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Abstract: Brain tissue is known to have elevated citrate levels, necessary to regulate ion chelation, neuron excitability, and are also necessary for the supply of necessary energy substrates to neurons. Importantly, citrate also acts as a central substrate in cancer metabolism. Recent studies have shown that extracellular citrate levels in the brain undergo significant changes during tumor development and may play a dual role in tumor progression, as well as cancer cell aggressiveness. In the present article, we review available literature describing changes of citrate levels in brain tissue, blood, and cerebrospinal fluid, as well as intracellular alterations during tumor development before and after metastatic progression. Based on the available literature and our recent findings, we hypothesize that changes in extracellular citrate levels may be related to the increased consumption of this metabolite by cancer cells. Interestingly, cancerassociated cells, including reactive astrocytes, might be a source of citrate. Extracellular citrate uptake mechanisms, as well as potential citrate synthesis and release by surrounding stroma, could provide novel targets for anti-cancer treatments of primary brain tumors and brain metastases.

Background Increasing knowledge about the genomic changes underpinning cancer development and growth has led to a rapidly expanding number of individualized therapies that specifically target these changes in a patient's tumor. Here we present a case report of a patient with metastatic esophageal carcinoma whose tumor harbored NTRK1 gene amplification and who received targeted systemic therapy with larotrectinib. At initial diagnosis, the patient presented with tumor obstruction of the middle esophagus, simultaneous liver and lung metastases, UICC IV and WHO performance status 3. Materials and Methods The solid tumor genomic profiling test FoundationOne CDx (F1CDx) was used to detect clinically relevant genomic alterations that, in turn, might identify a targeted therapeutic approach if suggested by the findings. The patient was then treated with larotrectinib and had subsequent follow‐up biopsies. Results Simultaneous biopsies of the primary tumor and liver lesions identified a metastatic squamous cell esophageal carcinoma. Comprehensive genomic profiling obtained from liver metastases identified numerous genomic alterations including amplification of NTRK1. Owing to the reduced performance status of the patient, chemotherapy could not be applied and was denied. Although larotrectinib is only approved for the treatment of cancers with NTRK gene fusions, treatment was started and led to a shrinkage of the primary tumor as well as the liver and lung metastases within 6 weeks according to RECIST criteria accompanied by tumor marker decrease. The NTRK1 gene amplification was below the limit of detection in a subsequent liver biopsy. Conclusion The use of comprehensive genomic profiling, specifically F1CDx, enabled the selection of a targeted therapy that led to a rapid reduction of the tumor and its metastases according to RECIST criteria. This case suggests that larotrectinib is not only effective in NTRK fusions but may be efficacious in cases with gene amplification. Key Points Advances in precision medicine have revolutionized the treatment of cancer and have allowed oncologists to perform more individualized therapy.This case shows that larotrectinib could also be effective in cases of NTRK amplification of cancer.Today, there is only limited knowledge about NTRK alterations in squamous epithelial carcinoma of the esophagus. Longitudinal tumor sequencing during the course of the disease may allow for the detection of a molecular genetic cause once the tumor progresses. Additional actionable gene alterations may then be identified, which may provide the rationale for a therapy switch., Knowledge of the efficacy of targeted therapy for TRK gene amplification is still lacking. This report presents the case of a patient with metastatic squamous cell esophageal carcinoma with NTRK1 gene amplification who received targeted therapy with larotrectinib with promising results.

It is well established that cancer cells acquire energy via the Warburg effect and oxidative phosphorylation. Citrate is considered to play a crucial role in cancer metabolism by virtue of its production in the reverse Krebs cycle from glutamine. Here, we review the evidence that extracellular citrate is one of the key metabolites of the metabolic pathways present in cancer cells. We review the different mechanisms by which pathways involved in keeping redox balance respond to the need of intracellular citrate synthesis under different extracellular metabolic conditions. In this context, we further discuss the hypothesis that extracellular citrate plays a role in switching between oxidative phosphorylation and the Warburg effect while citrate uptake enhances metastatic activities and therapy resistance. We also present the possibility that organs rich in citrate such as the liver, brain and bones might form a perfect niche for the secondary tumour growth and improve survival of colonising cancer cells. Consistently, metabolic support provided by cancer-associated and senescent cells is also discussed. Finally, we highlight evidence on the role of citrate on immune cells and its potential to modulate the biological functions of pro- and anti-tumour immune cells in the tumour microenvironment. Collectively, we review intriguing evidence supporting the potential role of extracellular citrate in the regulation of the overall cancer metabolism and metastatic activity.

Simple Summary Molecular tumor signatures are becoming increasingly important in the treatment of metastatic adenocarcinomas of the gastroesophageal junction (GEJ) and stomach (GC). There are few studies available analyzing data from the Caucasian population regarding molecular signatures and biomarkers. In the presented study, we investigated the distribution of gene variants in outpatients with advanced disease at the onset of diagnosis and correlated them with clinically relevant biomarkers according to ESCAT levels. In addition, we compared the results of conventional diagnostics (IHC/ISH) with NGS findings of gene amplifications. We were able to detect clinically relevant biomarkers according to ESCAT level I in approximately one-third of our patients, which have immediate therapeutic implications. The study highlights the importance of comprehensive molecular profiling for precision treatment of GEJ/GC and indicates that a biomarker evaluation should be performed for all patients with metastatic adenocarcinomas before the initiation of first-line treatment and during second-line or subsequent treatment. Abstract After several years of negative phase III trials in gastric and esophageal cancer, a significant breakthrough in the treatment of metastatic adenocarcinomas of the gastroesophageal junction (GEJ) and stomach (GC) is now becoming evident with the emerging of precision oncology and implementation of molecular targets in tumor treatment. In addition, new generation studies such as umbrella and basket trials are focused on these molecular targets, which makes an early molecular diagnosis based on IHC/ISH and NGS necessary. The required companion diagnostics of Her2neu overamplification or PD-L1 expression is based on immunohistochemistry (IHC) or additionally in situ hybridization (ISH) in case of an IHC Her2neu score of 2+. However, there are investigator-dependent differences in the assessment of Her2neu amplification and different PD-L1 scoring systems obtained by IHC/ISH. The use of high-throughput technologies such as next-generation sequencing (NGS) holds the potential to standardize the analysis and thus make them more comparable. In the presented study, real-world multigene sequencing data of 72 Caucasian patients diagnosed with metastatic adenocarcinomas of GEJ and stomach were analyzed. In the clinical companion diagnostics, we found ESCAT level I molecular targets in one-third of our patients, which directly determined the therapy. In addition, we found potential targets in 14/72 patients (19.4%) who potentially qualify for precision therapies in corresponding molecular studies. The study highlights the importance of comprehensive molecular profiling for precision treatment of GEJ/GC and indicates that a biomarker evaluation should be performed for all patients with metastatic adenocarcinomas before the initiation of first-line treatment and during second-line or subsequent treatment.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous diagnostic category with clinical manifestations in virtually every location. Here, we investigated the frequencies of mutations affecting C...

