrine signaling functions as a nonmutated growth pathway. In a panel of pleural mesothelioma cell lines, FGFR1 and FGF2 were coexpressed in three of seven cell lines and were significantly associated with sensitivity to the FGFR-active tyrosine kinase inhibitor (TKI), ponatinib, both in vitro and in vivo using orthotopically propagated xenografts. Furthermore, RNAi-mediated silencing confirmed the requirement for FGFR1 in specific mesothelioma cells and sensitivity to the FGF ligand trap, FP-1039, validated the requirement for autocrine FGFs. None of the FGFR1-dependent mesothelioma cells exhibited increased FGFR1 gene copy number, based on a FISH assay, indicating that increased FGFR1 transcript and protein expression were not mediated by gene amplification. Elevated FGFR1 mRNA was detected in a subset of primary MPM clinical specimens and like MPM cells; none harbored increased FGFR1 gene copy number. These results indicate that autocrine signaling through FGFR1 represents a targetable therapeutic pathway in MPM and that biomarkers distinct from increased FGFR1 gene copy number such as FGFR1 mRNA would be required to identify patients with MPM bearing tumors driven by FGFR1 activity. Implications: FGFR1 is a viable therapeutic target in a subset of MPMs, but FGFR TKI-responsive tumors will need to be selected by a biomarker distinct from increased FGFR1 gene copy number, possibly FGFR1 mRNA or protein levels. Mol Cancer Res; 12(10); 1460–9. ©2014 AACR.

Recently, the fibroblast growth factor receptor 1 (FGFR1) has been identified as the first actionable target in squamous cell lung cancer. Clinical trials testing specific FGFR inhibitors are in progress, and patients are selected based on their FGFR1 gene copy number status. Fluorescent in situ hybridization is the most commonly used method for detecting FGFR1 amplifications, but it has its limitations. In this paper, we describe a new non-fading and easy to assess assay for detecting FGFR1 amplification using a combination of chromogenic and silver in situ hybridization. We assessed 394 patients diagnosed with head and neck squamous cell carcinoma with the new assay and compared the results with those obtained by FGFR1 fluorescent in situ hybridization. We could assess copy number by the fluorescent in situ hybridization in 86.8 % (342/394) of cases, whereas with chromogenic and silver in situ hybridization, this was 79.4 % (313/394). By fluorescent in situ hybridization, a FGFR1 amplification was detected in 12.6 % (43/342) of cases, a low-level amplification (LLA) in 7.6 % (26/342) and a high-level amplification (HLA) in 5.0 % (17/342). By chromogenic and silver in situ hybridization, a FGFR1 amplification was found in 10.2 % (32/313) (5.7 % LLA, 4.5 % HLA). The two techniques showed highly concordant results (Pearson’s correlation coefficient = 0.971, p

The mediator complex is an evolutionary conserved key regulator of transcription of protein-coding genes and an integrative hub for diverse signaling pathways. In this study, we investigated whether the mediator subunit MED15 is implicated in castration-resistant prostate cancer (CRPC). MED15 expression and copy number/rearrangement status were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively on 718 prostate cancer (PCa) specimens and sequenced by Sanger on a subset. Furthermore, SMAD3 phosphorylation, androgen receptor (AR) and proliferation markers were evaluated by IHC. In PCa cells, siRNA/shRNA knockdown of MED15 was followed by proliferation assays with/without dihydrotestosterone (DHT), and treatments with recombinant TGF-β3. Our results show that MED15 is overexpressed in 76% of distant metastatic CRPC (CRPC(MET) ) and 70% of local-recurrent CRPC (CRPC(LOC) ), in contrast to low frequencies in androgen-sensitive PCa, and no expression in benign prostatic tissue. Furthermore, MED15 overexpression correlates with worse clinical outcome thus defining a highly lethal phenotype. Moreover, TGF-β signaling activation associates with MED15 overexpression in PCa tissues, and leads to increased expression of MED15 in PCa cells. MED15 knockdown effects phosphorylation and shuttling of p-SMAD3 to the nucleus as well as TGF-β-enhanced proliferation. In PCa tissues, MED15 overexpression associates with AR overexpression/amplification and correlates with high proliferative activity. MED15 knockdown decreases both androgen-dependent and -independent proliferation in PCa cells. Taken together, these findings implicate MED15 in CRPC, and as MED15 is evolutionary conserved, it is likely to emerge as a lethal phenotype in other therapeutic-resistant diseases, and not restricted to our disease model.

Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Recently, we characterized fibroblast growth factor receptor 1 amplification as a target for a rational therapy in lung squamous cell carcinoma. Patients harboring this genetic event are currently eligible for treatment with antifibroblast growth factor receptor small-molecule inhibitors in phase I clinical trials. This has the potential to significantly improve standard therapy for lung squamous cell carcinoma patients. The aim of this study was to elucidate whether fibroblast growth factor receptor 1 amplification is also a common genetic event in head and neck squamous cell carcinoma. For this purpose, we assembled a cohort of 555 patients, including 264 with metastatic disease and 147 with recurrent disease. Formalin-fixed, paraffin-embedded material of primary tumors, metastases and recurrences were assessed for fibroblast growth factor receptor 1 copy number status using fluorescence in situ hybridization. Human papilloma virus status was detected by p16 immunohistochemistry staining and PCR-ELISA. Molecular parameters were correlated with each other and with clinicopathological data. We found 15% of primary head and neck squamous cell carcinoma to display a fibroblast growth factor receptor 1 amplification. In nearly all cases, metastatic and recurrent tumor samples shared the same amplification status as the corresponding primary tumor. Fibroblast growth factor receptor 1 amplification was associated with nicotine and alcohol consumption, but was mutually exclusive with human papilloma virus infection. Amplification of the gene was associated with parameters of worse outcome. Our data identify fibroblast growth factor receptor 1 amplification as a frequent event in primary and metastatic head and neck squamous cell carcinoma and represents a potential biomarker for more aggressive disease. Fibroblast growth factor receptor 1-amplified tumors could potentially comprise a subset of head and neck squamous cell carcinoma against which targeted small-molecule inhibitors hold therapeutic efficacy.

Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non‐small cell tumors and secondary transitions from non‐small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of “small cell‐ness” based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT‐signaling in the context of mutual bi‐allelic RB1 and TP53 lesions. Additionaly, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH‐ASCL1‐RB‐p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon‐based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well., What's new? Using next generation sequencing and establishing features of ‘small cell‐ness’, we identified a NOTCH‐ASCL1‐RB1‐TP53 signaling axis driving small cell cancers. In contrast to the previously described bi‐allelic RB1/TP53 loss in neuroendocrine stem cells as origin of primary small cell neuroendocrine cancers, the NOTCH‐ASCL1 mediated signaling defines an alternative pathway driving secondary small cell neuroendocrine cancers arising from non‐small cell cancers. Moreover, we show a preclinical rational for therapeutically testing WNT‐inhibitors in small cell cancers.

ETS gene rearrangements are frequently found in prostate cancer. Several studies have assessed the rearrangement status of the most commonly found ETS rearranged gene ERG, and the less frequent genes, ETV-1, ETV-4, ETV-5, and ELK-4 in primary prostate cancer. However, frequency in metastatic disease is not well investigated. Recently, we have assessed the ERG rearrangement status in both primary and corresponding lymph node metastases and observed that ERG rearrangement in primary prostate cancer transfers into lymph node metastases, suggesting it to be a clonal expansion event during prostate cancer progression. As a continuation, we investigated in this study whether this observation is valid for the less frequent ETS rearranged genes. Using dual-color break-apart fluorescent in situ hybridization assays, we evaluated the status of all less frequent ETS gene rearrangements for the first time on tissue microarrays constructed from a large cohort of 86 patients with prostate cancer and composed of primary and corresponding lymph node metastases, as well as in a second cohort composed of 43 distant metastases. ETV-1, ETV-4, ETV-5, and ELK-4 rearrangements were found in 8 (10%) of 81, 5 (6%) of 85, 1 (1%) of 85, and 2 (2%) of 86 of primary prostate cancer, respectively, and in 6 (8%) of 73, 4 (6%) of 72, 1 (1%) of 75, and 1 (1%) of 78 of corresponding lymph node metastases, respectively. ETV-1 and ETV-5 rearrangements were not found in the distant metastases cases, whereas ETV-4 and ELK-4 rearrangements were found in 1 (4%) of 25 and 1 (4%) of 24, respectively. Our findings suggest that rearrangement of the less frequent ETS genes is a clonal event during prostate cancer progression.

