Numerous Tumor Entities Research Articles

The Pre-Treatment Platelet-to-Lymphocyte Ratio as a Prognostic Factor for Loco-Regional Control in Locally Advanced Rectal Cancer

Chronic inflammatory reactions have been proven to represent relevant mechanisms for the development and progression of cancer in numerous tumor entities. There is evidence that the platelet-to-lymphocyte ratio (PLR) is associated with the prognostic outcome. In rectal cancer, the prognostic role of this parameter has not yet been conclusively clarified. The aim of this study was to further clarify the prognostic significance of the pre-treatment PLR in patients with locally advanced rectal cancer (LARC). In the present study, 603 patients with LARC, who were treated with neoadjuvant chemoradiotherapy (nCRT) and subsequent surgical resection between 2004 and 2019, were retrospectively evaluated. The influence of clinico-pathological and laboratory factors on locoregional control (LC), metastasis-free survival (MFS) and overall survival (OS) was investigated. In univariate analyses, high PLR was significantly associated with worse LC (p = 0.017) and OS (p = 0.008). In multivariate analyses, the PLR remained an independent parameter for the LC (HR = 1.005, 95% CI: 1.000-1.009, p = 0.050). Pre-treatment lactate dehydrogenase (LDH) (HR: 1.005 95% CI:1.002-1.008; p = 0.001) and carcinoembryonic antigen (CEA) (HR: 1.006, 95% CI:1.003-1.009; p < 0.001) were independent predictors for MFS; additionally, age (HR: 1.052, 95% CI:1.023-1.081; p < 0.001), LDH (HR: 1.003, 95% CI:1.000-1.007; p = 0.029) and CEA (HR: 1.006, 95% CI:1.003-1.009; p < 0.001) independently predicted OS. Pre-treatment PLR before nCRT is an independent prognostic factor for LC in LARC, which could be used to further individualize tumor treatment.

Low-Dose Oxidant Toxicity and Oxidative Stress in Human Papillary Thyroid Carcinoma Cells K1

Medical gas plasmas are of emerging interest in pre-clinical oncological research. Similar to an array of first-line chemotherapeutics and physics-based therapies already approved for clinical application, plasmas target the tumor redox state by generating a variety of highly reactive species eligible for local tumor treatments. Considering internal tumors with limited accessibility, medical gas plasmas help to enrich liquids with stable, low-dose oxidants ideal for intratumoral injection and lavage. Pre-clinical investigation of such liquids in numerous tumor entities and models in vitro and in vivo provided evidence of their clinical relevance, broadening the range of patients that could benefit from medical gas plasma therapy in the future. Likewise, the application of such liquids might be promising for recurrent BRAF(V600E) papillary thyroid carcinomas, resistant to adjuvant administration of radioiodine. From a redox biology point of view, studying redox-based approaches in thyroid carcinomas is particularly interesting, as they evolve in a highly oxidative environment requiring the capability to cope with large amounts of ROS/RNS. Knowledge on their behavior under different redox conditions is scarce. The present study aimed to clarify resistance, proliferative activity, and the oxidative stress response of human papillary thyroid cancer cells K1 after exposure to plasma-oxidized DMEM (oxDMEM). Cellular responses were also evaluated when treated with different dosages of hydrogen peroxide and the RNS donor sodium nitroprusside (SNP). Our findings outline plasma-oxidized liquids as a promising approach targeting BRAF(V600E) papillary thyroid carcinomas and extend current knowledge on the susceptibility of cells to undergo ROS/RNS-induced cell death.

A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis.

