Profiling of syngeneic mouse HCC tumor models as a framework to understand anti-PD-1 sensitive tumor microenvironments.

BACKGROUND AND AIMS: The treatment of hepatocellular carcinoma (HCC) has been transformed by the use of immune checkpoint inhibitors. However, most patients with HCC do not benefit from treatment with immunotherapy. There is an urgent need to understand the mechanisms that underlie response or resistance to immunotherapy for patients with HCC. The use of syngeneic mouse models that closely recapitulate the heterogeneity of human HCC will provide opportunities to examine the complex interactions between cancer cells and nonmalignant cells in the tumor microenvironment. APPROACH AND RESULTS: We leverage a multifaceted approach that includes imaging mass cytometry and suspension cytometry by time of flight to profile the tumor microenvironments of the Hep53.4, Hepa 1-6, RIL-175, and TIBx (derivative of TIB-75) syngeneic mouse HCC models. The immune tumor microenvironments vary across these four models, and various immunosuppressive pathways exist at baseline in orthotopic liver tumors derived from these models. For instance, TIBx, which is resistant to anti-programmed cell death protein 1 therapy, contains a high proportion of "M2-like" tumor-associated macrophages with the potential to diminish antitumor immunity. Investigation of The Cancer Genome Atlas reveals that the baseline immunologic profiles of Hep53.4, RIL-175, and TIBx are broadly representative of human HCCs; however, Hepa 1-6 does not recapitulate the immune tumor microenvironment of the vast majority of human HCCs. CONCLUSIONS: There is a wide diversity in the immune tumor microenvironments in preclinical models and in human HCC, highlighting the need to use multiple syngeneic HCC models to improve the understanding of how to treat HCC through immune modulation.

The fibroblast growth factor receptor inhibitor, derazantinib, has strong efficacy in human gastric tumor models and synergizes with paclitaxel in vivo.

Derazantinib (DZB) is an inhibitor of fibroblast growth factor receptors 1-3 (FGFR1-3), with additional activity against colony-stimulating-factor-1 receptor (CSF1R). We have profiled the activity of DZB in gastric cancer (GC) as monotherapy and combined with paclitaxel, and explored means of stratifying patients for treatment. The antiproliferative potency of DZB in vitro was quantified in 90 tumor cell lines and shown to correlate significantly with FGFR expression (<0.01) but not with FGFR DNA copy-number (CN) or FGFR mutations. In four GC cell lines in vitro , little or no synergy was observed with paclitaxel. In athymic nude mice, bearing cell-line derived xenografts (CDX) or patient-derived xenograft (PDX) GC models, DZB efficacy correlated highly significantly with FGFR gene expression ( r2 = 0.58; P = 0.0003; n = 18), but not FGFR mutations or DNA-CN. In FGFR-driven GC models, DZB had comparable efficacy to three other FGFR inhibitors and was more efficacious than paclitaxel. DZB had dose-dependent plasma pharmacokinetics but showed low brain penetration at all doses. GC models (one CDX and six PDX) were tested for sensitivity to the combination of DZB and paclitaxel and characterized by immunohistochemistry. The combination showed synergy (5) or additivity (2), and no antagonism, with synergy significantly associated ( P < 0.05) with higher levels of M2-type macrophages. The association of strong efficacy of the combination in vivo with M2 macrophages, which are known to express CSF1R, and the absence of synergy in vitro is consistent with the tumor microenvironment also being a factor in DZB efficacy and suggests additional means by which DZB could be stratified for cancer treatment in the clinic.

Integration of whole-genome DNA methylation data with RNA sequencing data to identify markers for bull fertility.

Predicting bull fertility prior to breeding is a current challenge for the dairy industry. The use of molecular biomarkers has been previously assessed. However, the integration of this information has not been performed to extract biologically relevant markers. The goal of this study was to integrate DNA methylation data with previously published RNA-sequencing results in order to identify candidate markers for sire fertility. A total of 1765 differentially methylated cytosines were found between high- and low-fertility sires. Ten genes associated with 11 differentially methylated cytosines were found in a previous study of gene expression between high- and low-fertility sires. Additionally, two of these genes code for proteins found exclusively in bull seminal plasma. Collectively, our results reveal 10 genes that could be used in the future as a panel for predicting bull fertility.

