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Accurate stratification of tumors is imperative for adequate cancer management. In addition to staging, morphologic subtyping allows stratification of patients into additional prognostic groups. In this study, we used an image-based computational method on pan-cytokeratin IHC stainings to quantify tumor fragmentation (TF), a measure of tumor invasiveness of lung squamous cell carcinoma (LSCC). In two independent clinical cohorts from tissue microarrays (TMA: n = 208 patients) and whole sections (WS: n = 99 patients), TF was associated with poor prognosis and increased risk of blood vessel infiltration. A third cohort from The Cancer Genome Atlas (TCGA: n = 335 patients) confirmed the poor prognostic value of TF using a similar human-based score on hematoxylin-eosin staining. Integration of RNA-seq data from TCGA and LC-MS/MS proteomics from WS revealed an upregulation of extracellular matrix remodeling and focal adhesion processes in tumors with high TF, supporting their increased invasive potential. This proposed histologic parameter is an independent and unfavorable prognostic marker that could be established as a new grading parameter for LSCC. Cancer Res; 77(10); 2585–93. ©2017 AACR.

In solid tumors, the presence of lymph node–like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS develop in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multispectral microscopy, quantitative pathology, and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13+ perivascular and CXCL12+LTB+ and PD-L1+ epithelial cells supporting TLS formation. We also characterized sequential stages of TLS maturation in LSCC culminating in the formation of germinal centers (GC). In untreated patients, TLS density was the strongest independent prognostic marker. Furthermore, TLS density correlated with GC formation and expression of adaptive immune response–related genes. In patients treated with neoadjuvant chemotherapy, TLS density was similar, but GC formation was impaired and the prognostic value of TLS density was lost. Corticosteroids are coadministered with chemotherapy to manage side effects in LSCC patients, so we evaluated whether they impaired TLS development independently of chemotherapy. TLS density and GC formation were each reduced in chemotherapy-naïve LSCC patients treated with corticosteroids before surgery, compared with untreated patients, a finding that we confirmed in the experimental model of lung TLS induction. Overall, our results highlight the importance of GC formation in TLS during tumor development and treatment. Significance: Corticosteroid treatment during chemotherapy negatively affects the development of tertiary lymphoid structures and abrogates their prognostic value in patients with lung cancer. Cancer Res; 78(5); 1308–20. ©2018 AACR.

The von Hippel–Lindau (VHL) tumor suppressor protein pVHL is commonly mutated in clear cell renal cell carcinoma (ccRCC) and has been implicated in the control of multiple cellular processes that might be linked to tumor suppression, including promoting proper spindle orientation and chromosomal stability. However, it is unclear whether pVHL exerts these mitotic regulatory functions in vivo as well. Here, we applied ischemic kidney injury to stimulate cell division in otherwise quiescent mouse adult kidneys. We show that in the short term (5.5 days after surgery), Vhl-deficient kidney cells demonstrate both spindle misorientation and aneuploidy. The spindle misorientation phenotype encompassed changes in directed cell division, which may manifest in the development of cystic lesions, whereas the aneuploidy phenotype involved the occurrence of lagging chromosomes but not chromosome bridges, indicative of mitotic checkpoint impairment. Intriguingly, in the long term (4 months after the ischemic insult), Vhl-deficient kidneys displayed a heterogeneous pattern of ccRCC precursor lesions, including cysts, clear cell–type cells, and dysplasia. Together, these data provide direct evidence for a key role of pVHL in mediating oriented cell division and faithful mitotic checkpoint function in the renal epithelium, emphasizing the importance of pVHL as a controller of mitotic fidelity in vivo. Cancer Res; 74(9); 2422–31. ©2013 AACR.

Overexpression of the EVI1 oncogene is associated typically with aggressive myeloid leukemia, but is also detectable in breast carcinoma where its contributions are unexplored. Analyzing a tissue microarray of 608 breast carcinoma patient specimens, we documented EVI1 overexpression in both estrogen receptor–positive (ER+) and estrogen receptor–negative (ER−) breast carcinomas. Here, we report prognostic relevance of EVI1 overexpression in triple-negative breast carcinoma but not in the HER2-positive breast carcinoma subset. In human breast cancer cells, EVI1 silencing reduced proliferation, apoptosis resistance, and tumorigenicity, effects rescued by estrogen supplementation in ER+ breast carcinoma cells. Estrogen addition restored ERK phosphorylation in EVI1-silenced cells, suggesting that EVI1 and estradiol signaling merge in MAPK activation. Conversely, EVI1 silencing had no effect on constitutive ERK activity in HER2+ breast carcinoma cells. Microarray analyses revealed G-protein–coupled receptor (GPR) signaling as a prominent EVI1 effector mechanism in breast carcinoma. Among others, the GPR54-ligand KISS1 was identified as a direct transcriptional target of EVI1, which together with other EVI1-dependent cell motility factors such as RHOJ regulated breast carcinoma cell migration. Overall, our results establish the oncogenic contributions of EVI1 in ER- and HER2-negative subsets of breast cancer. Cancer Res; 77(8); 2148–60. ©2017 AACR.