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n=375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

Hyperforin and Myrtucommulone Derivatives Act as Natural Modulators of Wnt/β-Catenin Signaling in HCT116 Colon Cancer Cells

The therapeutic activities of natural plant extracts have been well known for centuries. Many of them, in addition to antiviral and antibiotic effects, turned out to have anti-tumor activities by targeting different signaling pathways. The canonical Wnt pathway represents a major tumorigenic pathway deregulated in numerous tumor entities, including colon cancer. Here, we investigated the acylphloroglucinols hyperforin (HF) from St. John’s wort (Hypericum perforatum L.) and myrtucommulone A (MC A) from myrtle (Myrtus communis) and semi-synthetic derivatives thereof (HM 177, HM 297, HM298) for their effects on Wnt/β-catenin signaling. None of these substances revealed major cytotoxicity on STF293 embryonic kidney and HCT116 colon carcinoma cells at concentrations up to 10 μM. At this concentration, HF and HM 177 showed the strongest effect on cell proliferation, whereas MC A and HM 177 most prominently inhibited anchorage-independent growth of HCT116 cells. Western blot analyses of active β-catenin and β-catenin/TCF reporter gene assays in STF293 cells revealed inhibitory activities of HF, MC A and HM 177. In line with this, the expression of endogenous Wnt target genes, Axin and Sp5, in HCT116 cells was significantly reduced. Our data suggest that the acylphloroglucinols hyperforin, myrtucommulone A and its derivative HM 177 represent potential new therapeutic agents to inhibit Wnt/β-catenin signaling in colon cancer.

Routine Molecular Pathology Diagnostics in Precision Oncology.

Technical advances in the field of molecular genetics permit precise genomic characterization of malignant tumors. This has not only improved our understanding of tumor biology but also paved the way for molecularly stratified treatment strategies in routine clinical practice. A selective search of PubMed to identify literature on molecular pathology methods, their indications, the challenges associated with molecular findings, and future developments. Tumors can be characterized with the aid of immunohistochemistry, in-situ hybridization, and sequencing of DNA or RNA. The benefits of molecularly stratified tumor treatment have been demonstrated by randomized clinical trials on numerous tumor entities, e.g., non-small-cell lung cancer, colorectal cancer, and breast cancer. Therefore, initiation of specific treatment for these entities should be preceded by molecular pathology biomarker analyses, generally carried out on tumor tissue. Randomized controlled trials and non-controlled studies show that enhanced progression-free survival ensues if the pharmacological treatment is oriented on the findings of molecular pathology diagnostics. In next-generation sequencing, numerous relevant gene sequences or even whole genes can be sequenced in parallel, dispensing with complex staged diagnostics and reducing the use of biomaterials. These new methods also complement the currently relevant predictive biomarkers by permitting the investigation of genetic alterations presently of interest in the context of clinical studies. Prior to widespread routine clinical application, however, sequencing of large gene panels or whole genomes or exomes need to be even more stringently validated. Quality-assured molecular pathology assays are universally available for the determination of currently relevant predictive biomarkers. However, the integration of extensive genomic analyses into routine molecular pathology diagnostics represents a future challenge in precision oncology.

Adoptive T Cell Therapy Is Complemented by Oncolytic Virotherapy with Fusogenic VSV-NDV in Combination Treatment of Murine Melanoma.