Aldehyde dehydrogenase activity plays a Key role in the aggressive phenotype of neuroblastoma.

BACKGROUND: The successful targeting of neuroblastoma (NB) by associating tumor-initiating cells (TICs) is a major challenge in the development of new therapeutic strategies. The subfamily of aldehyde dehydrogenases 1 (ALDH1) isoenzymes, which comprises ALDH1A1, ALDH1A2, and ALDH1A3, is involved in the synthesis of retinoic acid, and has been identified as functional stem cell markers in diverse cancers. By combining serial neurosphere passages with gene expression profiling, we have previously identified ALDH1A2 and ALDH1A3 as potential NB TICs markers in patient-derived xenograft tumors. In this study, we explored the involvement of ALDH1 isoenzymes and the related ALDH activity in NB aggressive properties. METHODS: ALDH activity and ALDH1A1/A2/A3 expression levels were measured using the ALDEFLUOR™ kit, and by real-time PCR, respectively. ALDH activity was inhibited using the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB), and ALDH1A3 gene knock-out was generated through the CRISPR/Cas9 technology. RESULTS: We first confirmed the enrichment of ALDH1A2 and ALDH1A3 mRNA expression in NB cell lines and patient-derived xenograft tumors during neurosphere passages. We found that high ALDH1A1 expression was associated with less aggressive NB tumors and cell lines, and correlated with favorable prognostic factors. In contrast, we observed that ALDH1A3 was more widely expressed in NB cell lines and was associated with poor survival and high-risk prognostic factors. We also identified an important ALDH activity in various NB cell lines and patient-derived xenograft tumors. Specific inhibition of ALDH activity with diethylaminobenzaldehyde (DEAB) resulted in a strong reduction of NB cell clonogenicity, and TIC self-renewal potential, and partially enhanced NB cells sensitivity to 4-hydroxycyclophosphamide. Finally, the specific knock-out of ALDH1A3 via CRISPR/Cas9 gene editing reduced NB cell clonogenicity, and mediated a cell type-dependent inhibition of TIC self-renewal properties. CONCLUSIONS: Together our data uncover the participation of ALDH enzymatic activity in the aggressive properties and 4-hydroxycyclophosphamide resistance of NB, and show that the specific ALDH1A3 isoenzyme increases the aggressive capacities of a subset of NB cells.

Multimodal immune phenotyping reveals microbial-T cell interactions that shape pancreatic cancer.

Microbes are an integral component of the tumor microenvironment. However, determinants of microbial presence remain ill-defined. Here, using spatial-profiling technologies, we show that bacterial and immune cell heterogeneity are spatially coupled. Mouse models of pancreatic cancer recapitulate the immune-microbial spatial coupling seen in humans. Distinct intra-tumoral niches are defined by T cells, with T cell-enriched and T cell-poor regions displaying unique bacterial communities that are associated with immunologically active and quiescent phenotypes, respectively, but are independent of the gut microbiome. Depletion of intra-tumoral bacteria slows tumor growth in T cell-poor tumors and alters the phenotype and presence of myeloid and B cells in T cell-enriched tumors but does not affect T cell infiltration. In contrast, T cell depletion disrupts the immunological state of tumors and reduces intra-tumoral bacteria. Our results establish a coupling between microbes and T cells in cancer wherein spatially defined immune-microbial communities differentially influence tumor biology.

Understanding persons with mental illness who are and are not criminal justice involved: a comparison of criminal thinking and psychiatric symptoms.