e13005 Background: Next generation sequencing (NGS) together with protein expression analysis is back bone of molecularly targeted therapy in precision medicine. The present study shows the retrospective analysis of our experience with NGS using a panel of 324 genes in combination with protein expression in patients with metastatic breast cancer (mBC) over the last 18 months. The primary objective of this study was to analyze the prevalence of individual genetic alterations combined with protein expression to define potential targets for an individualized therapy. Methods: We analyzed 41 patients with advanced BC using solid tumor genomic profiling test. The test was used to detect clinically relevant genomic alterations (point mutations, indels, rearrangements, CNAs, tumor mutation burden (TMB) and microsatellite instability (MSI)) and to support the selection of appropriate targeted therapy. The protein expression (hormon receptors, ERBB2 and PDL1) of tumors was analyzed by immunohistochemistry (IHC). Results: The most common BC subtypes were HR+/ERBB2- (n = 20), followed by triple-negative (n = 15), and HER2+ BC (n = 9). 41 different genetic alterations were reported. Most patients had more than one genetic alteration (n = 27), as determined by NGS. The most common alterations were PIK3CA (n = 14), out of which 11 (78%) were hormone-receptor positive/ERBB2 negative. Conclusions: The NGS of mBC supports the decision for the most promising treatment option in 58,5% of our patients with a Tier I evidence. For an appropriate treatment decision it is necessary to evaluate gene alterations und protein expression of metastases in account. For an appropriate interpretation of rare gene alterations in mBC basket trials on basis of real world data are necessary. We founded the OnkoVision alliance to bring MTB into clinically routine and to identify appropriate patients for basket trials.

Glycolysis and fatty acid synthesis are highly active in cancer cells through cytosolic citrate metabolism, with intracellular citrate primarily derived from either glucose or glutamine via the tricarboxylic acid cycle. We show here that extracellular citrate is supplied to cancer cells through a plasma membrane-specific variant of the mitochondrial citrate transporter (pmCiC). Metabolomic analysis revealed that citrate uptake broadly affected cancer cell metabolism through citrate-dependent metabolic pathways. Treatment with gluconate specifically blocked pmCiC and decreased tumor growth in murine xenografts of human pancreatic cancer. This treatment altered metabolism within tumors, including fatty acid metabolism. High expression of pmCiC was associated with invasion and advanced tumor stage across many human cancers. These findings support the exploration of extracellular citrate transport as a novel potential target for cancer therapy. Significance: Uptake of extracellular citrate through pmCiC can be blocked with gluconate to reduce tumor growth and to alter metabolic characteristics of tumor tissue. Cancer Res; 78(10); 2513–23. ©2018 AACR.

Inhibiting tumor growth by targeting the tumor vasculature was first proposed by Judah Folkman almost 40 years ago. Since then, different approaches and numerous drugs and agents have been developed to achieve this goal, either with the aim of inhibiting tumor neoangiogenesis or normalizing the tumor vasculature. Among the most promising therapeutic targets are receptor tyrosine kinases (RTKs), some of which are predominantly expressed on tumor endothelial cells, although they are sometimes also present on tumor cells. The majority of RTK inhibitors investigated over the past two decades competes with ATP at the active site of the kinase and therefore block the phosphorylation of intracellular targets. Some of these drugs have been approved for therapy, whereas others are still in clinical trials. Here, we discuss the scientific basis, current status, problems and future prospects of RTK inhibition in anti-tumor therapy.

Leiomyosarcomas remain challenging tumors to manage and novel therapy strategies besides radiation and conventional chemotherapy are needed. Targeting angiogenesis by inhibition of vascular endothelial growth factor (VEGF) receptor tyrosine kinases (RTKs) of the tumor vasculature with small molecules is a promising new therapy. It has been shown recently that these receptors are not only expressed on tumor endothelium but also on tumor cells themselves. Thus, we investigated the expression of members of the VEGF receptor (VEGFR) family and corresponding growth factors in leiomyosarcoma tissue specimens and in the leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1. We evaluated the influence of the VEGFR inhibitor PTK787/ZK222584 (PTK787) on cell growth, migration, apoptosis and phosphorylation of intracellular signalling molecules. In human leiomyosarcoma tissue specimens VEGFR-1/-2 and platelet-derived growth factor receptor (PDGFR-β) were strongly expressed. Both leiomyosarcoma cell lines expressed VEGFR-1/-3 and PDGFR-β but VEGFR-2 protein expression was positive only in SK-UT-1. SK-LMS-1 and SK-UT-1 cells secreted high and low amounts of VEGF-A, respectively, whereas PDGF-BB secretion was similar in both cell lines. Application of PTK787 led to partial inhibition of PDGF-BB-activated AKT/p90RSK and ERK1/2 signalling pathways. In contrast, protein phosphorylation was not affected by PTK787 in VEGF-A-treated cells. PTK787 turned out to inhibit cell migration even though no effects were observed upon stimulation with VEGF-A or PDGF-BB. In line, cell growth in leiomyosarcoma cell lines remained unchanged upon PTK787 treatment alone and with subsequent VEGF-A- or PDGF-BB-stimulation. However, VEGF-A, but not PDGF-BB-treated cells showed increased cell death upon PTK787 treatment. VEGFR family members are expressed in leiomyosarcomas in vivo and in vitro. Upon receptor stimulation, PTK787 is able to inhibit subsequent phosphorylation events and influences cell survival but not metabolic activity and migration. Thus, the inhibitor is possibly an additional option in the treatment of leiomyosarcomas.

Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene causing constitutive oncogenic protein activation have been shown to be frequent in papillary noninvasive bladder tumours and are associated with a low risk of progression and a favourable outcome. FGFR3 alterations have also been found in benign urothelial papilloma and flat urothelial hyperplasia suggesting FGFR3 alterations as an early event in bladder tumorigenesis. To date there is no data available on FGFR3 mutations in normal urothelium from patients with bladder cancer. We therefore analysed 64 samples of histopathological unsuspicious normal urothelium from 38 patients with FGFR3 mutated bladder tumours and 15 samples of urothelium from patients (n = 15) without any urothelial malignancy as a control group. Urothelial cells were microdissected from formalin-fixed, paraffin-embedded material. After DNA isolation whole genome amplification was done by I-PEP-PCR. FGFR3 mutations were detected using SNaPshot analysis. All samples could successfully be investigated. FGFR3 analyses did not reveal any mutation in the urothelium from neither the control group nor the bladder cancer group. All urothelial samples showed a wildtype sequence for FGFR3. These data suggest that mutations in the FGFR3 gene are not the earliest genetic alterations in bladder carcinogenesis and are associated with a hyperproliferative (hyperplastic) phenotype in the urothelium. Chromosomal alterations like deletions on chromosome 9q or aberrant promoter hypermethylation could play more important roles in early urothelial transformation than mutational FGFR3 activation. © 2009 UICC

Intensive lymph node involvement indicates poor prognosis in breast cancer patients. The significance of other molecular prognostic factors in this subgroup is unclear. Karyopherin alpha2 (KPNA2) has been reported as an important factor of tumorgenesis and progression of breast cancer. The aim of present study was to evaluate the impact of KPNA2 expression on prognosis of patients with high risk breast cancer (HRBC) and response intensive chemotherapy within the randomized WSG-AM-01 trial. KPNA2 nuclear expression (10% vs.10% of nuclei) was measured by immunohistochemistry on tissue arrays of 191 patients randomized to tandem high dose vs. conventional dose-dense chemotherapy in HRBC with9 positive lymph nodes and correlated with clinical outcome (median follow-up of 63.3 months) by Kaplan-Meier and multivariate Cox hazard model analysis, including, molecular subtypes determined by k-clustering (k = 5). KPNA2 overexpression (n = 74, 39%) significantly correlated with shorter event-free and overall survival (OS) in both therapy arms by univariate analysis. Multivariate analysis showed that the overexpression of KPNA2 was an independent prognostic factor of decreased OS HR = 1.86 [95% CI: 1.07-3.23, p = 0.03]. This predictive value was independent of basal-like/Her-2/neu subtypes, significantly associated with KPNA2 and was addressed particularly to G2 tumors. Our data suggest the use of KPNA2 nuclear expression as novel prognostic marker in node-positive patients, especially in determination of G2 tumors in 2 subgroups of different prognosis. KPNA2 expression may be also considered as a marker for global chemoresistance, which can not be overcome by conventional dose-modification of chemotherapy in advanced breast cancer.

The mammalian target of rapamycin (mTOR) has become an interesting target for cancer therapy through its influence on oncogenic signals, which involve phosphatidylinositol-3-kinase and hypoxia-inducible factor-1α (HIF-1α). Since mTOR is an upstream regulator of HIF-1α, a key mediator of gastric cancer growth and angiogenesis, we investigated mTOR activation in human gastric adenocarcinoma specimens and determined whether rapamycin could inhibit gastric cancer growth in mice. Expression of phospho-mTOR was assessed by immunohistochemical analyses of human tissues. For in vitro studies, human gastric cancer cell lines were used to determine S6K1, 4E-BP-1 and HIF-1α activation and cancer cell motility upon rapamycin treatment. Effects of rapamycin on tumor growth and angiogenesis in vivo were assessed in both a subcutaneous tumor model and in an experimental model with orthotopically grown tumors. Mice received either rapamycin (0.5 mg/kg/day or 1.5 mg/kg/day) or diluent per intra-peritoneal injections. In addition, antiangiogenic effects were monitored in vivo using a dorsal-skin-fold chamber model. Immunohistochemical analyses showed strong expression of phospho-mTOR in 60% of intestinal- and 64% of diffuse-type human gastric adenocarcinomas. In vitro, rapamycin-treatment effectively blocked S6K1, 4E-BP-1 and HIF-1α activation, and significantly impaired tumor cell migration. In vivo, rapamycin-treatment led to significant inhibition of subcutaneous tumor growth, decreased CD31-positive vessel area and reduced tumor cell proliferation. Similar significant results were obtained in an orthotopic model of gastric cancer. In the dorsal-skin-fold chamber model, rapamycin-treatment significantly inhibited tumor vascularization in vivo. In conclusion, mTOR is frequently activated in human gastric cancer and represents a promising new molecular target for therapy. © 2007 Wiley-Liss, Inc.