Simple SummaryCancer immunotherapy involves the application of strategies aimed at enhancing the body’s immune system to recognize and clear tumor cells. One such immunotherapeutic approach utilizes cytotoxic immune cells (T cells) harvested from the patient, which are expanded and activated, followed by re-infusion to the same patient. This process is termed adoptive cell therapy (ACT). Although effective in a limited setting, efforts are being made to improve these therapies through the development of rationally designed combination treatments. We have developed an approach, whereby tumors are pretreated with a virus, which has oncolytic effects on the tumor cells, in addition to modulating changes in the tumor microenvironment, thereby improving the recruitment of the adoptively transferred cytotoxic T cells and resulting in synergistic therapeutic responses in the tumor. This results in a substantial prolongation of survival, as demonstrated in an immune-competent mouse model of melanoma.Cancer immunotherapies have made major advancements in recent years and are becoming the prevalent treatment options for numerous tumor entities. However, substantial response rates have only been observed in specific subsets of patients since pre-existing factors determine the susceptibility of a tumor to these therapies. The development of approaches that can actively induce an anti-tumor immune response, such as adoptive cell transfer and oncolytic virotherapy, have shown clinical success in the treatment of leukemia and melanoma, respectively. Based on the immune-stimulatory capacity of oncolytic VSV-NDV virotherapy, we envisioned a combination approach to synergize with adoptive T cell transfer, in order to enhance tumor cell killing. Using the immune-competent B16 melanoma model, we demonstrate that combination treatment has beneficial effects on the suppressive microenvironment through upregulation of MHC-I and maintaining low expression levels of PD-L1 on tumor cells. The approach led to additive cytotoxic effects and improved the recruitment of T cells to virus-infected tumor cells in vitro and in vivo. We observed substantial delays in tumor growth and evidence of abscopal effects, as well as prolongation of overall survival time when administered at clinically relevant dosing conditions. Our results indicate that treatment with oncolytic VSV-NDV, combined with adoptive T cell therapy, induces multi-mechanistic and synergistic tumor responses, which supports the further development of this promising translational approach.

Loss of p16 and high Ki67 labeling index is associated with poor outcome in esophageal carcinoma.

The p16 tumor suppressor is coded by CDKN2A (9p21) and plays an important role during carcinogenesis and tumor progression in numerous tumor entities. The aim of our study was to evaluate the prognostic role of p16 expression and CDKN2A deletion in esophageal cancer (EC). Therefore, we analyzed p16 and KI67 expression by immunohistochemistry and 9p21 deletion by fluorescence in-situ hybridization on a tissue microarray including 398 adenocarcinomas (AC) and 293 squamous cell carcinomas (SCC) with clinical follow up-data. p16 positivity was found in 30.2% of AC and 13.9% of SCC and CDKN2A deletion in 32.1% of AC and 33.5% of SCC. In SCC p16 immunostaining correlated with low tumor stage (P = 0.014). In AC Ki67 positivity was associated with high tumor stage (P = 0.001), presence of lymph node metastasis (P = 0.009), high UICC stage (P = 0.001) and poor grading (P = 0.005). Overall survival (OS) was shorter for patients with high Ki67 labeling index (Ki67LI; P = 0.009) and negative p16 immunostaining (P = 0.026). In both histological tumor types, CDKN2A deletion showed no association with phenotype or outcome. Proportional cox-regression modeling revealed patients’ age, tumor stage, lymph node metastasis and Ki67 labeling index as independent prognostic markers in AC. In SCC, only patients’ age and tumor stage proved to be independent prognosticators. In summary, our study shows that loss of p16 expression and high Ki67LI is linked to shortened OS in AC. CDKN2A deletion shows no relevant association with tumor phenotype and patient outcome.

Cancer cell specific inhibition of Wnt/\u03b2-catenin signaling by forced intracellular acidification

Use of the diabetes type II drug Metformin is associated with a moderately lowered risk of cancer incidence in numerous tumor entities. Studying the molecular changes associated with the tumor-suppressive action of Metformin we found that the oncogene SOX4, which is upregulated in solid tumors and associated with poor prognosis, was induced by Wnt/β-catenin signaling and blocked by Metformin. Wnt signaling inhibition by Metformin was surprisingly specific for cancer cells. Unraveling the underlying specificity, we identified Metformin and other Mitochondrial Complex I (MCI) inhibitors as inducers of intracellular acidification in cancer cells. We demonstrated that acidification triggers the unfolded protein response to induce the global transcriptional repressor DDIT3, known to block Wnt signaling. Moreover, our results suggest that intracellular acidification universally inhibits Wnt signaling. Based on these findings, we combined MCI inhibitors with H+ ionophores, to escalate cancer cells into intracellular hyper-acidification and ATP depletion. This treatment lowered intracellular pH both in vitro and in a mouse xenograft tumor model, depleted cellular ATP, blocked Wnt signaling, downregulated SOX4, and strongly decreased stemness and viability of cancer cells. Importantly, the inhibition of Wnt signaling occurred downstream of β-catenin, encouraging applications in treatment of cancers caused by APC and β-catenin mutations.

Abstract 1352: Inactivation of factors of DNA double-strand break repair by homologous recombination or non-homologous end-joining leads to frequent catastrophic genomic events in murine and human tumors

Abstract Chromothripsis and chromoanasynthesis are two forms of genomic instability leading to complex genomic rearrangements that affect one or very few chromosomes. These one-off catastrophic events play a role in numerous tumor entities as well as in some congenital diseases. The availability of murine models recapitulating both phenomena would substantially facilitate the investigation of the mechanistic aspects underlying catastrophic genomic events. Homologous recombination repair (HR) and canonical Non-Homologous-End-Joining (cNHEJ) represent the two major processes for DNA double-strand break repair in mammalian cells. Conditional inactivation of key factors of either of these two pathways, such as Brca2 for HR and Xrcc4 or Lig4 for cNHEJ in nestin-expressing or Emx1-expressing murine neural progenitor cells leads to medulloblastomas and gliomas in a p53-deficient background. We showed by whole-genome sequencing that these tumors frequently display chromothripsis or chromoanasynthesis (33 to 73% of the analyzed tumors, n= 27) and that catastrophic rearrangements drive tumor development. FISH analysis identified a link between chromoanasynthesis and increased numerical and structural aberrations and with the presence of marker chromosomes. In addition, amplifications of c-Myc and n-Myc likely facilitate catastrophic events. Detailed analysis of the microhomologies at the breakpoint junctions on the chromosomes affected by complex genomic rearrangements identified cNHEJ and alternative end-joining as likely repair processes involved in chromothripsis and chromoanasynthesis. Treatment of cells derived from the mouse tumors with inhibitors of HR and/or alternative end-joining (e.g. RAD51 and PARP inhibitors, respectively) in combination with DNA damage revealed the dependence of these tumor cells on specific repair processes and showed that these DNA repair deficiencies can be utilized for synthetic lethality approaches. Comparison of the mouse tumors with whole-genome sequencing data from human medulloblastomas (n=68) and gliomas (n=32) identified an association between chromothripsis and deficiencies in repair processes, by analyzing copy-number level aberrations affecting repair factors and mutational signatures of DNA double-strand break repair defects. This link between DNA repair deficiency and chromothripsis was further confirmed in additional tumor entities such as breast cancer (n=356) and melanoma (n=69). In analogy to the clinical use of PARP inhibitors in the context of BRCA-deficient breast cancer, our findings point towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements. Citation Format: Manasi Ratnaparkhe, John Wong, Pei-Chi Wei, Mario Hlevnjak, Paul Northcott, David T. Jones, Marcel Kool, Anna Jauch, Agata Pastorczak, Andrey Korshunov, Rajiv Kumar, Susanna M. Downing, Stefan M. Pfister, Marc Zapatka, Peter J. McKinnon, Frederick W. Alt, Peter Lichter, Aurelie Ernst. Inactivation of factors of DNA double-strand break repair by homologous recombination or non-homologous end-joining leads to frequent catastrophic genomic events in murine and human tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1352.

PET/MRI

Magnet resonance imaging (MRI) is an excellent anatomical reference method for the combination with positron emission tomography (PET). But MRI does not produce data, which can be directly used for attenuation correction of PET data, potentially compromising quantitative accuracy of PET. Hybrid-positron emission tomography/computed tomography (PET/CT) is an established standard diagnostic tool, particularly for staging and restaging in oncology. Attenuation correction of PET data is performed with a µMAP derived from low-dose-CT, considered as a robust method. Using standardized MRI-sequences, tissue classes are segmented and attenuation maps are obtained, based on empirical density values. In addition, new reconstruction algorithms and the possibility to acquire PET and MRI simultaneously with MRI-based motion correction are available. These advances have improved image quality and quantitative accuracy of the PET-data in PET/MRI. In numerous oncological studies PET/CT and PET/MR were rated as equal in their diagnostic performance. The combination of functional-metabolic PET and multiparametric MRI with excellent soft tissue contrast complement each other with regard to their diagnostic information in numerous tumor entities. The standard diagnostic workup for lung cancer is currently still based on PET/CT. In numerous tumor entities, the combination of PET/MRI can provide additional diagnostic information.

Proteasome inhibitor bortezomib enhances the effect of standard chemotherapy in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive cancer showing a very poor prognosis because of metastasis formation at an early stage and acquisition of chemoresistance. One key driver of chemoresistance is the transcription factor Forkhead box protein M1 (FOXM1) that regulates cell cycle proliferation, maintenance of genomic stability, DNA damage response, and cell differentiation in numerous tumor entities. In this study we investigated the role of FOXM1 in SCLC progression and analyzed the effect of FOXM1 inhibition using two proteasome inhibitors, bortezomib and siomycin A. FOXM1 was strongly expressed in patient-derived SCLC samples (n=123) and its nuclear localization was associated with the proliferation marker Ki-67. Both proteasome inhibitors successfully inhibited FOXM1 expression leading to a significantly reduced proliferation and a decreased mitotic rate along with cell cycle arrest and apoptosis induction. These effects were further enhanced by addition of bortezomib to standard chemotherapy. Treatment of mice bearing chemoresistant SCLC xenografts with bortezomib reduced the mean bioluminescence signal of tumors by 54%. Similarly, treatment with cisplatin as a standard chemotherapy reduced the mean bioluminescence signal of tumors by 58%. However, in combination with standard chemotherapy bortezomib further reduced the mean bioluminescence signal by 93% (p=0.0258). In conclusion, we demonstrate the effect of bortezomib in inhibiting FOXM1 expression and thus in sensitizing resistant SCLC cells to standard chemotherapy. Thus, addition of bortezomib to standard chemotherapy might potently improve SCLC therapy, particularly in an extensive cancer stage.

Virucide properties of cold atmospheric plasma for future clinical applications.

Cold atmospheric plasma (CAP) has been repeatedly identified to bear powerful microbicidal efficacy on bacteria including multidrug resistant organisms and fungi on non-living surfaces, in biofilms as well as on contaminated and infected tissues. CAP furthermore was found to stimulate wound healing in chronic wounds and exerted anti-neoplastic effects on numerous tumor entities. Thus, CAP represents a promising medical tool for many clinical and therapeutic issues. Studies about CAP effects on virus particles recently were in arrears, but to date increasingly move into the focus of interest. Apparently, CAP treatment is followed by a promising virus inactivation and contributes to tissue regeneration. Here we review the current state of science concerning the so far investigated CAP effects on different virus species and virus-associated disorders. J. Med. Virol. 89:952-959, 2017. © 2017 Wiley Periodicals, Inc.

Oncolytic measles virus encoding interleukin-12 mediates potent antitumor effects through T cell activation

ABSTRACTCombination of oncolytic virotherapy with immunomodulators is emerging as a promising therapeutic strategy for numerous tumor entities. In this study, we developed measles Schwarz vaccine strain vectors encoding immunomodulators to support different phases in the establishment of antitumor immune responses. Therapeutic efficacy of the novel vectors was evaluated in the immunocompetent MC38cea tumor model. We identified vectors encoding an IL-12 fusion protein (MeVac FmIL-12) and an antibody against PD-L1 (MeVac anti-PD-L1), respectively, as the most effective. Treatment of established tumors with MeVac FmIL-12 achieved 90% complete remissions. Profiling of the tumor immune microenvironment revealed activation of a type 1 T helper cell-directed response, with MeVac FmIL-12 ensuring potent early natural killer and effector T cell activation as well as upregulation of the effector cytokines IFN-γ and TNF-α. CD8+ T cells were found to be essential for the therapeutic efficacy of MeVac FmIL-12. Results of this study present MeVac FmIL-12 as a novel approach for targeted IL-12 delivery and elucidate mechanisms of successful immunovirotherapy.

Recurrent somatic mutations in Ying Yang 1 (YY1) are found in a subgroup of sporadic insulinomas

Insulinomas represent the most common form of hormonally active neuroendocrine tumors of the pancreas. In the majority of cases their functional activity causes relevant morbidity with severe hypoglycemic episodes. Expression experiments led to the notion, that insulinomas have distinctively different features from other neuroendocrine tumors. Disease initiating mechanisms or genetic causes, however, are not well understood. Next generation sequencing techniques have allowed massive parallel identification of DNA alterations in numerous tumor entities in recent years. We applied this method to an initial cohort of 8 patients (5 females and 3 males) with sporadic insulinomas. Overall, 41 relevant somatic genetic alterations were detected, and a recurrent T372R mutation in YY1 (Ying Yang 1) was found in 2 different tumors. YY1 has been shown to be involved in cell regulatory processes. To confirm the relevance of this mutation, a total number of 47 insulinomas from three German centers (Düsseldorf, Marburg and München) were screened for this hotspot mutation, and were found positive in 13% (6/47 samples) of cases, which was much lower than described in a previous study. Interestingly, all affected patients were female (6/6, P = 0.04), and tended to be older compared to YY1 non-mutated cases (61.8 ± 13.0 vs. 47.6 ± 17.4 years of age; p = 0.06). However, other clinical parameters, including malignant or metastatic disease and severity of hyperinsulinism were not different between the two groups. In summary, we thus describe presence and prevalence of recurrent YY1 mutations in sporadic insulinomas for the first time in the Caucasian population.

External validation of a nomogram predicting overall survival of patients diagnosed with endometrial cancer

ObjectivesNomograms are predictive models that provide the overall probability of a specific outcome. Nomograms have shown better individual discrimination than currently used staging systems in numerous tumor entities. Recently, a nomogram for predicting overall survival (OS) in women with endometrial cancer was introduced by Memorial Sloan–Kettering Cancer Center (MSKCC). The aim of this study was to test the validity of the MSKCC endometrial cancer nomogram using an independent, external patient cohort. MethodsThe MSKCC nomogram is based on five readily available clinical characteristics. A multi-institutional endometrial cancer database was used to test the nomogram's validity. All consecutive patients treated for endometrial cancer between December 1995 and May 2011 and who had all nomogram variables documented were identified for analysis. ResultsSeven hundred sixty-five eligible patients were identified and used for external validation analysis. In the Austrian patient cohort, median OS was 134months, and 3-year and 5-year OS rates were 83.8% (95% CI, 80.6–86.5%) and 77.2% (95% CI, 43.5–80.5%), respectively. The nomogram concordance index was 0.71 (SE=0.017; 95% CI, 0.68–0.74). The correspondence between the actual OS and the nomogram predictions suggests a good calibration of the nomogram in the validation cohort. ConclusionThe MSKCC endometrial cancer nomogram was externally validated and was shown to be generalizable to a new and independent patient population. The nomogram provides a more individualized and accurate estimation of OS for patients diagnosed with endometrial cancer following primary therapy. The nomogram can be used for counseling patients more accurately and for better stratifying patients for clinical trials.

