Targeting HDACs in Pancreatic Neuroendocrine Tumor Models.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted.

PARP and CDK4/6 Inhibitor Combination Therapy Induces Apoptosis and Suppresses Neuroendocrine Differentiation in Prostate Cancer.

We analyzed the efficacy and mechanistic interactions of PARP inhibition (PARPi; olaparib) and CDK4/6 inhibition (CDK4/6i; palbociclib or abemaciclib) combination therapy in castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) models. We demonstrated that combined olaparib and palbociblib or abemaciclib treatment resulted in synergistic suppression of the p-Rb1-E2F1 signaling axis at the transcriptional and posttranslational levels, leading to disruption of cell-cycle progression and inhibition of E2F1 gene targets, including genes involved in DDR signaling/damage repair, antiapoptotic BCL-2 family members (BCL-2 and MCL-1), CDK1, and neuroendocrine differentiation (NED) markers in vitro and in vivo In addition, olaparib + palbociclib or olaparib + abemaciclib combination treatment resulted in significantly greater growth inhibition and apoptosis than either single agent alone. We further showed that PARPi and CDK4/6i combination treatment-induced CDK1 inhibition suppressed p-S70-BCL-2 and increased caspase cleavage, while CDK1 overexpression effectively prevented the downregulation of p-S70-BCL-2 and largely rescued the combination treatment-induced cytotoxicity. Our study defines a novel combination treatment strategy for CRPC and NEPC and demonstrates that combination PARPi and CDK4/6i synergistically promotes suppression of the p-Rb1-E2F1 axis and E2F1 target genes, including CDK1 and NED proteins, leading to growth inhibition and increased apoptosis in vitro and in vivo Taken together, our results provide a molecular rationale for PARPi and CDK4/6i combination therapy and reveal mechanism-based clinical trial opportunities for men with NEPC.

Targeting glycosphingolipids for cancer immunotherapy.

Aberrant expression of glycosphingolipids (GSLs) is a unique feature of cancer and stromal cells in tumor microenvironments. Although the impact of GSLs on tumor progression remains largely unclear, anticancer immunotherapies directed against GSLs are attracting growing attention. Here, we focus on GD2, a disialoganglioside expressed in tumors of neuroectodermal origin, and Globo H ceramide (GHCer), the most prevalent cancer-associated GSL overexpressed in a variety of epithelial cancers. We first summarize recent advances on our understanding of GD2 and GHCer biology and then discuss the clinical development of the first immunotherapeutic agent targeting a glycolipid, the GD2-specific antibody dinutuximab, its approved indications, and new strategies to improve its efficacy for neuroblastoma. Next, we review ongoing clinical trials on Globo H-targeted immunotherapeutics. We end with highlighting how these studies provide sound scientific rationales for targeting GSLs in cancer and may facilitate a rational design of new GSL-targeted anticancer therapeutics.

Phosphoprotein-based biomarkers as predictors for cancer therapy.

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies.

Malignant Gastrointestinal Neuroectodermal Tumor: Clinicopathologic, Immunohistochemical, and Molecular Analysis of 19 Cases.

A malignant gastrointestinal neuroectodermal tumor (GNET) is rare, and it is therefore yet to be completely understood. This study aimed to present the clinicopathologic features of GNET, including treatment information. We included 19 patients with GNET with a mean tumor size of 4.2 cm. The most common site of tumor origin was the small intestine (57.9%), followed by the stomach (15.8%), colon (10.5%), ileocecal junction (5.3%), lower esophagus (5.3%), and anal canal (5.3%). Microscopically, the tumors were composed of epithelioid cells with eosinophilic or clear cytoplasm arranged in nest, sheet-like, papillary, or pseudoalveolar patterns and/or spindle tumor cells with eosinophilic cytoplasm arranged in a fascicular pattern. Immunohistochemically, the tumor cells stained positively for S100 (19/19,100%), SOX10 (14/15, 93.3%), vimentin (17/17, 100%), synaptophysin (Syn) (7/17, 41.2%), CD56 (4/13, 30.8%), CD99 (1/5, 20%), and CD117 (1/15, 6.7%), and negatively for HMB45, Melan A, DOG1, CD34, AE1/AE3, CAM5.2, chromogranin A, smooth muscle actin, and desmin. In total, 14/15 (93.3%) cases showed split Ewing sarcoma breakpoint region 1 gene (EWSR1) signals consistent with a chromosomal translocation involving EWSR1. Within a mean follow-up of 29.7 months (range: 3 to 63 mo), 2/15 (13.3%) patients died of disease, 5 (33.3%) were alive with disease, and 8 (53.3%) had no evidence of disease. Two and 1 patients showed partial response to apatinib and anlotinib, respectively. In conclusion, GNET has distinctive morphologic, immunohistochemical, and molecular genetic features and should be distinguished from other gastrointestinal tract malignancies. Apatinib and anlotinib might be effective for the treatment of advanced GNET and could prolong patient survival.