Oncology reports 33(5), 2129-2134 (2015). doi:10.3892/or.2015.3851, Published by Spandidos Publ., Athens

The aim of the present population-based cohort study was to analyze the association between the prevalence of 32 types of human papilloma virus (HPV) in 615 female patients with abnormal cervical cytopathology findings. In total, 32 HPV types were screened by DNA array technology. HPV infection was detected in 470 women (76.42%), 419 of whom (89.15%) were infected with ≥1 high-risk (HR)-HPV type. HPV16, which is recognized as the main HR-HPV type responsible for the development of cervical cancer, was observed in 32.98% of HPV+ participants, followed by HPV42 (18.09%), HPV31 (17.66%), HPV51 (13.83%), HPV56 (10.00%), HPV53 (8.72%) and HPV66 (8.72%). The prevalence of HR-HPV types, which may be suppressed directly (in the case of HPV16 and 18), or possibly via cross-protection (in the case of HPV31) following vaccination, was considerably lower in participants ≤22 years of age (HPV16, 28.57%; HPV18, 2.04%; HPV31, 6.12%), compared with participants 23-29 years of age (HPV16, 45.71%; HPV18, 7.86%; HPV31, 22.86%), who were less likely to be vaccinated. Consequently, the present study hypothesizes that there may be a continuous shift in the prevalence of HPV types as a result of vaccination. Furthermore, the percentage of non-vaccine HR-HPV types was higher than expected, considering that eight HPV types formerly classified as 'low-risk' or 'probably high-risk' are in fact HR-HPV types. Therefore, it may be important to monitor non-vaccine HPV types in future studies, and an investigation concerning several HR-HPV types as risk factors for the development of cervical cancer is required.

Background. Development of cancer in transplant recipients may be influenced by different immunosuppressive agents. Recent publications suggest that rapamycin (RAPA), or possibly mycophenolate mofetil (MMF), may reduce established tumor growth; however, experimental data is lacking for de novo cancer prevention. Methods. We tested the effects of long-term immunosuppression on spontaneous tumor formation in p53 knock-out mice. Mice received no treatment, or were given RAPA, MMF, or cyclosporine (CsA) starting on week nine after birth, with the experimental endpoint being week 29. Results. All (9/9) untreated mice developed clinically evident tumors before week 26, as confirmed by histology (6 lymphomas, 2 sarcomas, 1 lymphoma+sarcoma). All CsA-treated mice (9/9) also developed clinical tumors before the endpoint (7 lymphomas, 1 sarcoma, 1 lymphoma + sarcoma). With MMF, 7/10 mice showed clinical evidence of tumor before the experimental endpoint (4 lymphomas, 2 sarcomas, 1 lymphoma+sarcoma), however, histologic tissue analysis revealed that the remaining three mice had subclinical cancer (3 lymphomas). In contrast, RAPA treatment resulted in only three mice with clinical tumors (all lymphomas), with histology revealing subclinical lymphomas in three additional mice, but no evidence of cancer in four animals. Statistically, cancer development was decreased with RAPA treatment (P=0.002), but was not affected with either MMF or CsA (P>0.10). Conclusion. These experiments are the first to show immunosuppression under RAPA can reduce spontaneous de novo cancer associated with p53 mutations. Although neither CsA nor MMF treatment affects p53-associated tumor incidence, MMF may have some tendency to reduce clinical tumor appearance.

Sarcomas are known to develop resistance to current chemotherapeutic strategies, displaying a multidrug-resistant phenotype. Mechanisms involved in drug resistance include reduced cellular drug accumulation, drug detoxification as well as alterations in drug target specificity. In seven sarcomas of the pulmonary artery (SPA) and ten leiomyosarcomas of other origin, we studied the immunohistochemical expression of P-glycoprotein (P-gp), multidrug-resistance protein (MRP), lung resistance protein (LRP), metallothionein (MT) and topoisomerase IIalpha. Upregulation was found in tumour cells for P-gp but not for MRP in SPA and other leiomyosarcomas. Topoisomerase IIalpha was expressed at high levels in tissue of primary tumours as well as recurrent tumours. Both P-gp and topoisomerase IIalpha were present in numerous tumour-associated vessels. LRP was expressed at high levels in SPA but to a lesser extent in the other leiomyosarcomas. MT was expressed at low levels but was markedly present at the border of necrosis. The overall survival and the relapse-free survival did not correlate with the expression of these factors. There was no significant relationship between treated and non-treated patients with respect to the expression of the examined molecules. P-gp, but not MRP, may play a role in the development of drug resistance. P-gp, LRP and topoisomerase IIalpha contribute to drug resistance through expression in tumour-associated vessels. Unique high levels of topisomerase IIalpha reflect the high proliferation rate of these tumours. MT seems to serve as a detoxifying agent of metabolites at the border of necrosis. Our findings underline the fact that multiple factors contribute to chemoresistance and that examination of a spectrum of relevant molecules is probably necessary to plan the best therapy.

Compared to other members of the fibroblast growth factor receptor (FGFR) family, only few studies investigate FGFR3 in tumour angiogenesis. We investigated the connection between angiogenesis and FGF/FGFR expression including FGFR3 mutation status in urothelial carcinomas. Immunohistochemistry was performed in invasive and non-invasive urothelial cancers of 61 patients. Protein expression of CD31, factor VIII (FVIII), FGF-1/2, FGFR1, FGFR3 and FGFR4 and FGFR3 mutation status were evaluated. Morphometric assessment of angiogenesis including microvessel count (MVC) and vascular surface area (VSA) was analysed. Correlation and survival analyses (overall survival (OS) and disease-free survival (DFS)) with univariate and multivariate analyses were performed. CD31 values (MVC and VSA) significantly correlated with OS and DFS. OS and DFS were significantly better in patients with FGFR3 overexpression. Multivariate analysis revealed FGFR3 protein expression and tumour grading (WHO classification 2004) as independent prognostic factors of OS and VSA of CD31 and FGFR3 protein expression of DFS. FGFR3 mutation status was correlated with VSA measured by FVIII. FGFR3 may be able to induce a pro-angiogenic phenotype in urothelial carcinomas and significantly influence prognosis. Consequently, FGFR3 is a potential therapeutic target also from the angiogenesis perspective.

