Targeted therapies in retinoblastoma: GD2-directed immunotherapy following autologous stem cell transplantation and evaluation of alternative target B7-H3.

BACKGROUND: GD2-directed immunotherapy is highly effective in the treatment of high-risk neuroblastoma (NB), and might be an interesting target also in other high-risk tumors. METHODS: The German-Austrian Retinoblastoma Registry, Essen, was searched for patients, who were treated with anti-GD2 monoclonal antibody (mAb) dinutuximab beta (Db) in order to evaluate toxicity, response and outcome in these patients. Additionally, we evaluated anti-GD2 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in retinoblastoma cell lines in vitro. Furthermore, in vitro cytotoxicity assays directed against B7-H3 (CD276), a new identified potential target in RB, were performed. RESULTS: We identified four patients with relapsed stage IV retinoblastoma, who were treated with Db following autologous stem cell transplantation (ASCT). Two out of two evaluable patients with detectable tumors responded to immunotherapy. One of these and another patient who received immunotherapy without residual disease relapsed 10 and 12 months after start of Db. The other patients remained in remission until last follow-up 26 and 45 months, respectively. In vitro, significant lysis of RB cell lines by ADCC and CDC with samples from patients and healthy donors and anti-GD2 and anti-CD276-mAbs were demonstrated. CONCLUSION: Anti-GD2-directed immunotherapy represents an additional therapeutic option in high-risk metastasized RB. Moreover, CD276 is another target of interest.

First-line Anti-GD2 Therapy Combined With Consolidation Chemotherapy in 3 Patients With Newly Diagnosed Metastatic Ewing Sarcoma or Ewing-like Sarcoma.

Despite multimodal therapy, the prognosis of patients with metastatic Ewing sarcoma (ES) remains poor, with new treatments urgently needed. The disialoganglioside GD2, a well-established tumor-associated antigen, is expressed in 40% to 90% of ES cells, making it a suitable therapeutic target. Here we report 3 cases with newly diagnosed, metastatic, GD2-positive ES or Ewing-like sarcoma treated with the anti-GD2 antibody dinutuximab beta in addition to standard chemotherapeutic regimens. Treatment was well-tolerated, and all patients achieved complete remission, without evidence of relapse. First-line anti-GD2 immunotherapy in patients with metastatic, GD2-positive ES or Ewing-like sarcoma represents a promising therapeutic option that warrants further clinical evaluation.

Cholesterol biosynthesis supports the growth of hepatocarcinoma lesions depleted of fatty acid synthase in mice and humans.

OBJECTIVE: Increased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC). DESIGN: We investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies. RESULTS: Ablation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture. CONCLUSION: Our study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

TEA Domain Transcription Factor 4 Is the Major Mediator of Yes-Associated Protein Oncogenic Activity in Mouse and Human Hepatoblastoma.

Hepatoblastoma (HB) is the most common type of pediatric liver cancer. Activation of yes-associated protein (YAP) has been implicated in HB molecular pathogenesis. The transcriptional co-activator Yap regulates downstream gene expression through interaction with the TEA domain (TEAD) proteins. Nonetheless, YAP also displays functions that are independent of its transcriptional activity. The underlying molecular mechanisms by which Yap promotes HB development remain elusive. In the current study, we demonstrated that blocking TEAD function via the dominant-negative form of TEAD2 abolishes Yap-driven HB formation in mice and restrains human HB growth in vitro. When TEAD2 DNA-binding domain was fused with virus protein 16 transcriptional activation domain, it synergized with activated ß-catenin to promote HB formation in vivo. Among TEAD genes, silencing of TEAD4 consistently inhibited tumor growth and Yap target gene expression in HB cell lines. Furthermore, TEAD4 mRNA expression was significantly higher in human HB lesions when compared with corresponding nontumorous liver tissues. Human HB specimens also exhibited strong nuclear immunoreactivity for TEAD4. Altogether, data demonstrate that TEAD-mediated transcriptional activity is both sufficient and necessary for Yap-driven HB development. TEAD4 is the major TEAD isoform and Yap partner in human HB. Targeting TEAD4 may represent an effective treatment option for human HB.

The mTORC2-Akt1 Cascade Is Crucial for c-Myc to Promote Hepatocarcinogenesis in Mice and Humans.