Malignant gastrointestinal neuroectodermal tumor (clear cell sarcoma-like tumor of the gastrointestinal tract) of the small intestine in a 12-year-old boy.

The aim of this paper is a clinical and anatomopathological demonstration of a malignant lesion, a gastrointestinal neuroectodermal tumor (GNET), as an exceedingly rare cause of ileus in the pediatric population. Specifically, we present the case of a 12-year-old boy who showed dramatic weight loss, hypochromic anemia, fever, dehydration, exaggerated granulation of the terminal ileum, and mechanical ileus due to the obstruction by an intramural tumor of the small intestine. A 50cm-long part of the small intestine with pathological stricture was surgically removed, sampled and routinely fixed and stained with hematoxylin and eosin. The additional immunostains that were preformed were: PAS, S-100, HMB-45, NSE, LCA, CK AE1 / AE3, desmin, SMA, vimentin, CD99, NSE, synaptophysin, WT-1, calretinin, and DOG-1. Moreover, fluorescent in situ hybridization (FISH) with the EWSR1 Break Apart FISH Probe was applied. The neoplasm was composed of nests and alveolar patterns of frankly malignant clear cells with immunoreactivity to S-100, vimentin, and CD 99. The FISH technique detected chromosomal breaking at 22q12. The tumor metastasized to both the mesenteric lymph nodes and a number of hepatic segments. With several chemotherapy protocols, repeat laparotomies, and liver thermal ablations, the patient had a 1.5-year-long survival from the moment of diagnosis. The diagnosis of this malignancy requires both histopathological evaluation and molecular analysis, and the follow-up is based on careful clinical imaging of the neoplastic spread in order to apply proper surgical and oncological treatments. In conclusion, the clinical course of GNET was highly aggressive.

Orbital Reconstruction in Neuroectodermal Tumor of the Orbit: Multimodal Treatment Approach.

INTRODUCTION: Primitive neuroectodermal tumors of peripheral origin are very rare, and orbital neuroectodermal tumors are even more uncommon. Only 25 patients with primary orbital involvement in the pediatric age group have been reported. METHODS: In this article, the authors describe their experience in the multimodality treatment approach to treat neuroectodermal tumor of the orbit. The authors also present a male patient 3-year old presenting with a neuroectodermal tumor of the right orbit causing rapidly progressive proptosis. The patient underwent an upper and lateral orbital marginotomy. The entire bone defect was reconstructed with a bone graft, allowing for the reconstruction of the floor and the lateral wall of the middle cranial fossa, the floor of the anterior cranial fossa, the upper and lateral orbital frame, and the right zygomatic bone. Over a period of 16 months, the patient was subjected to chemotherapy. RESULTS: In the postoperative period, a favorable evolution of the disease was observed, with growth in the reconstructed structures, good projection of the orbit and the eyeball, and stable results without tumor recurrence. CONCLUSIONS: The authors present the clinical analysis, surgical management, as well as the chemotherapy treatment established, with follow-ups at 1 and 2 and a half years. This experience shows the effectiveness of multimodality therapy in the treatment of rare tumors of difficult handling.

Activity of c-Met/ALK Inhibitor Crizotinib and Multi-Kinase VEGF Inhibitor Pazopanib in Metastatic Gastrointestinal Neuroectodermal Tumor Harboring EWSR1-CREB1 Fusion.