Department of Molecular Cell Biology, Max-Planck Institute for Physiology and Clinical Re-search, Bad Nauheim, Germany.The results of this article were presented at theannual Meeting of the German Society of Pathol-ogy, May 26–29, 1999, Jena, Germany.The authors acknowledge the excellent technicalsupport of Sandra Gerecht and Antonietta Ras-tiello, the photographic support of Peter Pulkowskiand Thomas Bo¨hm, and the helpful comments andcritical reading of the manuscript by Dr. NorbertSpeich (Imogen, Bonn, Germany) and Dr. Ron Un-ger.Address for correspondence: Andreas Gaumann,M.D., Department of Molecular Cell Biology, Max-Planck Institute for Physiology and Clincial Re-search, W. G. Kerckhoff Institut, Parkstrasse 1,D-61231 Bad Nauheim, Germany; Fax: 49-6032-705219; Email: a.gaumann@Kerckhoff.mpg.deReceived December 15, 2000; revision receivedApril 6, 2001; accepted April 10, 2001.

Immunosuppressive drugs are thought to cause the dramatically increased risk of carcinomas in sun-exposed skin of organ transplant recipients. These drugs differ in local effects on skin. We investigated whether this local impact is predictive of skin cancer risk and may thus provide guidance on minimizing the risk. Immunosuppressants (azathioprine, cyclosporine, tacrolimus, mycophenolate mofetil, and rapamycin) were assessed on altering the UV induction of apoptosis in human skin models and of p53 mutant cell clones (putative tumor precursors) and ensuing skin carcinomas (with mutant p53) in the skin of hairless mice. Rapamycin was found to increase apoptosis (three-fold), whereas cyclosporine decreased apoptosis (three-fold). Correspondingly, a 1.5- to five-fold reduction (P = 0.07) or a two- to three-fold increase (P < 0.001) was found in cell clusters overexpressing mutant p53 in chronically UV-exposed skin of mice that had been fed rapamycin or cyclosporine, respectively. Deep sequencing showed, however, that the allelic frequency (∼5%) of the hotspot mutations in p53 (codons 270 and 275) remained unaffected. The majority of cells with mutated p53 seemed not to overexpress the mutated protein. Unexpectedly, none of the immunosuppressants admixed in high dosages to the diet accelerated tumor development, and cyclosporine even delayed tumor onset by approximately 15% (P < 0.01). Thus, in contrast to earlier findings, the frequency of p53-mutant cells was not predictive of the incidence of skin carcinoma. Moreover, the lack of any accelerative effect on tumor development suggests that immunosuppressive medication is not the sole cause of the dramatic increase in skin cancer risk in organ transplant recipients. Cancer Prev Res; 6(2); 129–38. ©2012 AACR.

International audience; The goal of this analysis was to characterize the survival impact of angiogenesis in the patients with high-risk breast cancer, particularly the predictive impact on benefit from dose intensification of adjuvant chemotherapy. Formalin-fixed tissue sample of 152 patients treated as part of the WSG AM-01 trial by either high-dose or conventional dose-dense chemotherapy were analyzed. Angiogenic activity was measured using microvessel count and vascular surface area (VSA) determined by the expression of vascular markers CD31 ( = 128) and CD105/endoglin ( = 130). Protein molecular breast cancer subclasses were analyzed by -means clustering ( = 5). The univariate impact of factors on event-free (EFS) and overall survival (OS) was tested by log-rank statistics and quantified by univariate Cox analysis. Multivariate survival analysis included factors significant in univariate analysis, as well as interactions was performed for EFS. Both VSA/CD31 ( = 0.004) and VSA/CD105 ( = 0.003) were significantly higher among cases with increased Ki-67. A significant association with molecular subtypes was also found for VSA/CD105: in patients with basal-like/Her-2 subtypes, mean was 1.72 versus 1.24 in patients with other subtypes (

Increased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer generation immunosuppressive drugs, rapamycin (Rapa) and mycophenolate mofetil (MMF), have been shown to impair angiogenesis and outgrowth of tumor implants. To ascertain the overall effect on UV carcinogenesis, Rapa and MMF were admixed into the food pellets of hairless SKH1 mice receiving daily sub-sunburn UV dosages. With immunosuppressive blood levels neither of the drugs affected onset of tumors (2 mm), but in contrast to MMF, Rapa significantly increased latency of large tumors (or=4 mm, medians of 190 vs 125 days) and reduced their multiplicity (1.6 vs 4.5 tumors per mouse at 200 days). Interestingly, tumors (2 mm) from the Rapa-fed group showed a reduction in UV-signature p53 mutations (39% vs 90%) in favor of mutations from putative base oxidation. This shift in mutation spectrum was not essentially linked to the reduction in large tumors because it was absent in large tumors similarly reduced in number when feeding Rapa in combination with MMF, possibly owing to an antioxidant effect of MMF. Significantly fewer tumor cells were Vegf-positive in the Rapa-fed groups, but a correspondingly reduced expression of Hif1alpha target genes (Vegf, Ldha, Glut1, Pdk1) that would indicate altered glucose metabolism with increased oxidative stress was not found. Remarkably, we observed no effect of the immunosuppressants on UV-induced tumor onset, and with impaired tumor outgrowth Rapa could therefore strongly reduce skin carcinoma morbidity and mortality rates in organ transplant recipients.