Human melanoma is composed of distinct cell types reminiscent of neural crest derivatives and contains multipotent cells that express the neural crest stem cell markers CD271(p75NTR) and Sox10. When isolated from solid tumors by using a method that leaves intact cell surface epitopes, CD271-positive, but not CD271-negative, cells formed tumors on transplantation into nude or nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. These tumors fully mirrored the heterogeneity of the parental melanoma and could be passaged more than 5 times. In contrast, in more immunocompromised NOD/SCID/IL2rγnull mice, or in natural killer cell–depleted nude or NOD/SCID mice, both CD271-positive and CD271-negative tumor cell fractions established tumors. However, tumors resulting from either fraction did not phenocopy the parental tumors, and tumors derived from the CD271-negative cell fraction could not be passaged multiple times. Together, our findings identify CD271-positive cells as melanoma stem cells. Our observation that a relatively high frequency of CD271/Sox10-positive cells correlates with higher metastatic potential and worse prognosis further supports that CD271-positive cells within human melanoma represent genuine cancer stem cells. Cancer Res; 71(8); 3098–109. ©2011 AACR.

Recent insights into the role of the von-Hippel Lindau (VHL) tumor suppressor gene in hereditary and sporadic clear-cell renal cell carcinoma (ccRCC) have led to new treatments for patients with metastatic ccRCC, although virtually all patients eventually succumb to the disease. We performed an integrated, genome-wide analysis of copy-number changes and gene expression profiles in 90 tumors, including both sporadic and VHL disease-associated tumors, in hopes of identifying new therapeutic targets in ccRCC. We identified 14 regions of nonrandom copy-number change, including 7 regions of amplification (1q, 2q, 5q, 7q, 8q, 12p, and 20q) and 7 regions of deletion (1p, 3p, 4q, 6q, 8p, 9p, and 14q). An analysis aimed at identifying the relevant genes revealed VHL as one of three genes in the 3p deletion peak, CDKN2A and CDKN2B as the only genes in the 9p deletion peak, and MYC as the only gene in the 8q amplification peak. An integrated analysis to identify genes in amplification peaks that are consistently overexpressed among amplified samples confirmed MYC as a potential target of 8q amplification and identified candidate oncogenes in the other regions. A comparison of genomic profiles revealed that VHL disease-associated tumors are similar to a subgroup of sporadic tumors and thus more homogeneous overall. Sporadic tumors without evidence of biallelic VHL inactivation fell into two groups: one group with genomic profiles highly dissimilar to the majority of ccRCC and a second group with genomic profiles that are much more similar to tumors with biallelic inactivation of VHL. [Cancer Res 2009;69(11):4674–81]

Ecotropic viral integration site1 (EVI1) is a transcriptional regulator that is essential for the maintenance of hematopoietic stem cells and, when overexpressed in leukemia, associates with particularly aggressive disease. EVI1 expression has also been detected in a number of solid tumors including breast carcinoma (BC) where it was recently shown to predict poor survival in estrogen receptor negative (ER-) but not positive (ER+) tumors. The functional roles and molecular regulation of EVI1 in human BC are largely unexplored. Here we confirm that EVI1 is widely expressed in both ER- and ER+ BC in a tissue microarray containing 373 individual patient samples. Unlike in leukemia, overexpression of EVI1 in BC rarely resulted from gene amplification or translocation events at its chromosomal locus 3q26. Functionally, lentiviral knockdown of EVI1 reduced proliferation, motility and in vivo tumorigenicity of human BC cells while enhancing their apoptosis sensitivity. Interestingly, estrogen supplementation rescued growth and tumorigenicity in EVI1-knockdown ER+ (but not ER-) cells. Since microarray analyses of EVI1 knockdown cells revealed alterations of several regulators of cell cycle progression (e.g. CDKN1A, CDKN1C, CDK1), apoptosis (e.g. BIK, BMF, BBC3) and growth factor signaling (e.g. NRG2, EREG, PTK2B), we further investigated the canonical MAPK pathway as a major effector axis of EVI1 in BC. Indeed, knockdown of EVI1 in BC cells effectively impaired ERK1/2 phosphorylation (i.e. MAP kinase activation), which was phenotypically reflected by impaired in vitro proliferation and in vivo tumorigenicity rates. In ER+ BC cells, these effects were functionally restored by exogenous addition of estradiol, explaining why EVI1 gains particular clinical significance in the absence of estrogen signaling (i.e. in ER- BC). Supporting this notion, ectopic expression of EVI1 and estradiol treatment co-induced p-ERK1/2. Our microarray analysis further revealed GPCR signaling molecules (e.g. CCR1, GCGR, PTGFR) as a fourth category of genes significantly modulated by EVI1 in BC. Among these, the GPR54-ligand KISS1, previously implicated in cell migration, was most strongly modulated by EVI1 knockdown and also overexpression. A corresponding promoter analysis indicated several potential EVI1-binding sites within KISS1 regulatory elements, of which two showed robust recovery rates in chromatin immunoprecipitation (ChIP) assays. Functionally, EVI1 knockdown impaired BC cell migration, which was restored by addition of Kisspeptin, a soluble gene product of KISS1. Overall, our data characterize EVI1 as a novel oncogene in BC that impacts growth and migration via pERK and the GPR54-ligand KISS1, which we identified as a novel direct transcriptional target of EVI1-driven oncogenesis. Citation Format: Hui Wang, Thorsten Schaefer, Martina Konantz, Selina Reich, Martin Braun, Sven Perner, Zsuszanna Varga, Holger Moch, Tanja Fehm, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. Novel oncogenic roles of EVI1 in breast carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2015.