Abstract A21: MALAT-1 is essential for lung cancer metastasis in a novel human knockout model

Abstract The highly conserved long non-coding RNA MALAT-1 (Metastasis-Associated in Lung Adenocarcinoma Transcript 1) had been discovered as a prognostic marker associated with poor survival and development of distant metastasis in lung adenocarcinoma. Since then, it has been found to be deregulated in numerous tumor entities and has been linked to splicing. However, its functional relevance in tumor cells remains to be elucidated. Knockdown models for MALAT1 have been described but suffer from insufficient silencing efficiency of the highly abundant, nuclear non-coding RNA (ncRNA). In this project, we have developed a novel strategy to create ncRNA knockouts in human cancer cell lines. We have successfully used a synthetic Zinc Finger Nuclease engineered to target the 5′-region of MALAT1 to stably and biallelically integrate RNA-destabilizing elements into the genome of human lung cancer cells (A549). This approach resulted in a specific and more than 1000-fold silencing of MALAT1 in individual clones compared to a less than 5-fold silencing using siRNAs. Thus, this approach can be used to create functional knockouts of coding as well as non-coding genes also in human tumor cell lines allowing loss-of-function studies also of non-conserved ncRNAs in the future. Phenotypically, the MALAT1-Knockout cells (KO) greatly differ from their parental cell line and wildtype clones (WT): Next to morphological changes, the migration of the KO cells is largely impaired as shown in scratch assays. In xenograft assays after i.v. injection, the KO cells form significantly fewer and smaller lung metastases than their WT counterparts. Since no large difference was observed after subcutaneous injection of the WT and the KO cells, this indicates a specific, active and essential function of MALAT1 in metastasis. Exon microarrays of the WT and KO cell lines have revealed multiple migration- or metastasis-associated transcripts deregulated by loss of MALAT1 and hence potential target genes. These analyses also uncovered, that splicing is likely not the only functional mechanism of MALAT1 in the nucleus. Taken together, we have developed a novel, highly effective approach for the knockout of genes that can be used for non-coding as well as coding RNAs in human tumor cells as well as cells from other species. Knockout of MALAT1 in human lung cancer cells revealed its essential function in metastasis as well as genes directly and indirectly targeted by MALAT1. Citation Format: Tony Gutschner, Moritz Eißmann, Monika Hämmerle, Marion Stentrup, Catherina Hildenbrandt, Matthias Groß, Martin Zörnig, Sven Diederichs. MALAT-1 is essential for lung cancer metastasis in a novel human knockout model [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A21.

Angiogenese und Tumorhypoxie beim Mammakarzinom: Fakten, Fragen und therapeutische Möglichkeiten