The Ras/Raf/MEK pathway represents an important oncogenic signaling pathway in gastrointestinal malignancies, including pancreatic cancer. Although activating B-Raf mutations are infrequent in pancreatic cancer, we hypothesized that targeting Raf could be valuable for therapy of this cancer entity. Moreover, as vascular endothelial growth factor receptor 2 (VEGFR2) is involved in tumor angiogenesis, we sought to investigate the effects of dual inhibition of Raf and VEGFR2 on pancreatic tumor growth, vascularization, and metastasis. Effects of a Raf/VEGFR2 inhibitor (NVP-AAL881) on pancreatic cancer cells, endothelial cells, and vascular smooth muscle cells were determined by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and migration assays, respectively. Changes in the expression of VEGF-A or survivin were investigated by ELISA and/or real-time PCR. The growth-inhibitory effects of Raf/VEGFR2 inhibition were additionally evaluated in orthotopic tumor models. Results showed that various Raf isoforms were activated in pancreatic cancer cells and NVP-AAL881 diminished the activation of MEK, Akt, Erk, and also STAT3. Moreover, dual inhibition of Raf/VEGFR2 significantly reduced VEGF expression and impaired cancer cell migration. Importantly, besides blocking VEGF-induced Erk and SAPK phosphorylation in endothelial cells, the Raf inhibitor diminished STAT3 phosphorylation, independent of a VEGFR2 blockade, and reduced the expression of survivin. In addition, cell proliferation and migration of both endothelial cells and vascular smooth muscle cells were significantly reduced. In vivo, blocking Raf/VEGFR2 significantly inhibited orthotopic tumor growth and vascularization and reduced cancer metastasis. In conclusion, blocking Raf exerts growth-inhibitory effects on pancreatic tumor cells, endothelial cells, and pericytes and elicits antiangiogenic properties. Dual targeting of Raf and VEGFR2 appears to be a valid strategy for therapy of pancreatic cancer. [Mol Cancer Ther 2008;7(11):3509–18]

Tumor angiogenesis is a major step in tumor progression to clinically symptomatic cancer and thus a potential target for cancer therapy. It is essential to understand the fundamental mechanisms of the angiogenic processes to provide a rational for testing inhibitory strategies for cancer treatment. The dorsal skin fold chamber provides a suitable (chronic) model for intravital microscopy to monitor the same tumor in time-lapse imaging series and in real-time functional analysis e.g., of blood flow. Adaptation of this model to several rodent species and tumor types has led to numerous physical and drug based therapy options. With modification of implantation techniques, motility and invasion of individual cells can be visualized, in addition to angiogenesis and microcirculation. Modern fluorescent techniques such as ex vivo labelling of specific cell populations and the introduction of stably fluorescent protein expressing cell lines further enhance the suitability of this technique. In addition, laser scanning and multiphoton microscopy in combination with genetically altered mouse strains and cell lines are making the DCSF even more attractive for mechanistic and interventional studies in cancer research. Here we review the preparation as well as the applications of the DCSF in tumor angiogenesis.

Neoangiogenesis, driven by a variety of angiogenic factors, plays an essential role during development and progression of malignant tumors. Vascular endothelial growth factor (VEGF) and its receptors have been designated a central part in the angiogenic process during malignancy. We studied the vascular parameters by means of morphology and morphometry in 7 sarcomas of the pulmonary artery (SPA) and 10 poorly differentiated leiomyosarcomas of soft tissue. Immunohistochemical analysis of VEGF and VEGFR was related to survival and prognosis. The microvessel density (MVD) and intervascular distances (IVD) differed significantly only at sites of necrosis compared to non-necrotic areas in SPA but not for soft tissue leiomyosarcomas. MVD, IVD and vascular surface area (VSA) revealed no difference between SPA and leiomyosarcomas of different origin. We found a more pronounced expression of VEGF in most tumors at sites of necrosis. The receptors were present in a subset of tumor vessels mostly at the tumor border. VEGFR-2 expression was also seen in a subset of tumor cells whereas VEGFR-1 showed only weak expression in some tumors. Local hypoxia seems to induce a higher MVD and a lower IVD at sites of necrosis compared to those areas without necrosis. The presence of necrosis in both sarcoma groups was correlated with the presence of VEGF due to local tumor hypoxia and subsequent up-regulation of VEGFR-2 and VEGFR-1 in tumor vessels as well as tumor cells. Overall and relapse-free survival showed no difference concerning all examined parameters. Thus, microvessel density does not seem to be a prognostic factor in SPA and other sarcomas.

Neoangiogenesis is involved in the development and progression of malignant tumors. Vascular endothelial growth factor (VEGF) and its receptors have been designated a central part in this process. Since the significance of the assessment of angiogenesis in soft tissue tumors is still a matter of debate, we investigated the vascularisation of cardiac myxomas and compared it with pulmonary artery sarcomas (PAS). Angiogenesis in 18 PAS and 20 myxomas was assessed by morphometry. An immunohistochemical analysis of growth factors and their receptors, HIF-1alpha and tumor-associated macrophages (TAM) was performed. Results showed that microvessel density (MVD) in PAS was significantly higher at the border of necrosis versus the areas without necrosis but no difference was observed between PAS and myxomas. Vascular surface area (VSA) and intervascular distances showed a higher vascularisation at the border of necrosis compared to myxomas, which was not significant. VEGF expression was higher in PAS compared to myxomas and was prominent at the sites of necrosis. HIF-1alpha expression was marked at the border of necrosis in PAS but was absent in myxomas. Infiltration of the macrophages was significantly higher in myxomas compared to the sarcomas. VEGFR-2 expression was detected in a subset of tumor cells and in blood vessels mainly at the tumor periphery, whereas VEGFR-1 was weakly expressed in the tumors but prominent in the macrophages in cardiac myxomas. PDGF receptors and their ligands are strongly present in myxomas and to a lesser extent in the sarcomas. In conclusion, benign and malignant cardiovascular tumors with a different pathophysiology develop a comparable vascularisation. Hypoxia appears to be the strongest inducer of neoangiogenesis in the sarcomas. The expression of receptor tyrosine kinases of the VEGF family provides a basis for an adjuvant therapy.