Ecotropic viral integration site 1 (EVI1) is a nuclear transcription factor essential for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is found in ~10% of acute myeloid leukemia, where it associates with particularly aggressive disease. EVI1 is also expressed in solid tumors, but remains largely unstudied in this setting. A recent analysis of primary breast carcinoma (BC) samples suggests that enhanced EVI1 expression indicates poor outcome specifically in estrogen receptor (ER) negative tumors (Patel et al., 2011). Here, we explored expression, functional roles and molecular targets of EVI1 in human BC. EVI1 expression was analyzed by qRT-PCR and immunoblot analysis in 8 human BC cell lines and by immunohistochemistry in a cohort of 373 primary BC patient samples collected at the University Hospital Zurich. EVI1 was found to be expressed in most BC samples independently of their ER status. Long-term survival was influenced by EVI1 in ER-, but not ER+ patients, confirming previous results. To investigate the function of EVI1 in BC, cell lines with high and low endogenous EVI1 expression were transduced with EVI1 shRNA knockdown or inducible EVI1-expressing lentiviral particles. Growth, proliferation and cell cycle assays demonstrated that EVI1 promoted BC cell growth by accelerating cell cycle transition. Consistently, EVI1 knockdown BC cells showed reduced in vivo tumor growth compared to control cells in two xenotransplantation models (NSG mice and zebrafish embryo). Interestingly, estrogen supplementation rescued proliferation and tumorigenicity of EVI1-knockdown ER+ but not ER- BC cells, indicating that signaling through the ER pathway might override EVI1-dependent effects on proliferation. Conversely, EVI1-induced proliferation might be particularly important for tumors that are not regulated via the ER pathway, which is in line with its documented prognostic significance in such tumor types. In addition to its effects on proliferation, EVI1 was also found to inhibit BC cell apoptosis in response to both intrinsic and extrinsic stimuli, as previously shown by our group in acute lymphoblastic leukemia cells (Konantz et al, 2013). Moreover, in vitro migration assays showed impaired migration in EVI1 knockdown MDAMB231 cells suggesting that EVI1 regulates BC biology via different mechanisms. Currently, we are exploring the molecular mechanisms by which EVI1 mediates these different effects. Microarray analysis revealed molecules of the MAPK pathways, pro-apoptotic genes and, most interestingly, genes involved in GPCR signaling pathway (e.g. KISS1) as most significantly regulated by EVI1. Analysis of the KISS1 promoter revealed several potential EVI1-binding sites, and a direct association of EVI1 protein with one of these could be confirmed by CHIP. We are currently further exploring the relevance of this binding site for EVI1-mediated activation of KISS1 using luciferase KISS1 reporter assays and site-directed-mutagenesis. In ER- BC, KISS1 expression was reported to enhance cell motility and invasiveness and to reduce cell adhesion. Interestingly, the impaired migration observed in EVI1 knockdown MDAMB231 cells could be indeed rescued by stimulation with Kisspeptin. Neither EVI1 overexpression nor stimulation with Kisspeptin could influence the poorly migrating ER+ MCF7 cells. Taken together, our data identify EVI1 as a novel oncogene particularly important for ER- BC biology. Next to stimulation of BC cell proliferation and reduction of apoptosis sensitivity, we show that EVI1 stimulates ER- BC cell migration via activation of its direct molecular target KISS1. Citation Format: Hui Wang, Thorsten Schäfer, Martina Konantz, Selina Reich, Martin Braun, Sven Perner, Zsuzsanna Varga, Holger Moch, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. EVI1 – a novel oncogene in breast carcinoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A45.

Mutations of the von Hippel-Lindau (VHL) gene are frequent in clear cell renal cell carcinomas (ccRCC). Nonsense and frameshift mutations abrogate the function of the VHL protein (pVHL), whereas missense mutations can have different effects. To identify those missense mutations with functional consequences, we sequenced VHL in 256 sporadic ccRCC and identified 187 different VHL mutations of which 65 were missense mutations. Location and destabilizing effects of VHL missense mutations were determined in silico. The majority of the thermodynamically destabilizing missense mutations were located in exon 1 in the core of pVHL, whereas protein surface mutations in exon 3 affected the interaction domains of elongin B and C. Their impact on pVHL's functionality was further investigated in vitro by stably reintroducing VHL missense mutations into a VHL null cell line and by monitoring the green fluorescent protein (GFP) signals after the transfection of a hypoxia inducible factor (HIF)α-GFP expression vector. pVHL's functionality ranged from no effect to complete HIF stabilization. Interestingly, Asn78Ser, Asp121Tyr, and Val130Phe selectively influenced HIF1α and HIF2α degradation. In summary, we obtained three different groups of missense mutations: one with severe destabilization of pVHL; a second without destabilizing effects on pVHL but relevance for the interaction with HIFα, elongin B, and elongin C; and a third with pVHL functions comparable with wild type. We therefore conclude that the specific impact of missense mutations may help to distinguish between driver and passenger mutations and may explain responses of ccRCC patients to HIF-targeted therapies. Cancer Res; 71(16); 5500–11. ©2011 AACR.