Schon vor über 35 Jahren postulierte Judah Folkman als Pionier der Angiogeneseforschung, dass das Wachstum und die Metastasierung von Tumoren entscheidend von ihrer Blutgefäßversorgung abhängt. Seitdem ist der Wachstumsfaktor VEGF (Vascular Endothelial Growth Factor), der über seine Rezeptoren VEGFR-1 (Flt-1) und VEGFR-2 (KDR) wirkt, als Schlüsselmolekül der Angiogenese identifiziert worden. VEGF bewirkt das Aussprossen neuer Blutgefäße aus vorbestehenden Gefäßen in der Tumorumgebung. Diese Tumorblutgefäße sind abnormal und durch einen chaotischen Verlauf sowie eine unreife Struktur mit einer höheren Permeabilität charakterisiert. Dies bewirkt einen schlechteren Blutfluss, sodass beispielsweise Chemotherapeutika einen Tumor nicht in optimaler Dosierung erreichen können. Darüber hinaus induziert VEGF auch direkt die Tumorzell-Proliferation. VEGF wird von zahlreichen Tumoren und auch beim Mammakarzinom gebildet. Beim Mammakarzinom hat VEGF einen negativen prognostischen und prädiktiven Wert. In den letzten Jahren sind zahlreiche antiangiogen wirkende Substanzen (z. B. Antikörper, Rezeptor-Tyrosinkinase-Inhibitoren, lösliche Rezeptoren) gegen VEGF, seine Rezeptoren oder den VEGF-Signalweg entwickelt worden. Die am weitesten entwickelte Substanz ist Bevacizumab (Avastin®), ein monoklonaler humanisierter Antikörper gegen VEGF, der die Bindung des Liganden an seine Rezeptoren und somit die nachgeschaltete angiogene Signalwirkung verhindert. In Phase-III-Studien konnte die klinische Wirksamkeit von Bevacizumab in Kombination mit einer Chemotherapie (Paclitaxel bzw. Docetaxel) beim unvorbehandelten metastasierten Mammakarzinom (first line) gezeigt werden. In der klinischen Erprobung sind unter anderem auch sogenannte Multikinase-Inhibitoren (z. B. Sunitinib, Sorafenib), die verschiedene proangiogene Rezeptoren gleichzeitig hemmen. Trotz aller Fortschritte hat sich die klinische Umsetzung antiangiogener Therapiestrategien als schwieriger als erwartet herausgestellt. In der Zukunft wird es wichtig sein, diejenigen Patientinnen zu identifizieren, die maximal von einer bestimmten antiangiogenen Therapie profitieren. Neben der Identifikation geeigneter Tumoren und Erkrankungsstadien müssen auch prädiktive Biomarker entwickelt werden, die eine Vorhersage zulassen, welche Substanz bei einer Patientin optimal wirksam ist, sodass eine individualisierte maßgeschneiderte Therapie möglich ist. In den nächsten Jahren werden die Daten zahlreicher Studien zur Antiangiogenese verfügbar, die zum besseren Verständnis dieser Zusammenhänge beitragen könnten. Dabei kann uns insbesondere die translationale Forschung helfen, den klinischen Einsatz der antiangiogenen Therapien zu optimieren.

Humoral Immune Responses Against the Oncofetal-Antigen/Immature Laminin Receptor (OFA/iLR) in Patients with Chronic Lymphocytic Leukemia.

The 37 kD oncofetal antigen/immature laminin receptor (OFA/iLR) is a strongly overexpressed tumor antigen on malignant cells of numerous tumor entities including several hematological malignancies but it is not present on the cell surface of normal differentiated tissues. OFA/iLR is widely expressed on chronic lymphocytic leukemia (CLL) cells while absent on different normal B-cell subsets. The aim of this study was to evaluate and characterize humoral immune responses towards OFA/iLR in patients with CLL. 38 healthy individuals and 68 untreated patients with chronic lymphocytic leukemia were analysed for the presence of anti-OFA/iLR-antibodies. Sera were screened in an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM anti-OFA/iLR-antibodies. Detectable IgG or IgM responses to OFA protein were present in 32 of 68 patients (47,0%) but only in 4 of 38 (10,5%) healthy donors. IgG subtype analysis revealed a predominant IgG1 and IgG3 response. Utilizing 42 peptides deduced from the complete OFA/iLR amino acid sequence, 24 (35,3%) samples with significant anti-OFA/iLR IgG and/or IgM titers were also reactive with at least one OFA/iLR epitope. Patient-derived anti-OFA/iLR antibodies were capable of recognizing and selectively killing OFA/iLR expressing CLL cells in complement-mediated and antibody-dependent cellular cytotoxicity (ADCC) assays. Kaplan-Meier plots for progression-free survival (PFS) revealed a tendendy for longer PFS in patients with detectable humoral responses compared patients with negative ELISA (p=0,0165).In addition, sera from eight CLL patients who had undergone allogeneic hematopoetic stem cell transplantion (allo-HSCT) were analysed. Six out of eight patients showed high values for IgG or IgM anti-OFA/iLR-antibodies after allo-HSCT as long as remaining in complete remission suggesting a potential role of anti-OFA/iLR-directed Graft-versus-Leukemia effects. For the first time, these data suggest that anti-OFA/iLR antibodies might be involved in the immunological control of OFA/iLR expressing CLL cells in vivo.