The insulin-like growth factor-I receptor (IGF-IR) is frequently overexpressed and constitutively activated in pancreatic cancer, thus representing a promising target for therapy. We investigated the impact of a novel inhibitor of IGF-IR (NVP-AEW541) on signalling and growth of pancreatic cancer. Human pancreatic cancer cells and endothelial cells were employed, and effects of NVP-AEW541 on signalling pathways investigated by Western blotting. NVP-AEW541 diminished the activation of IGF-IR, IRS-1, Erk, Akt and STAT3. Furthermore, NVP-AEW541 reduced cancer cell proliferation and abrogated migratory effects of IGF-I. NVP-AEW541 elicited a direct effect on endothelial cells in terms of reducing endothelial cell migration. In vivo, treatment of mice with NVP-AEW541 significantly reduced orthotopic pancreatic tumour growth, vascularisation, and VEGF expression. Interestingly, NVP-AEW541 lowered serum levels of IGF-binding-protein-3 (IGFBP-3). In conclusion, the IGF-IR inhibitor NVP-AEW541 effectively disrupts IGF-I signalling and reduces pancreatic tumour growth. Hence, blocking IGF-IR could prove valuable for targeted therapy of pancreatic cancer.

We recently showed that inhibition of heat shock protein 90 (Hsp90) decreases tumor growth and angiogenesis in gastric cancer through interference with oncogenic signaling pathways. However, controversy still exists about the antimetastatic potential of Hsp90 inhibitors. Moreover, in vitro studies suggested that blocking Hsp90 could overcome p53-mediated resistance of cancer cells to oxaliplatin. We therefore hypothesized that blocking oncogenic signaling with a Hsp90 inhibitor would impair metastatic behavior of colon cancer cells and also improve the efficacy of oxaliplatin in vivo. Human colon cancer cells (HCT116, HT29, and SW620) and the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) were used for experiments. In vitro, 17-DMAG substantially inhibited phosphorylation of epidermal growth factor receptor, c-Met, and focal adhesion kinase, overall resulting in a significant decrease in cancer cell invasiveness. Importantly, 17-DMAG led to an up-regulation of the transcription factor activating transcription factor-3, a tumor suppressor and antimetastatic factor, on mRNA and protein levels. In a cell death ELISA, 17-DMAG markedly induced apoptosis in both p53-wt and p53-deficient cells. In vivo, 17-DMAG significantly reduced tumor growth and vascularization. Furthermore, blocking Hsp90 reduced hepatic tumor burden and metastatic nodules in an experimental model of hepatic colon cancer growth. Importantly, combining oxaliplatin with 17-DMAG in vivo significantly improved growth inhibitory and proapoptotic effects on p53-deficient cells, compared with either substance alone. In conclusion, inhibition of Hsp90 abrogates the invasive properties of colon cancer cells and modulates the expression of the antimetastatic factor activating transcription factor-3. Hence, targeting Hsp90 could prove valuable for treatment of advanced colorectal cancer by effectively inhibiting colon cancer growth and hepatic metastasis and improving the efficacy of oxaliplatin. [Mol Cancer Ther 2007;6(11):2868–78]

Purpose: Inhibitors of heat-shock protein 90 (Hsp90) may interfere with oncogenic signaling pathways, including Erk, Akt, and hypoxia-inducible factor-1α (HIF-1α). Because insulin-like growth factor-I receptor (IGF-IR) and signal transducer and activator of transcription 3 (STAT3) signaling pathways are implicated in the progression of pancreatic cancer, we hypothesized that blocking Hsp90 with geldanamycin derivates [17-allylamino-geldanamycin (17-AAG), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)] would impair IGF-I– and interleukin-6–mediated signaling and thus reduce pancreatic tumor growth and angiogenesis in vivo. Experimental Design: Human pancreatic cancer cells (HPAF-II, L3.6pl) were used for experiments. Changes in signaling pathway activation upon Hsp90 blockade were investigated by Western blotting. Effects of Hsp90 inhibition (17-AAG) on vascular endothelial growth factor were determined by ELISA and real-time PCR. Effects of 17-DMAG (25 mg/kg; thrice a week; i.p.) on tumor growth and vascularization were investigated in a s.c. xenograft model and in an orthotopic model of pancreatic cancer. Results: 17-AAG inhibited IGF-IR signaling by down-regulating IGF-IRβ and directly impairing IGF-IR phosphorylation. Hypoxia- and IL-6–mediated activation of HIF-1α or STAT3/STAT5 were substantially inhibited by 17-AAG. Moreover, a novel IL-6/STAT3/HIF-1α autocrine loop was effectively disrupted by Hsp90 blockade. In vivo, 17-DMAG significantly reduced s.c. tumor growth and diminished STAT3 phosphorylation and IGF-IRβ expression in tumor tissues. In an orthotopic model, pancreatic tumor growth and vascularization were both significantly reduced upon Hsp90 inhibition, as reflected by final tumor weights and CD31 staining, respectively. Conclusions: Blocking Hsp90 disrupts IGF-I and IL-6–induced proangiogenic signaling cascades by targeting IGF-IR and STAT3 in pancreatic cancer, leading to significant growth-inhibitory effects. Therefore, we suggest that Hsp90 inhibitors could prove to be valuable in the treatment of pancreatic cancer.

Oncogenic signaling through activation of epidermal growth factor receptor (EGFR), HER-2, and hypoxia inducible-factor-1α (HIF-1α) has been implicated in gastric cancer growth and angiogenesis through up-regulation of vascular endothelial growth factor (VEGF). Recently, heat shock protein 90 (Hsp90) has been identified as a critical regulator of oncogenic protein stability, including EGFR, HER-2, and HIF-1α. We hypothesized that inhibition of Hsp90 impairs EGF- and hypoxia-mediated angiogenic signaling in gastric cancer cells and consequently inhibits angiogenesis and tumor growth. In vitro, the geldanamycin derivate 17-allylamino-17-demethoxygeldanamycin (17-AAG) led to marked reduction in constitutive and inducible activation of extracellular signal-regulated kinase 1/2, Akt, and signal transducer and activator of transcription 3 and decreased nuclear HIF-1α protein. In addition, EGFR and HER-2 were down-regulated after Hsp90 inhibition. With respect to regulation of angiogenic molecules, 17-AAG significantly reduced EGF-mediated VEGF secretion. Phosphorylation of focal adhesion kinase and paxillin were both abrogated by 17-AAG, which resulted in significant impairment of cancer cell motility. Interestingly, cytotoxic effects of 17-AAG in vitro were higher on cancer cells and gastric fibroblasts than on pericytes. In vivo, the water-soluble compound 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; 25 mg/kg, thrice per week) significantly reduced s.c. xenografted tumor growth. By immunohistochemistry, 17-DMAG significantly reduced vessel area and numbers of proliferating tumor cells in sections. Furthermore, similar significant growth-inhibitory effects of 17-DMAG were achieved when administered as low-dose therapy (5 mg/kg, thrice per week). In conclusion, blocking Hsp90 disrupts multiple proangiogenic signaling pathways in gastric cancer cells and inhibits xenografted tumor growth in vivo. Hence, gastric cancer harbors attractive molecular targets for therapy with Hsp90 inhibitors, which could lead to improved efficacy of antineoplastic therapy regimens. [Mol Cancer Ther 2007;6(3):1123–32]