Hepatocellular carcinoma (HCC) is a deadly form of liver cancer with limited treatment options. The c-Myc transcription factor is a pivotal player in hepatocarcinogenesis, but the mechanisms underlying c-Myc oncogenic activity in the liver remain poorly delineated. Mammalian target of rapamycin complex 2 (mTORC2) has been implicated in cancer by regulating multiple AGC kinases, especially AKT proteins. In the liver, AKT1 and AKT2 are widely expressed. While AKT2 is the major isoform downstream of activated phosphoinositide 3-kinase and loss of phosphatase and tensin homolog-induced HCC, the precise function of AKT1 in hepatocarcinogenesis is largely unknown. In the present study, we demonstrate that mTORC2 is activated in c-Myc-driven mouse HCC, leading to phosphorylation/activation of Akt1 but not Akt2. Ablation of Rictor inhibited c-Myc-induced HCC formation in vivo. Mechanistically, we discovered that loss of Akt1, but not Akt2, completely prevented c-Myc HCC formation in mice. Silencing of Rictor or Akt1 in c-Myc HCC cell lines inhibited phosphorylated forkhead box o1 expression and strongly suppressed cell growth in vitro. In human HCC samples, c-MYC activation is strongly correlated with phosphorylated AKT1 expression. Higher expression of RICTOR and AKT1, but not AKT2, is associated with poor survival of patients with HCC. In c-Myc mice, while rapamycin, an mTORC1 inhibitor, had limited efficacy at preventing c-Myc-driven HCC progression, the dual mTORC1 and mTORC2 inhibitor MLN0128 effectively promoted tumor regression by inducing apoptosis and necrosis. Conclusion: Our study indicates the functional contribution of mTORC2/Akt1 along c-Myc-induced hepatocarcinogenesis, with AKT1 and AKT2 having distinct roles in HCC development and progression; targeting both mTORC1 and mTORC2 may be required for effective treatment of human HCC displaying c-Myc amplification or overexpression.

Evaluation of the prognostic role of co-morbidities on disease outcome in renal cell carcinoma patients.

BACKGROUND: Co-morbidities may induce local and systemic tumor progression of renal cell carcinoma (RCC); however, the prognostic impact of co-morbidities has not yet been well characterized. PATIENTS AND METHODS: RCC patients (n = 2206) surgically treated at three academic institutions in the US and Europe were included in the analysis. Presence of diabetes mellitus, hypertension, chronic kidney disease, chronic obstructive pulmonary disease, coronary heart disease, and hypothyroidism were investigated for their association with clinicopathological features and cancer-specific survival. RESULTS: Hypertension was associated with less advanced T stages (p = 0.025), a lower risk of lymph-node (p = 0.026) and distant metastases (p = 0.001), and improved cancer specific survival in univariable analysis (HR 0.81 95% CI 0.69-0.96, p = 0.013). However, hypertension was not an independent prognostic factor after adjustment for TNM stages, grading, and ECOG performance status (HR 0.95, 95% CI 0.80-1.12; p = 0.530). A correlation between the use of concomitant anti-hypertensive medications and improved survival outcome was not identified. All other investigated co-morbidities did not show significant associations with clinicopathological features or cancer-specific survival. CONCLUSION: Although the investigated co-morbidities are capable or inducing pathophysiological changes that are predisposing factors for tumor progression, none is an independent prognostic factor in patients with RCC.

Transcriptomic and Proteomic Analysis of Clear Cell Foci (CCF) in the Human Non-Cirrhotic Liver Identifies Several Differentially Expressed Genes and Proteins with Functions in Cancer Cell Biology and Glycogen Metabolism.

Clear cell foci (CCF) of the liver are considered to be pre-neoplastic lesions of hepatocellular adenomas and carcinomas. They are hallmarked by glycogen overload and activation of AKT (v-akt murine thymoma viral oncogene homolog)/mTOR (mammalian target of rapamycin)-signaling. Here, we report the transcriptome and proteome of CCF extracted from human liver biopsies by laser capture microdissection. We found 14 genes and 22 proteins differentially expressed in CCF and the majority of these were expressed at lower levels in CCF. Using immunohistochemistry, the reduced expressions of STBD1 (starch-binding domain-containing protein 1), USP28 (ubiquitin-specific peptidase 28), monad/WDR92 (WD repeat domain 92), CYB5B (Cytochrome b5 type B), and HSPE1 (10 kDa heat shock protein, mitochondrial) were validated in CCF in independent specimens. Knockout of Stbd1, the gene coding for Starch-binding domain-containing protein 1, in mice did not have a significant effect on liver glycogen levels, indicating that additional factors are required for glycogen overload in CCF. Usp28 knockout mice did not show changes in glycogen storage in diethylnitrosamine-induced liver carcinoma, demonstrating that CCF are distinct from this type of cancer model, despite the decreased USP28 expression. Moreover, our data indicates that decreased USP28 expression is a novel factor contributing to the pre-neoplastic character of CCF. In summary, our work identifies several novel and unexpected candidates that are differentially expressed in CCF and that have functions in glycogen metabolism and tumorigenesis.

Loss of Fbxw7 synergizes with activated Akt signaling to promote c-Myc dependent cholangiocarcinogenesis.