Malignant gastrointestinal neuroectodermal tumor (GNET) is an aggressive rare tumor, primarily occurring in young adults with frequent local-regional metastases and recurrence after local control. The tumor is characterized by the presence of EWSR1-ATF1 or EWSR1-CREB1 and immunohistochemical positivity for S-100 protein without melanocytic marker positivity. Due to poor responses to standard sarcoma regimens, GNET has a poor prognosis, and development of effective systemic therapy is desperately needed to treat these patients. Herein, we present a patient with a small bowel GNET who experienced recurrent hepatic and skeletal metastases after a primary resection. Comprehensive genomic profiling (CGP) in the course of clinical care with DNA and RNA sequencing demonstrated the presence of an exon 7 to exon 6 EWSR1-CREB1 fusion in the context of a diploid genome with no other genomic alterations. In a clinical trial, the patient received a combination of 250 mg crizotinib with 600 mg pazopanib quaque die and achieved partial response and durable clinical benefit for over 2.8 years, and with minimal toxicity from therapy. Using a CGP database of over 50,000 samples, we identified 11 additional cases that harbor EWSR1-CREB1 and report clinicopathologic characteristics, as these patients may also benefit from such a regimen.

Open removal of a retained retrohepatic inferior vena cava filter with a residual primary neuroectodermal renal tumoral thrombus.

Primary neuroectodermal renal tumours (PNET) are rare and aggressive neoplasms; thrombosis of the inferior vena cava (IVC) is associated with this entity. We report here the case of a 19-year-old man who experienced a new onset of abdominal pain. A CT scan revealed a large left renal mass, perirenal haematoma and IVC thrombosis. Owing to an acute drop in haemoglobin and subsegmentary pulmonary embolism, he underwent emergency selective renal artery angiography and embolisation of bleeding vessels and IVC filter (IVCF) placement. Once stable, he underwent a left radical nephrectomy and IVC thrombectomy; the pathology report confirmed PNET. 6 months later, imaging revealed a residual tumoral thrombus in the IVCF located in the retrohepatic IVC. The patient underwent removal of this device and the thrombus via a right thoracoabdominal approach. He recovered well and at 4 months, he continues his chemotherapy cycles.

Efficacy of cabazitaxel in mouse models of pediatric brain tumors.

BACKGROUND: There is an unmet need in the treatment of pediatric brain tumors for chemotherapy that is efficacious, avoids damage to the developing brain, and crosses the blood-brain barrier. These experiments evaluated the efficacy of cabazitaxel in mouse models of pediatric brain tumors. METHODS: The antitumor activity of cabazitaxel and docetaxel were compared in flank and orthotopic xenograft models of patient-derived atypical teratoid rhabdoid tumor (ATRT), medulloblastoma, and central nervous system primitive neuroectodermal tumor (CNS-PNET). Efficacy of cabazitaxel and docetaxel were also assessed in the Smo/Smo spontaneous mouse medulloblastoma tumor model. RESULTS: This study observed significant tumor growth inhibition in pediatric patient-derived flank xenograft tumor models of ATRT, medulloblastoma, and CNS-PNET after treatment with either cabazitaxel or docetaxel. Cabazitaxel, but not docetaxel, treatment resulted in sustained tumor growth inhibition in the ATRT and medulloblastoma flank xenograft models. Patient-derived orthotopic xenograft models of ATRT, medulloblastoma, and CNS-PNET showed significantly improved survival with treatment of cabazitaxel. CONCLUSION: These data support further testing of cabazitaxel as a therapy for treating human pediatric brain tumors.

Extraskeletal Ewing sarcomas in late adolescence and adults: a study of 37 patients.

BACKGROUND: Extraskeletal Ewing sarcoma (EES)/primitive neuroectodermal tumours (PNET) are rare soft tissue sarcomas. Prognostic factors and optimal therapy are still unconfirmed. MATERIALS AND METHODS: We performed a retrospective analysis on patients to explore the clinic characteristics and prognostic factors of this rare disease. A total of 37 patients older than 15 years referred to our institute from Jan., 2002 to Jan., 2012 were reviewed. The characteristics, treatment and outcome were collected and analyzed. RESULTS: The median age was 28 years (range 15-65); the median size of primary tumours was 8.2 cm (range 2-19). Sixteen patients (43%) had metastatic disease at the initial presentation. Wide surgical margins were achieved in 14 cases (38%). Anthracycline or platinum-based chemotherapy was performed on 29 patients (74%). Radiotherapy was delivered in 13 (35%). At a median follow-up visit of 24 months (range 2-81), the media event-free survival (EFS) and overall survival (OS) were 15.8 and 30.2 months, respectively. The 3-year EFS and OS rates were 24% and 43%, respectively. Metastases at presentation and wide surgical margins were significantly associated with OS and EFS. Tumour size was significantly associated with OS but not EFS. There were no significant differences between anthracycline and platinum based chemotherapy regarding EFS and OS. CONCLUSIONS: EES/PNET is a malignant tumour with high recurrence and frequent distant metastasis. Multimodality therapy featuring wide surgical margins, aggressive chemotherapy and adjuvant local radiotherapy is necessary for this rare disease. Platinum-based chemotherapy can be used as an adjuvant therapy.

Is there a role for high dose chemotherapy with hematopoietic stem cell rescue in patients with relapsed supratentorial PNET?

Supratentorial primitive neuroectodermal tumors (sPNET) are rare childhood brain tumors. There is no standard strategy for treating relapsed sPNETs. The role of high dose chemotherapy with hematopoietic stem cell rescue (HDC with HSCR) in treating relapsed sPNET is controversial. A systematic review of the literature regarding outcome of patients with relapsed sPNET treated with HDC and HSCR was performed to examine the potential predictive factors that would justify its use in this subset of patients. Forty-six patients were identified fulfilling the inclusion criteria. Of those, 15 patients were infants and 15 were pineoblastomas. With a median follow-up of 40 months (range 3-123 months) 15 patients were reported alive. Thirteen patients out of the 15 survivors did not receive craniospinal irradiation (CSRT). The 12 month overall survival (OS) of the cohort was 44.2 ± 7.5 months. Twelve-month OS for children less than 36 months was 66.7 ± 12.2 months while for older children it was 27.8 ± 10.6 (P = 0.003). Twelve-month OS was 20.0 ± 10.3 for those patients with pineoblastoma versus 54.6 ± 9.0 for those with non-pineal sPNETs (P < 0.001). Cox regression analysis revealed pineal location as the only independent adverse prognostic factor. In conclusion high dose chemotherapy with HSCR might lead to survival primarily in younger children with relapsed sPNET even in the absence of concomitant use of radiotherapy, whereas the outcome in older children and/or in pineal location is extremely poor with this modality.

Detection of N-glycolyl GM3 ganglioside in neuroectodermal tumors by immunohistochemistry: an attractive vaccine target for aggressive pediatric cancer.

The N-glycolylated ganglioside NeuGc-GM3 has been described in solid tumors such as breast carcinoma, nonsmall cell lung cancer, and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in pediatric neuroectodermal tumors by immunohistochemistry. Twenty-seven archival cases of neuroblastoma and Ewing sarcoma family of tumors (ESFT) were analyzed. Formalin-fixed, paraffin-embedded tumor samples were cut into 5 µm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Presence of NeuGc-GM3 was evident in 23 of 27 cases (85%), with an average of about 70% of positive tumors cells. Immunoreactivity was moderate to intense in most tumors, showing a diffuse cytoplasmic and membranous staining, although cases of ESFT demonstrated a fine granular cytoplasmic pattern. No significant differences were observed between neuroblastoma with and without NMYC oncogene amplification, suggesting that expression of NeuGc-GM3 is preserved in more aggressive cancers. Until now, the expression of N-glycolylated gangliosides in pediatric neuroectodermal tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of neuroectodermal tumors, suggesting its potential utility as a specific target of immunotherapy.

SAE: an R package for early stopping rules in clinical trials.