Comprehensive expression analysis using microarrays has identified a number of differentially expressed genes in smoke-exposed bronchial epithelium and non-small cell lung cancers (NSCLCs). To evaluate the prognostic relevance of these proteins in NSCLCs, we used immunohistochemistry to investigate the expression of beta-catenin (CTNNB1), dickkopf, Xenopus, homolog of 3 (DKK3 gene), fibroblast growth factor receptor 3 (FGFR3), fragile histidine triad (FHIT), tumor protein p53 (TP53), mucin1 (MUC1), topoisomerase II alpha (TOP2A), and glutathione S-transferase-Pi (GST) in a cohort of patients (n = 125). We correlated the expression data with clinicopathologic features and clinical outcome. In addition, SNaPshot multiplex assays (Applied Biosystems, Darmstadt, Germany) and restriction fragment length polymorphism analysis were used to screen for activating point mutations at the hot spots of FGFR3 in a cohort of 30 samples of NSCLC. Using Kaplan-Meier analysis, we observed significantly better overall survival in adenocarcinomas compared with squamous cell cancers (P = .049). Loss of FHIT expression showed a strong association with shorter overall survival in both histologic types of NSCLC (squamous cell cancers, P < .001; adenocarcinomas, P = .001). In adenocarcinomas, the cytoplasmic expression of beta-catenin was associated with shorter survival (P = .012); MUC1 expression was associated with worse prognosis in patients with squamous cell cancers (P = .002). The nuclear staining of TP53 (P = .008) and TOP2A (P = .059) was associated with cancers without lymphonodal metastases. A correlation with positive staining of TOP2A (P = .03) and FGFR3 positivity (P = .057) was found in adenocarcinomas of male patients. Positive MUC1 stainings were associated with squamous cell cancers of male patients (P = .03). DKK3 expression did not show any significant association with clinical outcome or pathologic features. The screening of the FGFR3 sequence in lung cancers showed only wild-type sequences and did not detect mutations in the known hot spots for FGFR3 mutations. We conclude that the immunohistochemical loss of FHIT expression and the positivity for beta-catenin and MUC1 in NSCLC are useful prognostic markers, whereas the variable expression of TP53, TOP2A, and FGFR3 in relation to the different histologic types of NSCLC and sex of the patients is suggestive for different underlying molecular pathways.

Purpose: Several angiogenic factors have been reported to influence prognosis of breast cancer and spread to regional lymph nodes. CD105 (Endoglin) is expressed by human vascular endothelial cells and plays a major role in angiogenesis. Similarly, CD31 is a platelet endothelial cell adhesion molecule involved in normal and pathologic vessel function. In the prospective randomized WSG-AM-01 trial HRBC patients with more than 9 positive lymph nodes derived particular benefit from tandem high-dose (HD) rather than dose-dense (DD) chemotherapy with regard to both event-free (EFS) and overall survival (OS), particularly among tumors carrying a basal-like or HER2 molecular phenotype. The goal of the present study was to examine the interaction between angiogenic factors and efficacy of different dose intensification strategies in the WSG-AM-01 trial among both unselected patients as well as among distinct molecular disease subtypes.Methods: Immunohistochemical staining for CD31 (n=128) and CD105 (n=130) was performed to assess vascular surface area (VSA) separately for both factors using the Chalkley count method.Expression of estrogen (ER) and progesterone receptor (PR), HER2, Ki67, and epidermal growth factor receptor (EGFR) was analyzed immunohistochemically using tissue microarrays, allowing for stratification into molecular breast cancer subclasses (k-clustering k=5 by expression of 24 proteins). Correlation analysis between CD31 and CD105 expression and molecular subtypes was carried out using Pearson correlations. Univariate survival estimates were determined by Kaplan-Meier analysis and tested for significance by log rank statistics. Multivariate survival modeling was performed by a generalized Cox model, with linear proportional hazards terms in the first block and time-varying interactions in the second block.Results: Both VSA/CD 31 and VSA/CD105 showed significantly higher expression among HER-2 and basal-like breast cancer subtypes (p=0.007), as well as among cases wit increased Ki67 (p=0.005).Increased VSA/CD105 (above a median of 1.295) was associated with significantly decreases in both EFS (p=0.01) and OS (p=0.02). In multivariate analysis (including therapy, tumor size, age and grade as well as expression of ER, PR, HER-2 and Ki-67) increased VSA/CD 105 was an independent prognostic factor for decreased EFS (HR=1.68, p=0.05). Dose intensification showed significantly increased efficacy (for OS and EFS) only among patients with low VSA/CD105 tumors.Discussion: Expression of angiogenic markers may mirror/confer resistance to chemotherapy among patients with breast cancer, particularly in the context of dose intensified chemotherapy and may explain differences observed among distinct molecular breast cancer phenotypes. The correlation observed between angiogenic markers and distinct molecular subtypes may serve as a rationale for exploring antiangiogenic treatment options in patients carrying these tumors. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2154.