Research has begun to elucidate that persons with mental illness become involved in the criminal justice system as a result of criminality and not merely because of their mental illness. This study aims to clarify the similarities and differences in criminal thinking and psychiatric symptomatology between persons with mental illness who are and are not criminal justice involved. Male and female (n = 94) participants admitted to an acute psychiatric facility completed measures to assess criminal thinking (i.e., Psychological Inventory of Criminal Thinking Styles and Criminal Sentiments Scale-Modified) and psychiatric symptomatology (Millon Clinical Multiaxial Inventory-Third Edition). In addition to the inpatient sample, 94 incarcerated persons with mental illness from a previously conducted study were selected based on their match with the current sample on several key demographic and psychiatric variables. The results of this study indicated that hospitalized persons with mental illness with a history of criminal justice involvement evidenced similar thinking styles to persons with mental illness who were incarcerated. Persons with mental illness without criminal justice involvement evidenced fewer thinking styles supportive of a criminal lifestyle than the incarcerated sample. Furthermore, the persons with mental illness sample with no history of criminal justice involvement showed significantly lower levels of psychopathology shown to be risk factors for criminal justice involvement (e.g., antisocial personality, drug dependence, alcohol dependence). These findings have implications for offender-type classification, development of targeted treatment interventions, and program placement.

CyTOF protocol for immune monitoring of solid tumors from mouse models.

Techniques for robust immune profiling of mouse tumor and blood are key to understanding immunological responses in mouse models of cancer. Here, we describe mass cytometry (cytometry by time-of-flight) procedures to facilitate high-parameter profiling of low-volume survival blood samples and end-of-study tumor samples. We employ live-cell barcoding systems to mark all cells from each tumor and blood to improve cost-effectiveness and minimize batch effects. For complete details on the use and execution of this protocol, please refer to Charmsaz et al. (2021).1.

The microtubule-targeted agent lisavanbulin (BAL101553) shows anti-tumor activity in lymphoma models.

Microtubules are major components of the cellular cytoskeleton, ubiquitously founded in all eukaryotic cells. They are involved in mitosis, cell motility, intracellular protein and organelle transport, and maintenance of cytoskeletal shape. Avanbulin (BAL27862) is a microtubule-targeted agent (MTA) that promotes tumor cell death by destabilization of microtubules. Due to its unique binding to the colchicine site of tubulin, differently from other MTAs, avanbulin has previously shown activity in solid tumor cell lines. Its prodrug, lisavanbulin (BAL101553), has shown early signs of clinical activity, especially in tumors with high EB1 expression. Here, we assessed the preclinical anti-tumor activity of avanbulin in diffuse large B cell lymphoma (DLBCL) and the pattern of expression of EB1 in DLBCL cell lines and clinical specimens. Avanbulin showed a potent in vitro anti-lymphoma activity, which was mainly cytotoxic with potent and rapid apoptosis induction. Median IC50 was around 10 nM in both ABC and GCB-DLBCL. Half of the cell lines tested showed an induction of apoptosis already in the first 24 h of treatment, the other half in the first 48 h. EB1 showed expression in DLBCL clinical specimens, opening the possibility for a cohort of patients that could potentially benefit from treatment with lisavanbulin. These data show the basis for further preclinical and clinical evaluation of lisavanbulin in the lymphoma field.

Indocyanine green angiography-guided photodynamic therapy for treatment of chronic central serous chorioretinopathy: a pilot study. 2003.