In the case of an unexpected high frequency of serious adverse events (SAE), statistical methods are needed to help in the decision making process as to continuation of accrual to the trial. This paper describes an R package, named SAE that implements a method recently developed by defining stopping rules after each observed SAE. The package function control for excessive toxicity either during the trial at the observation of each SAE (function SAE) or during the planning phase of a clinical trial (function DESIGN). This description and the package documentation are complementary to help the users to apply the method. The main difficulty in the implementation of the method is the choice of a priori parameters. Data from an ongoing clinical trial are presented as an example to improve the understanding and the use of the package.

Case report of a poorly differentiated uterine tumour with t(10;17) translocation and neuroectodermal phenotype.

Endometrial stromal sarcoma (ESS) with primitive neuroectodermal differentiation is a very uncommon entity. Such a case presenting as stage IIIc (International Federation of Gynaecology and Obstetrics (FIGO) 2010) disease in a 51-year-old female is described. Microscopy suggested a small blue round cell tumour. Cytogenetic and multicolour fluorescent in situ hybridisation (M-FISH) analysis revealed a complex karyotype with the presence of unbalanced t(10;17)(q22;p13) translocation, indicating ESS. Peripheral Ewing´s sarcoma was excluded based on FISH and RT-PCR fusion transcripts analysis. After surgical staging, the patient received bleomycin-etoposide-cisplatin combination chemotherapy. A detailed analysis of the histopathology and genetic findings forms the basis of this report.

Primary cutaneous Ewing's sarcoma/primitive neuroectodermal tumor manifesting numerous small and huge ulcerated masses: its complete remission by chemotherapy and magnetic resonance imaging findings.

Extraskeletal Ewing's sarcoma (ES) and primitive neuroectodermal tumor (PNET) are widely regarded as clinically and histologically identical tumors which consist of small blue round cells. Extraskeletal ESs/PNETs usually occur in the deep soft tissues of the paraspinal region, chest wall, or lower extremities. However, superficially located cases, so-called cutaneous ESs/PNETs, are exceedingly rare, and the vast majority of the reported cases present as a single small mass. We present magnetic resonance imaging (MRI) findings and clinical course of a unique case of primary cutaneous ES/PNET presenting as numerous huge masses with severe ulceration on them.

Regulation of endoplasmic reticulum stress-induced cell death by ATF4 in neuroectodermal tumor cells.

The neuroectodermal tumors neuroblastoma and melanoma represent biologically aggressive and chemoresistant cancers. The chemotherapeutic agents fenretinide and bortezomib induce apoptosis through endoplasmic reticulum (ER) stress in these tumor types. The aim of this study was to test the hypothesis that the early events of ER stress signaling and response pathways induced by fenretinide and bortezomib are mediated by the eukaryotic initiation factor 2alpha (eIF2alpha)-ATF4 signaling pathway. Treatment of neuroblastoma and melanoma cell lines with fenretinide, bortezomib, or thapsigargin resulted in induction of eIF2alpha signaling, characterized by increased expression of phosphorylated eIF2alpha, ATF4, ATF3, and GADD34. These events correlated with induction of the pro-apoptotic protein Noxa. The cytotoxic response, characterized by up-regulation of Noxa and cell death, was dependent on ATF4, but not the ER-related pro-death signaling pathways involving GADD153 or IRE1. Although PERK-dependent phosphorylation of eIF2alpha enhanced ATF4 protein levels during ER stress, cell death in response to fenretinide, bortezomib, or thapsigargin was not abrogated by inhibition of eIF2alpha phosphorylation through PERK knockdown or overexpression of wild-type eIF2alpha. Furthermore, ATF4 induction in response to ER stress was dependent primarily on transcriptional activation, which occurred in a PERK- and phosphorylated eIF2alpha-independent manner. These results demonstrate that ATF4 mediates ER stress-induced cell death of neuroectodermal tumor cells in response to fenretinide or bortezomib. Understanding the complex regulation of cell death pathways in response to ER stress-inducing drugs has the potential to reveal novel therapeutic targets, thus allowing the development of improved treatment strategies to overcome chemoresistance.