The prognosis of patients with renal cell carcinoma (RCC) is poor. A full understanding of the molecular genetics and signaling pathways involved in renal cancer development and in the metastatic process is of central importance for developing innovative and novel treatment options. In this study, BD Atlas Human Cancer 1.2 cDNA microarrays were used to identify genes involved in renal tumorigenesis. By analyzing gene expression patterns of four clear cell RCC (cRCC) cell lines and normal renal tissue, 25 genes were found differentially expressed. To determine the relevance of these genes, RNA in situ hybridization was performed on a tissue microarray generated from 61 snap-frozen primary renal cell carcinomas and 12 normal renal cortex biopsies. B-cell translocation gene 2 (BTG2), a negative cell cycle regulator, which was expressed in normal renal tissue but down-regulated in cRCC cell lines and primary cRCCs, was selected for additional experiments. Quantitative BTG2 mRNA expression analysis in 42 primary cRCCs and 18 normal renal cortex biopsies revealed up to 44-fold reduced expression in the tumor tissues. Decrease of BTG2 expression was not associated with tumor stage, grade, and survival. Cell culture experiments demonstrated that BTG2 expression was weakly inducible by the phorbolester 12-O-tetradecanoylphorbol-13-acetate in one of four cRCC cell lines. In contrast, increasing cell density led to elevated BTG2 mRNA expression in three of four cRCC cell lines. In both experiments, BTG2 mRNA levels did not reach values observed in normal renal tissue. These data suggest that down-regulation of BTG2 is an important step in renal cancer development.

Background Recurrent mutations in the Speckle-Type POZ Protein (SPOP) gene have been reported in up to 15% of prostate cancers. However, the frequency and features of cancers with these mutations across different populations is unclear. In addition, detection of point mutations in archival samples can be technically challenging, making mutational analysis of well annotated archival tissue difficult. We set out to investigate SPOP mutations across diverse cohorts, and validate a series of assays employing High Resolution Melting Analysis (HRM) and Sanger sequencing for mutational analysis of FFPE material. Methods: An assay employing HRM and Sanger sequencing was optimized to screen for somatic mutations in recurrently altered areas of the SPOP gene. 720 prostate cancer samples from six international cohorts spanning Caucasian, African American, and Asian patients, including both PSA-screened and unscreened populations, were screened for their SPOP mutation status. Status of SPOP was correlated to molecular features (ERG rearrangement, PTEN deletion, CHD1 deletion) as well as clinical and pathologic features. Results: The overall frequency of SPOP mutations was 8.1% in 720 patient's samples, ranging from 4.6% in the African American cohort to 14.4% in the WCMC cohort. There were no significant differences in frequency between ethnicities or cohorts (p=0.14). SPOP mutation was inversely associated with ERG rearrangement (p Conclusions: In this study we were able to show the high sensitivity of HRM followed by Sanger Sequencing to detect mutated SPOP in 720 cases consisting of fresh frozen as well as FFPE material. Using next generation sequencing did not rescue samples which were determined as “assay failure” by HRM and or Sanger Sequencing. SPOP is mutated in 4.6-14.4% of prostate cancer patients across different ethnic and demographic backgrounds. There was no significant association between SPOP mutations with ethnicity, clinical or pathologic parameters. Mutual exclusivity of SPOP mutation with ERG rearrangement as well as a high association with CHD1 deletion reinforces SPOP mutation as defining a distinct molecular subclass of prostate cancer. Citation Format: Mirjam Blattner, Daniel Lee, Catherine O´Reilly, Kyung Park, Theresa Y. MacDonald, Francesca Khani, Kevin Turner, Peter J. Wild, Haley Hieronymus, Charles L. Sawyers, Ashutosh K. Tewari, Holger Moch, Ghil Suk Yoon, Ove Andrén, Katja Fall, Juan Miguel Mosquera, Brian D. Robinson, Andrea Sboner, Christopher E. Barbierie, Mark A. Rubin. SPOP mutations in prostate cancer across demographically diverse patient cohorts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2244. doi:10.1158/1538-7445.AM2014-2244

Type II endometrial carcinomas are a highly aggressive group of tumour subtypes that are frequently associated with inactivation of the TP53 tumour suppressor gene. We show that mice with endometrium-specific deletion of Trp53 initially exhibited histological changes that are identical to known precursor lesions of type II endometrial carcinomas in humans and later developed carcinomas representing all type II carcinoma subtypes. The PI3K-Akt-mTOR signalling pathway was frequently activated in these precursor lesions and tumours, suggesting a genetic cooperation between this pathway and Trp53 deficiency in tumour initiation. Consistent with this idea, immunohistochemical analyses of 521 human endometrial carcinomas identified frequent mTOR pathway activation in type I as well as type II endometrial carcinoma subtypes and mTOR pathway activation and p53 expression each independently predicted poor patient survival. We suggest that molecular alterations in p53 and the PI3K-mTOR pathway play different roles in the initiation of the different endometrial cancer subtypes, but that combined p53 inactivation and PI3K-mTOR pathway activation are unifying pathogenic features among histologically diverse subtypes of late stage aggressive endometrial tumours. 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 3293. doi:1538-7445.AM2012-3293

Background Lung cancer is the leading cause of all cancer deaths worldwide with suboptimal prognosis and treatment options. Therefore we aimed to identify molecular characteristics with a predictive clinical utility that at the same time might represent novel therapeutic targets for human lung adenocarcinoma. Within this study we investigated KRAS mutations, a gene frequently mutated in lung adenocarcinoma and their association with enzymatic activities of members of the serine hydrolase superfamily, a large class of enzymes that have previously been linked to cancer. Methods Participating individuals underwent surgery for lung adenocarcinoma at the University Hospital Zurich between 2003 and 2006. Formalin fixed, paraffin embedded as well as snap-frozen lung adenocarcinoma samples were histologically reviewed by a pathologist. Mutation detection was performed via a custom service by Sequenom. Cell extracts derived from human snap-frozen biopsies were processed prior to mass spectrometric analysis as previously described to analyze serine hydrolase activities. Results Mutation analysis of 40 human lung adenocarcinoma specimens identified 4 patients (10%) harbouring a cysteine for glycine substitution at position 12 (G12C) in the KRAS gene. By employing two-sided unpaired t-test we found a statistical significant (p = 0.01) activity difference of the enzyme myeloblastin between patients harbouring a G12C mutation in the KRAS gene and patients harbouring no KRAS mutation. Conclusions The results of this study revealed that the activity of myeloblastin is significantly altered in lung adenocarcinoma biopsies harboring a KRAS gene mutation. Based on the results of this study we conclude that the combination of activity-based proteomics with mutational analysis is a valid approach for the discovery of novel biomarkers for human lung adenocarcinoma. 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 4794. doi:1538-7445.AM2012-4794

Introduction: Lung neuroendocrine tumors (NET) comprise typical (TC) and atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC) and small cell lung carcinoma (SCLC). Phosphatase and tensin homolog (PTEN) protein is a known cytosolic tumor suppressor with recently described novel nuclear functions. We investigated this differential PTEN protein compartmentalization together with its genomic status in pulmonary NETs. Patients and Methods: A retrospective multicenter cohort of 192 patients with lung NET was investigated on a tissue microarray (TMA, surgical resections = 183/autopsies = 9) by immunohistochemistry (IHC, cytoplasm and nucleus) and fluorescence in-situ hybridization (FISH, nucleus). Results were correlated with markers of neuroendocrine differentiation (synaptophysin, chromogranin A), proliferation (mib-1), cell cycle (p27, cyclin D1) and survival data. Results: The histotypes represented were TC in 58 patients, AC in 42, LCNEC in 32 and SCLC in 60. All NETs showed higher PTEN immunoreactivity in the cytoplasm than in the nucleus, whereby the greatest nucleo-cytoplasmic difference was found for SCLC. In both compartments, immunoreactivity was higher in the carcinoids than in LCNEC and SCLC. Loss of cytosolic or nuclear PTEN protein was correlated with increased mitotic rate, with low synaptophysin or chromogranin A and with PTEN genomic deletion. Loss of nuclear PTEN protein correlated with high nuclear TTF1, whereas loss of cytosolic protein correlated with low p27 and low cyclin D1 (all p-values 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 4159. doi:1538-7445.AM2012-4159

Rationale: Metastatic malignant melanoma patients have a median survival of 8.5 months and a probability of surviving 5 years after the diagnosis of less than 5%. Melanoma patients have been treated with immunotherapeutic approaches including vaccination which induced durable clinical responses in a small subset of patients. Here we present a thorough characterisation of a patient living almost 10 years with metastatic melanoma, who was treated with standard therapy followed by two anti-NY-ESO-1 vaccinations, surgeries and finally Ipilimumab. The patient (ZH-311) was closely immune monitored for the expression of NY-ESO-1 in the tumor and for NY-ESO-1 specific immune response during the course of disease. Methods: The clinical course of the patient was followed from the beginning of diagnosis (03/01) to his death in 07/10. NY-ESO-1-specific CD8+ T cell responses in peripheral blood mononuclear cells (PBMC) were analyzed by intracellular cytokine staining. Antibodies against NY-ESO-1 were monitored by ELISA. Each operated tumor and tumor site found in autopsy was analyzed for NY-ESO-1 by immunohistochemistry. Results: The clinical course of ZH-311 is remarkable, because he lived for more than 6y with immunotherapy and 5 surgical interventions for metastases. 4 surgeries were performed due to progressing disease and the liver metastasis were stable at the time of operation. The early lesions displayed strong NY-ESO-1 expression and a very strong, polyfunctional CD8+ T-cell response against NY-ESO-1 was observed before immunotherapy, which increased upon VF vaccination. The cellular response was mirrored by the antibody response. However, the NY-ESO-1-specific CD8+ T cell response decreased upon vaccination with NY-ESO-1 protein plus CpG, whereas the antibody response remained high for one more year. Examination of later lesions revealed a loss of NY-ESO-1 expression, which is suggestive of escape as a result of immunological pressure. Conclusion: Before and after the vaccination with NY-ESO-1 the patient had an integrated immune response with NY-ESO-1 including antibodies as well as polyfunctional CD8+ T cells. All tumors analysed after the anti-NY-ESO-1 vaccination were NY-ESO-1 negative, suggesting outgrowing escape variants. The stable liver metastases were NY-ESO-1 positive and were apparently immunologically controlled. The persisting antibody levels against NY-ESO-1 dropped after the excision of the NY-ESO-1+ liver metastasis, which suggests a correlation between antigen load and antibody titers. The study we present here provides direct clinical evidence for initial control and susbsequent sculpting of the tumor by the adaptive immune response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5530. doi:10.1158/1538-7445.AM2011-5530

Rationale: It is accepted that the immune system can control cancer and a favorable clinical course correlates with high infiltration by CD8+ T cells in different cancer entities. Thus, boosting cancer-specific immunity is an interesting therapeutic modality. Cancer-testis antigens (CTA) are promising target antigens due to their tumor-restricted expression pattern and high immunogenicity. NY-ESO-1 is one of the best characterized CTA, and spontaneous anti-NY-ESO-1 humoral and cellular responses were described in patients with various cancers. We propose that antibodies (Abs) against intracellular CTA have therapeutic potential because they may facilitate the uptake and presentation of antigens by dendritic cells (DC), which supports cancer-specific T cell responses. This effect may be more pronounced when Abs are used in combination with therapies that result in death, and thus antigen release, of cancer cells. We report here the characterization of the first human anti-NY-ESO-1 monoclonal Ab. Methods: Human-derived monoclonal Ab 12D7 was obtained by molecular cloning from memory B cells of a melanoma patient, ZH-311. To characterize the binding of 12D7 we performed ELISA, Biacore analysis and tissue staining. In addition, we used 12D7 to immunoprecipitate native NY-ESO-1 from human melanoma cell line SK-MEL-37. As a proof of concept, we compared the activation of NY-ESO-1-specific CD8+ T cell clones by DC fed with NY-ESO-1 vs. NY-ESO-1/12D7 immune complexes (IC) in vitro. To determine the therapeutic effect in vivo, we treated BALB/c mice bearing syngeneic CT26/NY-ESO-1 colorectal tumors with 5-FU +/- 12D7. To investigate Ab accumulation in established tumors 12D7 was labeled with FITC and injected into mice 2 days after 5-FU treatment. Results: Human monoclonal 12D7 specifically bound to NY-ESO-1 with high affinity (pM range) and precipitated endogenous NY-ESO-1 from SK-MEL-37. The uptake and cross-presentation of NY-ESO-1 by DC was greatly enhanced when NY-ESO-1 was complexed with 12D7 before feeding. Importantly, uptake of NY-ESO-1/12D7 IC, but not NY-ESO-1, resulted in maturation of DCs as determined by upregulation of co-stimulatory molecules. Finally, 12D7 significantly improved the therapeutic efficacy of 5-FU treatment in established CT26/NY-ESO-1 tumors. Ab accumulation in CT26/NY-ESO-1 tumors was enhanced when mice were treated with 5-FU as compared with untreated mice. Conclusion: We propose that the release of intracellular tumor antigens by chemotherapy and the accumulation of Ab to such antigens results in the local formation of IC. These IC are efficiently cross-presented to tumor-specific CD8+ T cells by DCs and may induce in situ maturation of the latter, both of which support tumor-specific effector function and thus contributes to tumor control. Our results suggest that targeting intracellular antigens, more specifically CTA, with Abs is a useful strategy for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4741. doi:10.1158/1538-7445.AM2011-4741

Introduction and Objective Y chromosome losses have been associated with various malignant tumors including bladder cancer, kidney cancer and prostate cancer. Conflicting data exist on the potential prognostic role of Y chromosome losses in these urological malignancies and on whether Y losses could be age-related. The aim of this study was to further clarify the potential relevance of Y chromosome losses in these tumors with respect to tumor phenotype, clinical outcome and patient age. Methods Preexisting tissue microarrays (TMA) of bladder (573 tissue samples from male patients), kidney (447 tissue samples from male patients) and prostate cancer (3261 tissue samples) with long-term clinical follow-up data were used in this study. Y chromosome losses were analyzed by dual-labeling FISH (fluorescence in situ hybridization) using centromere Y and X probe. Results Y losses were seen in 22% of bladder cancers. There was no significant age difference between patients having tumors with (67.4 +/-2.1years) or without (67.3 +/-1.2 years) Y losses (p=0.6294). Y losses were unrelated to tumor grade (p=0.7831) and stage (p=0.6140). There was no correlation between Y losses and survival in 224 invasive urothelial cancers (pT2-4; p=0.2324). Y losses were not associated with an increased risk for recurrences in 197 analyzed pTa tumors (p=0.7649) or increased progression risk in 76 analyzed pT1 tumors (p=0.4582). In kidney cancer Y losses were seen in 23% of cases. Likewise, there was no significant age difference between patients having tumors with (59.2 +/-3.6 years) or without (61.2+/-1.5 years) Y losses (p=0.5873). Y losses were unrelated to tumor grade (p=0.3111) and tumor stage (p=0.2511). There was also no correlation between Y losses and survival (p=0.7431). Remarkably, Y losses were seen in only 13 of 2826 (0.5%) analyzable cases of prostate cancers. The number of Y chromosome losses was too low for further statistical analyses. Conclusions These data show, that Y losses are frequent in urinary bladder cancer and kidney cancer, but lack association with tumor phenotype, clinical outcome and patient age. While the mechanism for Y losses in these cancers remains unclear, our data do not suggest clinical significance. In contrast to bladder and kidney cancer, Y chromosome losses were almost absent in prostate cancer. This may suggest that prostate epithelial cells are highly dependent on proper function of at least one chromosome Y gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 322.

Aims: Previously, we identified Calcium-activated nucleotidase 1 (CANT1) mRNA to be overexpressed in human prostate cancer, however, so far no involvement of CANT1 in the progression of human tumours was reported. Therefore the protein expression level of CANT1 in prostate tumours was determined, followed by the elucidation of a functional implication of CANT1 overexpression in prostate cancer progression. Methods: Two tissue microarrays including 1100 prostate samples from two different institutions were constructed to examine the protein expression level of CANT1. The role of CANT1 in tumour progression was assessed in two prostate cancer cell lines in an RNA interference based approach. Cell number and DNA synthesis rate were determined by in vitro assays to measure cell proliferation, further cell migration was studied in transwell chamber assays. Results: A recurrent overexpression of CANT1 protein in human prostate cancers was confirmed on the tissue microarrays. Functionally, CANT1 knockdown induced a reduction of LNCaP as well as PC-3 cell number, which was caused by a diminished DNA synthesis rate. Moreover, a considerable decrease of cell migration towards a fibronectin gradient was observed in both cell lines upon CANT1 knockdown. This decreased cell mobility was also accompanied by morphological cell changes. Conclusions: Two cellular processes that are directly linked to tumour progression are markedly impaired upon CANT1 knockdown in prostate cancer cell lines, demonstrating for the first time a tumourbiological relevance of CANT1 expression. In future experiments the mechanism how CANT1 exerts its effects on proliferation and migration will be elucidated as well as the pathways that lead to CANT1 overexpression in prostate cancer. This will contribute to a better understanding of prostate carcinogenesis and to the evaluation of the potential of CANT1 as a therapeutic target. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1772.

Aims: At codon 72, the human p53 gene contains a single nucleotide polymorphism (SNP), where the sequence can either read CGC, which encodes the amino acid arginine (R), or CCC which codes for proline (P). This SNP, p53-R72P, was found to affect numerous carcinogenesis related cell functions, like apoptosis, cell cycle progression, DNA repair, growth arrest and transcriptional activation. P53-R72 was found to be more effective at protecting stressed cells from neoplastic development. Significant associations of this variant to cancer development were found in various cell types, e.g. breast, gastric and lung cancer. No data are available for renal cell carcinoma (RCC). Methods: DNA extracted from formalin-fixed, paraffin-embedded normal tissue of 334 caucasian renal tumor patients (among them 157 affected by clear cell, 54 by papillary, 70 by chromophobe carcinoma and 41 by benign oncocytoma) and 196 controls was examined by restriction fragment length polymorphism (RFLP) using primers for PCR and the restriction enzyme BSTUI. After digestion, the length of the resulting bands was estimated by gel electrophoresis. Genotype distribution of both groups was compared by two sided exact Chi2 test. We also tested for associations of any genotype to histologic subtype, stage, nuclear grade, age at diagnosis, tumor-related survival and gender. Survival analysis was performed using log rank statistics. For examination of the potential influence of the presence or absence of the C allele, analyses were repeated with a combined C/C+CG group vs. G/G. Results: Genotype distribution of RCC or benign oncocytoma carriers and controls did not differ significantly, and there was no correlation of genotype and age of onset. We did not observe a significant acceleration of a certain genotype relative to gender, age group, subtype, stage or nuclear grade. Both tumor-related survival and progression free survival were not affected by genotype. Similar results were obtained when the two C allele carrying groups were pooled for analysis. Conclusions: The p53 R72P polymorphism does not seem to affect the risk of developing RCC or benign oncocytoma, the course of the disease, or histopathologic features in Caucasians. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2838.