BACKGROUND: Most patients with central serous chorioretinopathy (CSC) have spontaneous resolution of exudative macular detachments and a good visual prognosis. Patients with CSC have a primary choroidal hyperpermeability problem evident as multifocal areas of hyperpermeability during indocyanine green (ICG) angiography. A small percentage of patients develop chronic or progressive disease with widespread decompensation of the retinal pigment epithelium and severe vision loss. There is no known treatment for this variant of the disorder. PURPOSE: To study ICG-guided photodynamic therapy (PDT) with verteporfin as a potential treatment for patients with chronic CSC. METHODS: Twenty eyes of 15 patients were studied with fluorescein angiography, optical coherence tomography, and ICG angiography to diagnose the maculopathy, monitor the detachments, and localize the choroidal hyperpermeability of the disorder. PDT with ICG guidance was applied to areas of choroidal hyperpermeability, and the patients were observed to determine the anatomic and functional outcomes. RESULTS: Photodynamic therapy guided by ICG was associated with complete resolution of exudative macular detachments in 12 patients and incomplete resolution in the remaining eight eyes. The vision improved in six eyes and remained unchanged in 14 eyes during a mean follow-up of 6.8 months. Six weeks after treatment, the mean visual acuity improved by 0.55 lines, an amount that was marginally significant. There was a significant inverse correlation between the baseline visual acuity and the amount of improvement in acuity at 6 weeks. No patient had any treatment-related side effects. CONCLUSIONS: Indocyanine green angiography-guided PDT with verteporfin seems to aid in the resolution of exudative detachments in patients with chronic CSC. This treatment was associated with a rapid reduction in subretinal fluid and improvement in visual acuity. Although the follow-up time and number of patients in this pilot study were limited, the encouraging results and lack of complications suggest that further study is indicated.

Integrated T cell cytometry metrics for immune-monitoring applications in immunotherapy clinical trials.

Mass cytometry, or cytometry by TOF (CyTOF), provides a robust means of determining protein-level measurements of more than 40 markers simultaneously. While the functional states of immune cells occur along continuous phenotypic transitions, cytometric studies surveying cell phenotypes often rely on static metrics, such as discrete cell-type abundances, based on canonical markers and/or restrictive gating strategies. To overcome this limitation, we applied single-cell trajectory inference and nonnegative matrix factorization methods to CyTOF data to trace the dynamics of T cell states. In the setting of cancer immunotherapy, we showed that patient-specific summaries of continuous phenotypic shifts in T cells could be inferred from peripheral blood-derived CyTOF mass cytometry data. We further illustrated that transfer learning enabled these T cell continuous metrics to be used to estimate patient-specific cell states in new sample cohorts from a reference patient data set. Our work establishes the utility of continuous metrics for CyTOF analysis as tools for translational discovery.

Chemokines in neuroectodermal cancers: the crucial growth signal from the soil.

Although chemokines and their receptors were initially identified as regulators of cell trafficking during inflammation and immune response, they have emerged as crucial players in all stages of tumor development, primary growth, migration, angiogenesis, and establishment as metastases in distant target organs. Neuroectodermal tumors regroup neoplasms originating from the embryonic neural crest cells, which display clinical and biological similarities. These tumors are highly malignant and rapidly progressing diseases that disseminate to similar target organs such as bone marrow, bone, liver and lungs. There is increasing evidence that interaction of several chemokine receptors with corresponding chemokine ligands are implicated in the growth and invasive characteristics of these tumors. In this review we summarize the current knowledge on the role of CXCL12 chemokine and its CXCR4 and CXCR7 receptors in the progression and survival of neuroectodermal tumors, with particular emphasis on neuroblastoma, the most typical and enigmatic neuroectodermal childhood tumor.

A global live cell barcoding approach for multiplexed mass cytometry profiling of mouse tumors.

With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a potentially novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex 10 tumors from s.c. MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the 2 models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.

Systemic inhibition of PTPN22 augments anticancer immunity.

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a novel small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD1-PDL1 axis in the setting of PTPN22 inhibition could be further leveraged with PD1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.

Multi-omic profiling of lung and liver tumor microenvironments of metastatic pancreatic cancer reveals site-specific immune regulatory pathways.

BACKGROUND: The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies have limited activity with a dismal 5-year survival rate of only 8%. The liver and lung are the most common sites of PDAC metastasis, and each have been differentially associated with prognoses and responses to systemic therapies. A deeper understanding of the molecular and cellular landscape within the tumor microenvironment (TME) metastasis at these different sites is critical to informing future therapeutic strategies against metastatic PDAC. RESULTS: By leveraging combined mass cytometry, immunohistochemistry, and RNA sequencing, we identify key regulatory pathways that distinguish the liver and lung TMEs in a preclinical mouse model of metastatic PDAC. We demonstrate that the lung TME generally exhibits higher levels of immune infiltration, immune activation, and pro-immune signaling pathways, whereas multiple immune-suppressive pathways are emphasized in the liver TME. We then perform further validation of these preclinical findings in paired human lung and liver metastatic samples using immunohistochemistry from PDAC rapid autopsy specimens. Finally, in silico validation with transfer learning between our mouse model and TCGA datasets further demonstrates that many of the site-associated features are detectable even in the context of different primary tumors. CONCLUSIONS: Determining the distinctive immune-suppressive features in multiple liver and lung TME datasets provides further insight into the tissue specificity of molecular and cellular pathways, suggesting a potential mechanism underlying the discordant clinical responses that are often observed in metastatic diseases.

Derazantinib: an investigational drug for the treatment of cholangiocarcinoma.

INTRODUCTION: This review evaluates the clinical role of fibroblast growth factor receptor 2 (FGFR2) inhibition with derazantinib in patients with intrahepatic cholangiocarcinoma (iCCA) harboring actionable oncogenic FGFR2 fusions/rearrangements, mutations and amplifications. FGFR inhibitors such as derazantinib are currently being evaluated to address the unmet medical need of patients with previously treated, locally advanced or metastatic iCCA harboring such genetic aberrations. AREAS COVERED: We summarize the pharmacokinetics, and the emerging safety and efficacy data of the investigational FGFR inhibitor derazantinib. We discuss the future directions of this novel therapeutic agent for iCCA. EXPERT OPINION: Derazantinib is a potent FGFR1‒3 kinase inhibitor which also has activity against colony stimulating factor-1‒receptor (CSF1R) and vascular endothelial growfth factor receptor‒2 (VEGFR2), suggesting a potentially differentiated role in the treatment of patients with iCCA. Derazantinib has shown clinically meaningful efficacy with durable objective responses, supporting the therapeutic potential of derazantinib in previously treated patients with iCCA harboring FGFR2 fusions/rearrangements, mutations and amplifications. The clinical safety profile of derazantinib was well manageable and compared favorably to the FGFR inhibitor class, particularly with a low incidence of drug-related hand-foot syndrome, stomatitis, retinal and nail toxicity. These findings support the need for increased molecular profiling of cholangiocarcinoma patients.

Neoadjuvant Cabozantinib and Nivolumab Converts Locally Advanced HCC into Resectable Disease with Enhanced Antitumor Immunity.

A potentially curative hepatic resection is the optimal treatment for hepatocellular carcinoma (HCC), but most patients are not candidates for resection and most resected HCCs eventually recur. Until recently, neoadjuvant systemic therapy for HCC has been limited by a lack of effective systemic agents. Here, in a single arm phase 1b study, we evaluated the feasibility of neoadjuvant cabozantinib and nivolumab in patients with HCC including patients outside of traditional resection criteria (NCT03299946). Of 15 patients enrolled, 12 (80%) underwent successful margin negative resection, and 5/12 (42%) patients had major pathologic responses. In-depth biospecimen profiling demonstrated an enrichment in T effector cells, as well as tertiary lymphoid structures, CD138+ plasma cells, and a distinct spatial arrangement of B cells in responders as compared to non-responders, indicating an orchestrated B-cell contribution to antitumor immunity in HCC.

The Intergenerational Impacts of Paternal Diet on DNA Methylation and Offspring Phenotypes in Sheep.

Knowledge of non-genomic inheritance of traits is currently limited. Although it is well established that maternal diet influences offspring inheritance of traits through DNA methylation, studies on the impact of prepubertal paternal diet on DNA methylation are rare. This study aimed to evaluate the impact of prepubertal diet in Polypay rams on complex traits, DNA methylation, and transmission of traits to offspring. A total of 10 littermate pairs of F0 rams were divided so that one ram was fed a control diet, and the other was fed the control diet with supplemental methionine. Diet was associated with earlier age at puberty in treatment vs. control F0 rams. F0 treatment rams tended to show decreased pubertal weight compared to control rams; however, no differences were detected in overall growth. A total of ten F0 rams were bred, and the entire F1 generation was fed a control diet. Diet of F0 rams had a significant association with scrotal circumference (SC) and weight at puberty of F1 offspring. The paternal diet was not significantly associated with F1 ram growth or age at puberty. The DNA methylation of F0 ram sperm was assessed, and genes related to both sexual development (e.g., DAZAP1, CHD7, TAB1, MTMR2, CELSR1, MGAT1) and body weight (e.g., DUOX2, DUOXA2) were prevalent in the data. These results provide novel information about the mechanisms through which the prepubertal paternal diet may alter body weight at puberty and sexual development.

Alleged predisposing dietary factors fail to increase the incidence of osteochondrosis-like lesions in growing pigs at 14 and 24 wk of age.

Early lesions of osteochondrosis (OC) are exhibited by regions of cartilage retention along the growth plate and articular cartilage. Progression of OC lesions may impair locomotion and necessitate euthanasia in adherence to animal welfare guides. Little is known about the role of nutrition in the initiation and early stages of OC. However, dietary components are commonly implicated as predisposing factors. In this study, diets were altered as an attempt to induce early stage OC lesions under controlled conditions. At 8 wk of age, 96 crossbred gilts (body weight [BW] = 17.4 ± 0.18 kg) were randomly assigned to one of four corn-soybean meal-based diets (four pens per diet, six pigs per pen) to assess diet effects on the number and volume of OC lesions in the distal femur. Diets included a non-pelleted control diet (Ctl); Ctl plus 20% glucose (Glc); the Ctl with increased concentrations of lysine, Ca, and P (+CaP); and the +CaP diet in a pelleted form (PEL). Femurs were collected from pigs euthanized at either 14-wk (Wk 14) or 24-wk (Wk 14) of age for assessments of OC lesions. Based on a mixed model analysis with pen as the experimental unit, dietary treatments did not affect final BW (129.3 ± 3.8 kg) or average daily gain (ADG) (1.00 ± 0.03 kg/d) over the trial. As expected, pigs fed PEL and Glc diets were more efficient (P < 0.05) in feed conversion compared with Ctl and +CaP. Using femurs as the experimental unit at Wk 14 (collected from two of the six pigs per pen), bone mineral content, determined by dual-energy x-ray absorptiometry scans, was greater (P < 0.05) in pigs fed +CaP and PEL than Ctl or Glc diets; however, only +CaP group differed (P < 0.05) at Wk 24 (collected from four pigs per pen). Computed tomography (CT) scans of femurs were reconstructed as three-dimensional images to allow detection of the number, volume, and surface area of lesions in distal growth plates. At Wk 14, pigs fed Ctl had fewer number of lesions (P < 0.05); however, no differences were detected among dietary treatments in lesion volume or lesion surface area. Pigs had fewer lesions at Wk 24 than Wk 14; however, differences were not detected among dietary treatments. At Wk 24, pigs fed Ctl diets had the greatest lesion volume among dietary treatments (P < 0.05). In conclusion, none of the pigs exhibited symptoms of lameness regardless of dietary treatment or OC lesion traits. Diet modifications due to pelleting or inclusion of rapidly digestible ingredients, such as glucose, did not increase prevalence or size of OC lesions. Image analysis of CT scans was a reliable method to quantify the number, size, and location of OC lesions.

Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable MYCN-Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group.

PURPOSE: For localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome. PATIENTS AND METHODS: Diagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children's Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN) Biology Group. RESULTS: Patients with stage 1 tumors had an excellent outcome (5-year event-free survival [EFS] ± standard deviation [SD], 95% ± 2%; 5-year overall survival [OS], 99% ± 1%). In contrast, patients with stage 2 tumors had a reduced EFS in both age groups (5-year EFS ± SD, 84% ± 3% in patients < 18 months of age and 75% ± 7% in patients ≥ 18 months of age). However, OS was significantly decreased only in the latter group (5-year OS ± SD in < 18months and ≥ 18months, 96% ± 2% and 81% ± 7%, respectively; P = .001). In < 18months, relapses occurred independent of segmental chromosome aberrations (SCAs); only 1p loss decreased EFS (5-year EFS ± SD in patients 1p loss and no 1p loss, 62% ± 13% and 87% ± 3%, respectively; P = .019) but not OS (5-year OS ± SD, 92% ± 8% and 97% ± 2%, respectively). In patients ≥ 18 months, only SCAs led to relapse and death, with 11q loss as the strongest marker (11q loss and no 11q loss: 5-year EFS ± SD, 48% ± 16% and 85% ± 7%, P = .033; 5-year OS ± SD, 46% ± 22% and 92% ± 6%, P = .038). CONCLUSION: Genomic aberrations of resectable non-MYCN-amplified stage 2 neuroblastomas have a distinct age-dependent prognostic impact. Chromosome 1p loss is a risk factor for relapse but not for diminished OS in patients < 18 months, SCAs (especially 11q loss) are risk factors for reduced EFS and OS in those > 18months. In older patients with SCA, a randomized trial of postoperative chemotherapy compared with observation alone may be indicated.

Fenretinide-induced caspase-8 activation and apoptosis in an established model of metastatic neuroblastoma.

BACKGROUND: Resistance of high-risk metastatic neuroblastoma (HR-NB) to high dose chemotherapy (HD-CT) raises a major therapeutic challenge in pediatric oncology. Patients are treated by maintenance CT. For some patients, an adjuvant retinoid therapy is proposed, such as the synthetic retinoid fenretinide (4-HPR), an apoptotic inducer. Recent studies demonstrated that NB metastasis process is enhanced by the loss of caspase-8 involved in the Integrin-Mediated Death (IMD) process. As the role of caspase-8 appears to be critical in preventing metastasis, we aimed at studying the effect of 4-HPR on caspase-8 expression in metastatic neuroblasts. METHODS: We used the human IGR-N-91 MYCN-amplified NB experimental model, able to disseminate in vivo from the primary nude mouse tumor xenograft (PTX) into myocardium (Myoc) and bone marrow (BM) of the animal. NB cell lines, i.e., IGR-N-91 and SH-EP, were treated with various doses of Fenretinide (4-HPR), then cytotoxicity was analyzed by MTS proliferation assay, apoptosis by the propidium staining method, gene or protein expressions by RT-PCR and immunoblotting and caspases activity by colorimetric protease assays. RESULTS: The IGR-N-91 parental cells do not express detectable caspase-8. However the PTX cells established from the primary tumor in the mouse, are caspase-8 positive. In contrast, metastatic BM and Myoc cells show a clear down-regulation of the caspase-8 expression. In parallel, the caspases -3, -9, -10, Bcl-2, or Bax expressions were unchanged. Our data show that in BM, compared to PTX cells, 4-HPR up-regulates caspase-8 expression that parallels a higher sensitivity to apoptotic cell death. Stable caspase-8-silenced SH-EP cells appear more resistant to 4-HPR-induced cell death compared to control SH-EP cells. Moreover, 4-HPR synergizes with drugs since apoptosis is restored in VP16- or TRAIL-resistant-BM cells. These results demonstrate that 4-HPR in up-regulating caspase-8 expression, restores and induces apoptotic cell death in metastatic neuroblasts through caspase-8 activation. CONCLUSION: This study provides basic clues for using fenretinide in clinical treatment of HR-NB patients. Moreover, since 4-HPR induces cell death in caspase-8 negative NB, it also challenges the concept of including 4-HPR in the induction of CT of these patients.