BACKGROUND & AIMS: The ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7) is recognized as a tumor suppressor in many cancer types due to its ability to promote the degradation of numerous oncogenic target proteins. Herein, we aimed to elucidate its role in intrahepatic cholangiocarcinoma (iCCA). METHODS: Herein, we first confirmed that FBXW7 gene expression was reduced in human iCCA specimens. To identify the molecular mechanisms by which FBXW7 dysfunction promotes cholangiocarcinogenesis, we generated a mouse model by hydrodynamic tail vein injection of Fbxw7ΔF, a dominant negative form of Fbxw7, either alone or in association with an activated/myristylated form of AKT (myr-AKT). We then confirmed the role of c-MYC in human iCCA cell lines and its relationship to FBXW7 expression in human iCCA specimens. RESULTS: FBXW7 mRNA expression is almost ubiquitously downregulated in human iCCA specimens. While forced overexpression of Fbxw7ΔF alone did not induce any appreciable abnormality in the mouse liver, co-expression with AKT triggered cholangiocarcinogenesis and mice had to be euthanized by 15 weeks post-injection. At the molecular level, a strong induction of Fbxw7 canonical targets, including Yap, Notch2, and c-Myc oncoproteins, was detected. However, only c-MYC was consistently confirmed as a FBXW7 target in human CCA cell lines. Most importantly, selected ablation of c-Myc completely impaired iCCA formation in AKT/Fbxw7ΔF mice, whereas deletion of either Yap or Notch2 only delayed tumorigenesis in the same model. In human iCCA specimens, an inverse correlation between the expression levels of FBXW7 and c-MYC transcriptional activity was observed. CONCLUSIONS: Downregulation of FBXW7 is ubiquitous in human iCCA and cooperates with AKT to induce cholangiocarcinogenesis in mice via c-Myc-dependent mechanisms. Targeting c-MYC might represent an innovative therapy against iCCA exhibiting low FBXW7 expression. LAY SUMMARY: There is mounting evidence that FBXW7 functions as a tumor suppressor in many cancer types, including intrahepatic cholangiocarcinoma, through its ability to promote the degradation of numerous oncoproteins. Herein, we have shown that the low expression of FBXW7 is ubiquitous in human cholangiocarcinoma specimens. This low expression is correlated with increased c-MYC activity, leading to tumorigenesis. Our findings suggest that targeting c-MYC might be an effective treatment for intrahepatic cholangiocarcinoma.

Hippo Cascade Controls Lineage Commitment of Liver Tumors in Mice and Humans.

Primary liver cancer consists mainly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). A subset of human HCCs expresses a ICC-like gene signature and is classified as ICC-like HCC. The Hippo pathway is a critical regulator of normal and malignant liver development. However, the precise function(s) of the Hippo cascade along liver carcinogenesis remain to be fully delineated. The role of the Hippo pathway in a murine mixed HCC/ICC model induced by activated forms of AKT and Ras oncogenes (AKT/Ras) was investigated. The authors demonstrated the inactivation of Hippo in AKT/Ras liver tumors leading to nuclear localization of Yap and TAZ. Coexpression of AKT/Ras with Lats2, which activates Hippo, or the dominant negative form of TEAD2 (dnTEAD2), which blocks Yap/TAZ activity, resulted in delayed hepatocarcinogenesis and elimination of ICC-like lesions in the liver. Mechanistically, Notch2 expression was found to be down-regulated by the Hippo pathway in liver tumors. Overexpression of Lats2 or dnTEAD2 in human HCC cell lines inhibited their growth and led to the decreased expression of ICC-like markers, as well as Notch2 expression. Altogether, this study supports the key role of the Hippo cascade in regulating the differentiation status of liver tumors.

Inhibition of MELK Protooncogene as an Innovative Treatment for Intrahepatic Cholangiocarcinoma.

Background and Objectives: Intrahepatic cholangiocarcinoma (iCCA) is a pernicious tumor characterized by a dismal outcome and scarce therapeutic options. To substantially improve the prognosis of iCCA patients, a better understanding of the molecular mechanisms responsible for development and progression of this disease is imperative. In the present study, we aimed at elucidating the role of the maternal embryonic leucine zipper kinase (MELK) protooncogene in iCCA. Materials and Methods: We analyzed the expression of MELK and two putative targets, Forkhead Box M1 (FOXM1) and Enhancer of Zeste Homolog 2 (EZH2), in a collection of human iCCA by real-time RT-PCR and immunohistochemistry (IHC). The effects on iCCA growth of both the multi-kinase inhibitor OTSSP167 and specific small-interfering RNA (siRNA) against MELK were investigated in iCCA cell lines. Results: Expression of MELK was significantly higher in tumors than in corresponding non-neoplastic liver counterparts, with highest levels of MELK being associated with patients' shorter survival length. In vitro, OTSSP167 suppressed the growth of iCCA cell lines in a dose-dependent manner by reducing proliferation and inducing apoptosis. These effects were amplified when OTSSP167 administration was coupled to the DNA-damaging agent doxorubicin. Similar results, but less remarkable, were obtained when MELK was silenced by specific siRNA in the same cells. At the molecular level, siRNA against MELK triggered downregulation of MELK and its targets. Finally, we found that MELK is a downstream target of the E2F1 transcription factor. Conclusion: Our results indicate that MELK is ubiquitously overexpressed in iCCA, where it may represent a prognostic indicator and a therapeutic target. In particular, the combination of OTSSP167 (or other, more specific MELK inhibitors) with DNA-damaging agents might be a potentially effective therapy for human iCCA.