Aim: The cell adhesion molecule L1 (L1-CAM) is overexpressed in a variety of human cancers and is correlated to bad prognosis. So far only lung cancer cell lines have been tested for L1-CAM expression. Therefore, we aimed to evaluate the diagnostic and prognostic value of this molecule in non-small cell lung cancer (NSCLC) tissue. Methods: We studied tumours of 509 surgically treated NSCLC patients, assembled on a tissue microarray. The cell membrane protein expression of L1-CAM was immunohistochemically determined and semi-quantitatively scored by two pathologists. Correlations of protein expression with clinico-pathologic parameters were calculated by chi-squared tests, overall survival by the Kaplan-Meier method. Results: L1-CAM protein was overexpressed in 8.7% of the cases. Histological subtype analysis showed that of squamous cell carcinomas 12.9%, of adenosquamous carcinomas 33.3% and of adenocarcinomas 6.7% expressed high membranous L1-CAM. High L1-CAM protein expression was significantly correlated with higher grade, larger tumour size, higher stage and vessel infiltration but not with nodal state. High L1-CAM was significantly correlated with decreased overall survival on univariate analysis and found to be an independent prognostic factor on multivariate cox regression hazard models (including pT, pN and grade, all p-values Conclusions: This is the first comprehensive study on L1-CAM expression in NSCLC. Particularly, epithelial expression of L1-CAM is significantly associated with shortened patient survival defining a subset of tumours with dismal prognosis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2324.

Background Following an accidental observation, we aimed to analyse expression, regulation and function of myoglobin (Mb) in breast cancer. Methods Immunohistochemistry/Immunofluorescence, microdissection, transmission electron microscopy, Western blot, qPCR, cell culture (under normoxia and hypoxia), shRNA and siRNA knockdown, in silico data mining, high resolution respirometry. Functional assays: proliferation, migration and colony formation. Results Mb protein is de novo expressed in 71% of invasive breast carcinomas (n=917) in association with the hypoxia inducible factor 2α (HIF-2 α), carbonic anhydrase IX and a better prognosis. Expression of Mb in breast cancer was confirmed on mRNA level and occurred irrespective of rhabdomyoid differentiation and was inducible by prolonged hypoxia in breast cancer cell lines (MDA-MB231, MDA-MB468, MCF7) with a median change fold of 3.4 after 72 hours. Interestingly, hypoxically induced MB mRNA originated from a novel, alternative transcription start site 6 kb upstream of the genuine ATG codon. Functionally, stable myoglobin knockdown in MDA-MB468 cells was associated with a stimulated O2 uptake during mild hypoxia, yet did not impact the O2 diffusion gradient in these small cells during limiting conditions. Knockdown of Mb also conferred a significant retardation of the cells’ in vitro motility to close an inflicted wound within the normoxic culture. Conclusions Regarding cancer cells, unconventional functions of myoglobin, not directly related to the transport of oxygen, thus need to be envisioned. These unprecedented findings may have fundamental implications for our understanding of non-muscle Mb in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 440.

Purpose: The identification of novel biomarkers for the detection of localized cancers will have a profound impact on human health, especially for tumors with silent progression such as prostate cancer. Current prostate cancer biomarkers such as Prostate Specific Antigen (PSA) have limited accuracy and there is a need for improved non-invasive diagnostic tests. Material and Methods: Blood is thought to pick up molecular cues as it circulates through the various tissues, collectively reflecting molecular processes active in those different tissues. To increase the proteomic detection sensitivity, we have developed a method for the selective analysis of N-glycosites from tissue and serum. The analysis of glycoproteins is ideally suited for the detection of such signatures because they are preferentially released from tissue and deposited in the bloodstream where they can be detected in a non-invasive fashion. In our study we initially used the Pten conditional knock-out mouse model for prostate cancer progression. This model in which the different stages of the disease can be deliberately and reproductively induced and followed, allowed us to simulate with very high fidelity the early steps of the human disease. Under the assumption that proteins of interest are enriched in the tissue, we performed label-free comparative proteomics of Pten−/− and control tissue at different time points. Interesting candidates were then monitored in the corresponding mice sera by targeted mass spectrometry using Multiple Reaction Monitoring (MRM) at high sensitivity and selectivity. Results: This approach enabled us to identify 785 N-linked glycoproteins, mostly located to relevant cellular compartments such as the extracellular space, the plasma membrane or the secretory pathway. Quantitative proteomic comparison of mice with homozygous Pten deletion and constitutive PKB activation with the corresponding control animals led to the identification of 153 candidate biomarkers differentially regulated in a Pten dependent manner. The validation phase was carried out using 76 sera collected from patients with either benign prostatic hyperplasia (n=35) or localized prostate cancer (n=41). Candidate biomarkers were monitored by immunoassays and/or targeted mass spectrometry. Using a cross-validated logistic regression approach in our ongoing study, we have identified a 4-biomarker signature that correctly predicts 71% of cases (sens. 78%/spec. 63%). In comparison, PSA correctly predicted 67% (sens. 89%/spec. 32%) at a cutoff of 2 ng/ml. By combining PSA with the 4-biomarker signature, accuracy was greatly improved and 87% of the cases were correctly predicted (sens. 90/spec. 83%). We further extended our analysis to the discovery of novel candidate serum biomarkers for the discrimination of aggressive cancers (Gleason score>7) from slow progressing cancers (Gleason score Conclusion: The use of a translational approach which relies on a genetically defined mouse model together with novel proteomics techniques and validation in human sera is thus an ideal starting point for the discovery of novel biomarkers. Citation Information: Cancer Res 2009;69(23 Suppl):C28.