The recently detected TMPRSS2-ERG fusion gene was revealed as a recurrent and prevalent prostate cancer (PCa)-specific event, potentially qualifying it for clinical use. To detect this alteration, fluorescence in situ hybridization (FISH) is the method of choice. However, FISH has some disadvantages for widespread adoption in clinical practice. Subsequently, chromogenic in situ hybridization, which uses organic chromogens, and enzymatic metallography silver in situ hybridization have emerged as promising bright-field alternatives. Compared with chromogenic in situ hybridization, silver in situ hybridization signals are very distinct and superior with regard to signal clarity and resolution, but the method excludes multicolor protocols. Based on the ERG break-apart FISH assay, we established a dual-color ERG break-apart assay using combined chromogenic in situ hybridization and silver in situ hybridization (CS-ISH) and compared these results with those obtained by FISH. We assessed 178 PCa and 10 benign specimens for their ERG rearrangement status by applying dual-color FISH and CS-ISH ERG break-apart assays to consecutive sections. We observed a highly significant concordance (97.7%) between FISH- and CS-ISH-based results (Pearson's correlation coefficient = 0.955, P0.001). Our findings demonstrate that the ERG rearrangement status can reliably be assessed by CS-ISH. Further, the CS-ISH technique combines the accuracy and precision of FISH with the ease of bright-field microscopy. This tool allows a much broader spectrum of applications in which to study the biological role and clinical use of ERG rearrangements in PCa.

Next generation sequencing (NGS) technologies have revolutionized cancer research allowing the comprehensive study of cancer using high throughput deep sequencing methodologies. These methods detect genomic alterations, nucleotide substitutions, insertions, deletions and copy number alterations. SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) is a promising technology generating billions of 50 bp sequencing reads. This robust technique, successfully applied in gene identification, might be helpful in detecting novel genes associated with cancer initiation and progression using formalin fixed paraffin embedded (FFPE) tissue. This study’s aim was to compare the validity of whole exome sequencing of fresh-frozen vs. FFPE tumor tissue by normalization to normal prostatic FFPE tissue, obtained from the same patient. One primary fresh-frozen sample, corresponding FFPE prostate cancer sample and matched adjacent normal prostatic tissue was subjected to exome sequencing. The sequenced reads were mapped and compared. Our study was the first to show comparable exome sequencing results between FFPE and corresponding fresh-frozen cancer tissues using SOLiD sequencing. A prior study has been conducted comparing the validity of sequencing of FFPE vs. fresh frozen samples using other NGS platforms. Our validation further proves that FFPE material is a reliable source of material for whole exome sequencing.

FISH (fluorescent in situ hybridization) is a molecular cytogenetic method to detect large-scale genetic alterations in tissue and/or cells. Numerical aberrations (deletions and amplifications) and structural aberrations (translocations and fusions) are detectable. Probes bind complementary to the DNA strand of the region of interest. Subsequently, the probes are detected via fluorochromes and appear as colored dots that can be assessed under the fluorescence microscope.In situ hybridization is divided into three steps: pretreatment, hybridization, and posthybridization. Pretreatment opens up the cell membranes for hybridization, so that the probe can bind to the complementary DNA target. Posthybridization includes washing steps to remove excessive probes and detection of probes via secondary marked fluorochromes. DAPI stains nuclei and serves as mounting media.

FISH (fluorescent in situ hybridization) is a molecular cytogenetic method to detect large-scale genetic alterations in tissue and/or cells. Numerical aberrations (deletions and amplifications) and structural aberrations (translocations and fusions) are detectable. Probes bind complementary to the DNA strand of the region of interest. Subsequently, the probes are detected via fluorochromes and appear as colored dots that can be assessed under the fluorescence microscope.In situ hybridization is divided into three steps: pretreatment, hybridization, and posthybridization. Pretreatment opens up the cell membranes for hybridization, so that the probe can bind to the complementary DNA target. Posthybridization includes washing steps to remove excessive probes and detection of probes via secondary marked fluorochromes. DAPI stains nuclei and serves as mounting media.

Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies.

Aims Pancreatic ductal adenocarcinomas (PDACs) are chemoresistant, resulting in extremely poor survival of patients; therefore, novel molecular targets, even in small subsets of genetically characterized tumours, are urgently needed. Tyrosine kinase receptor inhibitors (TKIs) are already in clinical use. The aims of this study were to examine the gene copy number and expression of fibroblast growth factor receptor 1 (FGFR1) in 155 patients with PDAC, and investigate the effects of the FGFR-specific inhibitor BGJ398 on FGFR1-amplified pancreatic tumour cells in vitro. Methods and results Fluorescence in-situ hybridization (FISH) and immunohistochemical analysis of 155 PDACs were performed using tissue microarrays. Amplification of FGFR1 was found in 2.6% (4/155) of cases. Four per cent of tumours (5/125) were shown to express FGFR1 by immunohistochemistry. Sequence analysis demonstrated an activating KRAS mutation (exon 2) in all FGFR1-amplified cases. The FGFR1-amplified pancreatic carcinoma cell line PT45P1 showed high levels of FGFR1 mRNA and protein expression. Proliferation of this cell line can be inhibited using the FGFR1 inhibitor BGJ398. Conclusions FGFR1 represents a potential new therapeutic target in a subset of patients harbouring FGFR1-amplified tumours. Identification of pancreatic cancers harbouring FGFR1 amplification may be important in preselecting patients and/or interpreting clinical studies using TKIs.

Background We previously identified amplification of the fibroblast growth factor receptor 1 gene (FGFR1) as a potential therapeutic target for small-molecule inhibitor therapy in squamous cell lung cancer (L-SCC). Currently, clinical phase I trials are underway to examine whether patients with FGFR1-amplified L-SCC benefit from a targeted therapy approach using small-molecule inhibitors. Because most patients with lung cancer present with metastatic disease, we investigated whether lymph node metastases in L-SCC share the FGFR1 amplification status of their corresponding primary tumor. Methods The study cohort consisted of 72 patients with L-SCC, 39 with regional lymph node metastases. Tissue microarrays were constructed from formalin-fixed, paraffin-embedded tissue of the primary tumors and, where present, of the corresponding lymph node metastasis. A biotin-labeled target probe spanning the FGFR1 locus (8p11.22-23) was used to determine the FGFR1 amplification status by fluorescence in situ hybridization. Results FGFR1 amplification was detected in 16% (12 of 72) of all primary L-SCCs. In metastatic tumors, 18% (seven of 39) of the lymph node metastases displayed FGFR1 amplification with an exact correlation of FGFR1 amplification status between tumor and metastatic tissue. Conclusions FGFR1 amplification is a common genetic event occurring at a frequency of 16% in L-SCCs. Moreover, lymph node metastases derived from FGFR1-amplified L-SCCs also exhibit FGFR1 amplification. Therefore, we suggest that the FGFR1 amplification is a clonal event in tumor progression. Beyond this biologically relevant observation, the findings carry potential therapeutic implications in that small-molecule inhibitors may be applicable to the treatment of a subset of patients with metastatic L-SCC.

Aim: Castration resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) with a poor prognosis, and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNA) by whole exome sequencing on five CRPC/normal paired formalin fixed paraffin embedded (FFPE) samples using the SOLiD4 next generation sequencing platform. Methods: Genomic DNA was extracted from 5 CRPC/normal paired FFPE samples and sequenced using the SOLiD4 next generation sequencing platform. The sequencing output was mapped, sorted, filtered and annotated using human genome databases. Among a set of prominent amplified/deleted genes in PCa, the 8q12.2-24.22 region encoding PTK2 and YWHAZ was found to be amplified. The data was validated using fluorescence in-situ hybridization (FISH) assays with a PCa progression cohort containing 137 localized PCa samples, 105 primary tumors, 71 corresponding lymph node metastases and 39 CRPC samples. Additionally PTK2 and YWHAZ amplification status, mRNA expression (LightCycler) and protein expression (Western Blot) were determined in selected PCa cell lines. Within PC-3 cells, inhibition of PTK2 by the specific inhibitor TAE226 (Novartis), as well as knock down of YWHAZ were analyzed. The effect of PTK2 inhibition and YWHAZ knock down on cell proliferation (MTT assay) and cell-invasion (Matrigel invasion chambers) were determined. Results: Whole exome sequencing analysis identified regions of deletion and amplification, which included well-known genes such as PTEN, CMYC and AR. Furthermore, we identified PTK2 and YWHAZ on chromosome 8 to be amplified in the sequenced PCa samples. The FISH analysis of the region showed an increasing amplification frequency of PTK2 and YWHAZ with progression of the disease. PTK2 was amplified in 1% of the localized PCa and in 35% of the CRPC samples. YWHAZ was amplified in 4% of the localized PCa and in 48% of the CRPC samples. The FISH analysis revealed a high level amplification for PTK2/YWHAZ, in the PC-3 cells, which also exhibited a high PTK2/YWHAZ protein expression. PTK2 inhibition by TAE226 and YWHAZ knock down had a significant effect on cell proliferation and migration in the PC-3 cells in vitro. PC-3 cells treated with TAE226 or YWHAZ knocked down cells had a significantly reduced cell proliferation and cell migration ability compared to untreated cells. Conclusion: Our findings suggest that the inhibition of PTK2 and YWHAZ could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. The validated whole exome sequencing data shows that FFPE tissue could be a promising alternative for SCNA screening using next generation sequencing technologies. Citation Format: Roopika Menon, Kerstin Rüenauver, Mario Deng, Friedrich Kunze, Diana Boehm, Wenzel Vogel, Veit Scheble, Falko Fend, Glen Kristiansen, Nicolas Wernert, Nicole Oberbeckmann, Saskia Biskup, Mark Rubin, Zaki Shaikhibrahim, Sven Perner. Somatic copy number alterations by whole-exome sequencing reveals YWHAZ and PTK2 as potential therapeutic targets in castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B97.

Introduction and Objective: Overexpression of the ERG protein is highly prevalent in prostate cancer (PCa) and most commonly results from gene fusions involving the ERG gene. Recently, an N-terminal epitope targeted mouse and a C-terminal epitope targeted rabbit monoclonal anti-ERG antibody have been introduced for the detection of the ERG protein. Independent studies reported that immunohistochemical (IHC) stains with both monoclonal anti-ERG antibodies (ERG-MAbs) highly correlate with the underlying ERG gene rearrangement status. However, a comparative study of both antibodies has not been provided so far. Here, we are the first to compare the mouse ERG-MAb to the rabbit ERG-MAb for their concordance on the same PCa cohort. Furthermore, we assessed if the ERG protein expression is conserved in lymph node and distant PCa metastases, of which a subset underwent decalcification. Methods: We evaluated tissue microarrays of 278 specimens containing 265 localized PCa, 29 lymph node, 30 distant metastases, and 13 normal prostatic tissues. We correlated the ERG protein expression with the ERG rearrangement status using an ERG break-apart fluorescence in-situ hybridization (FISH) assay and IHC of both ERG antibodies. Results: ERG protein expression and ERG rearrangement status were highly concordant regardless of whether the mouse or rabbit ERG-MAb was used (97.8% versus 98.6%, respectively). Of interest, both ERG antibodies reliably detected the ERG expression in lymph node and distant PCa metastases, of which a subset underwent decalcification. If an ERG protein expression was present in localized PCa, we observed the same pattern in the corresponding lymph node metastases. Conclusions: This is the first study to comprehensively compare the two available ERG-MAbs. By demonstrating a broad applicability of IHC to study ERG protein expression using either antibody, this study adds an important step towards a facilitated routine clinical application. Further, we demonstrate that the clonal nature of the ERG rearrangement is not restricted to the genomic level, but proceeds in the proteome. Together, our results simplify future efforts to further elucidate the biological role of ERG in PCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1698. doi:1538-7445.AM2012-1698

Aim: Castration resistant prostate cancer (CR-PCa) is the most aggresive form of prostate cancer (PCa) having a poor prognosis, and is a significant therapeutic challenge. The key to the development of novel therapeutic targets for CR-PCa is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to perform whole exome sequencing and gene copy number analysis on 5 CR-PCa/normal paired formalin fixed paraffin embedded (FFPE) samples using the SOLiD4 next generation sequencing platform. Methods: Genomic DNA was extracted from 5 CR-PCa/normal paired FFPE samples. The DNA was subjected to targeted exon capture using the Agilent Sure Select kit. The captured DNA was sequenced using the SOLiD4 next generation sequencing platform. The sequencing output was mapped, sorted, filtered and annotated using well-known human genome databases. The results were further analyzed for SNPs and copy number variations. A set of amplified/deleted genes were validated using fluorescence in-situ hybridization (FISH) assays with a PCa progression cohort. The cohort consisted of 138 cases for localized cancer, 105 patients with primary PCa and corresponding LN metastasis, and 39 samples for castration resistant tumors. Results: Whole exome sequencing analysis identified focal regions of deletion, which included well-known tumor suppressors such as NKX3.1 and PTEN. Focal regions of amplification included well-known genes such as CMYC and AR that are known to play a role in PCa. Furthermore, we identified several amplified genes as druggable targets e.g. HDAC6, NTRK1, PLD1, SPHK1, and SIRT7. NTRK1 is a kinase that plays an active role in cell proliferation. HDAC6, PLD1, SPHK1 and SIRT7 regulate numerous complex cellular processes including signal transduction, transcription and apoptosis. Conclusions: This is the first study to use whole exome sequencing approaches on FFPE CR-PCa material to identity novel therapeutic targets. Validation studies would further shed light into the biological understanding of the disease and its plausible treatment options. Citation Format: Roopika Menon, Mario Deng, Diana Boehm, Falko Fend, Detlef Boehm, Saskia Biskup, Sven Perner. Whole-exome sequencing identifies potential therapeutic targets for castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C27.

Introduction and Objective: Overexpression of the ERG protein is highly prevalent in prostate cancer (PCa) and most commonly results from gene fusions involving the ERG gene. Recently, an N-terminal epitope targeted mouse and a C-terminal epitope targeted rabbit monoclonal anti-ERG antibody have been introduced for the detection of the ERG protein. Independent studies reported that immunohistochemical (IHC) stains with both monoclonal anti-ERG antibodies (ERG-MAbs) highly correlate with the underlying ERG gene rearrangement status. However, a comparative study of both antibodies has not been provided so far. Here, we are the first to compare the mouse ERG-MAb to the rabbit ERG-MAb for their concordance on the same PCa cohort. Furthermore, we assessed if the ERG protein expression is conserved in lymph node and distant PCa metastases, of which a subset underwent decalcification. Methods: We evaluated tissue microarrays of 278 specimens containing 265 localized PCa, 29 lymph node, 30 distant metastases, and 13 normal prostatic tissues. We correlated the ERG protein expression with the ERG rearrangement status using an ERG break-apart fluorescence in-situ hybridization (FISH) assay and IHC of both ERG antibodies. Results: ERG protein expression and ERG rearrangement status were highly concordant regardless of whether the mouse or rabbit ERG-MAb was used (97.8% versus 98.6%, respectively). Of interest, both ERG antibodies reliably detected the ERG expression in lymph node and distant PCa metastases, of which a subset underwent decalcification. If an ERG protein expression was present in localized PCa, we observed the same pattern in the corresponding lymph node metastases. Conclusions: This is the first study to comprehensively compare the two available ERG-MAbs. By demonstrating a broad applicability of IHC to study ERG protein expression using either antibody, this study adds an important step towards a facilitated routine clinical application. Further, we demonstrate that the clonal nature of the ERG rearrangement is not restricted to the genomic level, but proceeds in the proteome. Together, our results simplify future efforts to further elucidate the biological role of ERG in PCa. Citation Format: Taduru Sreenath, Falko Fend, Nicolas Wernert, Glen Kristiansen, Sven Perner, Shyh-Han Tan, Sr., Martin Braun, Diane Goltz, Zaki Shaikibrahim, Wenzel Vogel, Diana Boehm, Veit Scheble, Albert Dobi. ERG protein expression and genomic rearrangement status in primary and metastatic prostate cancer a comparative study of two monoclonal antibodies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A30.