A functional mammalian target of rapamycin complex 1 signaling is indispensable for c-Myc-driven hepatocarcinogenesis.

Amplification and/or activation of the c-Myc proto-oncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc-dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of mTORC1, strongly inhibits c-Myc liver tumor formation. Also, the p70 ribosomal S6 kinase/ribosomal protein S6 and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed up-regulation of multiple amino acid transporters, including solute carrier family 1 member A5 (SLC1A5) and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1 as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human hepatocellular carcinoma specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6. CONCLUSION: Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis; thus, targeting the mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of hepatocellular carcinoma with activated c-Myc signaling. (Hepatology 2017;66:167-181).

Microvascular and lymphovascular tumour invasion are associated with poor prognosis and metastatic spread in renal cell carcinoma: a validation study in clinical practice.

OBJECTIVE: To validate microvascular (MVI) and lymphovascular (LVI) invasion as prognostic factors in patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: Data of patients with RCC who underwent radical or nephron-sparing surgery were prospectively collected from three academic centres. The occurrence of MVI and LVI was determined with standard staining protocols by experienced pathologists at the time of diagnosis. The association of MVI and LVI with clinicopathological data, metastatic spread, and cancer-specific survival (CSS) were evaluated with Fisher's exact tests, binary logistic regression analyses, and univariable and multivariable Cox proportional hazard regression models. RESULTS: MVI was present in 201 of 747 patients (26.9%) and was associated with advanced Tumour-Node-Metastasis (TNM) stages, high Fuhrman grades, and sarcomatoid features (all P < 0.001). MVI was associated with a higher rate of metastatic spread. LVI was present in 32 of 573 patients (5.5%) and was associated with advanced TNM stages, high Fuhrman grade, and sarcomatoid features (all P < 0.001). Two-thirds of LVI-positive patients died (P < 0.001). Both LVI and MVI were significantly associated with CSS in all patients, clear cell RCC (ccRCC), and localised RCC in univariable analysis (all P < 0.001). On multivariable analysis, presence of MVI was identified as an independent prognostic factor (hazard ratio 2.09; P = 0.001). Moreover, MVI [odds ratio (OR) 2.7; P = 0.001] and not macrovascular invasion (P = 0.895) was an independent predictor of sychronuous metastatic spread. LVI was the strongest factor associated with sychronous metastatic spread (OR 4.73, 95% confidence interval 1.84-12.14; P = 0.001) in all patients and in the subgroup of patients with ccRCC (P = 0.001). CONCLUSIONS: The present study validated LVI and MVI as prognostic factors for poor outcome in RCC. These findings endorse an evaluation of both variables in the clinical routine setting to facilitate survival prognostication in follow-up protocols and for assignment to adjuvant treatment trials.

Gastrointestinal Stromal Tumors: Clinical Symptoms, Location, Metastasis Formation, and Associated Malignancies in a Single Center Retrospective Study.

BACKGROUND AND AIMS: Gastrointestinal stromal tumors (GISTs) are rare malignancies but the most common mesenchymal tumors of the digestive tract. Recent advances in diagnostic imaging and an increasing incidence will confront us more frequently with stromal tumors. This single center study aimed to characterize GIST patients in terms of tumor location, clinical presentation, metastasis formation, as well as associated secondary malignancies. METHODS: In a retrospective study, 104 patients with a histologically confirmed diagnosis of GIST, collected between 1993 and 2011, were characterized for several clinical features. RESULTS: The most common GIST location was the stomach (67.6%) followed by the small intestine (16.2%). Gastrointestinal bleeding (55.8%) and abdominal pain (38.5%) were the most frequently reported symptoms whereas about one-third of patients remained clinically asymptomatic (31.6%); 14.4% of patients had either synchronous or metachronous metastases and there was a significant prevalence also in the low risk group. The proportion of secondary malignant associated neoplasms was 31% in our GIST cohort, among which gastrointestinal, genitourinary tumors, and breast cancer were the most prevalent. CONCLUSION: There was a considerable risk for metastasis formation and the development of secondary neoplasias that should encourage discussion about the appropriate surveillance strategy after surgery for GIST.

NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice.

Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91phox KO mice. Only infected gp91phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis.