Neoadjuvant immunotherapy represents a pioneering approach in the preoperative treatment of cancer, providing new strategies for tumor reduction and improved patient outcomes by modulating the immune response. This study investigated neoadjuvant immunotherapy using intratumoral administration of mannan-BAM, Toll-like receptor ligands, and anti-CD40 antibody (MBTA therapy) followed by surgery in murine models of MTT pheochromocytoma, B16-F10 melanoma, and 4T1 and E0771.lmb mammary carcinomas. In the MTT pheochromocytoma model, it was found that neoadjuvant MBTA therapy followed by surgery could prevent the development of distant metastases in 100% of treated animals, compared to a 60% mortality rate in the control group due to metastatic disease after surgery. These outcomes were achieved even in tumors three times larger than those in the control group. In the aggressive 4T1 model, neoadjuvant MBTA therapy resulted in slower tumor progression and a significant prolongation of survival. In the B16-F10 and E0771.lmb models, neoadjuvant MBTA therapy also protected animals from metastases development and tumor recurrence upon rechallenge with tumor cells after surgery. Transcriptomic analysis revealed enhanced effector immune cell infiltration, cytotoxicity, and antigen presentation in retransplanted tumors from MBTA-treated mice, indicating robust immune memory. Notably, the exclusion of the anti-CD40 antibody from the neoadjuvant MBTA therapy (MBT therapy) yielded comparable outcomes in protection against metastases development. These findings advocate for further investigation of intratumoral neoadjuvant MBTA therapy for immunologically "cold" tumors, including those at high risk of metastases or recurrence.

Despite enormous progress, advanced cancers are still one of the most serious medical problems in current society. Although various agents and therapeutic strategies with anticancer activity are known and used, they often fail to achieve satisfactory long-term patient outcomes and survival. Recently, immunotherapy has shown success in patients by harnessing important interactions between the immune system and cancer. However, many of these therapies lead to frequent side effects when administered systemically, prompting treatment modifications or discontinuation or, in severe cases, fatalities. New therapeutic approaches like intratumoral immunotherapy, characterized by reduced side effects, cost, and systemic toxicity, offer promising prospects for future applications in clinical oncology. In the context of locally advanced or metastatic cancer, combining diverse immunotherapeutic and other treatment strategies targeting multiple cancer hallmarks appears crucial. Such combination therapies hold promise for improving patient outcomes and survival and for promoting a sustained systemic response. This review aims to provide a current overview of immunotherapeutic approaches, specifically focusing on the intratumoral administration of drugs in patients with locally advanced and metastatic cancers. It also explores the integration of intratumoral administration with other modalities to maximize therapeutic response. Additionally, the review summarizes recent advances in intratumoral immunotherapy and discusses novel therapeutic approaches, outlining future directions in the field.

Immunotherapy represents a revolutionary advancement in cancer treatment, which has traditionally focused on T cells; however, the role of B cells in cancer immunotherapy has gained interest because of their role in antigen presentation, antibody production, and cytokine release. In this study, we examined the role of B cells in previously developed intratumoral MBTA therapy (mannan-BAM, TLR ligands, and anti-CD40 antibody) in murine models of MTT pheochromocytoma. The results indicated that B cells significantly enhance the success of MBTA therapy, with wild-type mice exhibiting a lower tumor incidence and smaller tumors compared with B cell-deficient mice. Increased IL-6 and TNF-alpha levels indicated severe inflammation and a potential cytokine storm in B cell-deficient mice. Neutralization of TNF-alpha ameliorated these complications but resulted in increased tumor recurrence. The results highlight the important role of B cells in enhancing the immune response and maintaining immune homeostasis during MBTA therapy. Our findings offer new insights into improving therapeutic outcomes.

BACKGROUND:Renal cell carcinoma (RCC) is a disease typified by anomalies in cell metabolism. The function of mitochondria, including subunits of mitochondrial respiratory complex II (CII), in particular SDHB, are often affected. Here we investigated the state and function of CII in RCC patients. METHODS:We evaluated tumour tissue as well as the adjacent healthy kidney tissue of 78 patients with RCC of different histotypes, focusing on their mitochondrial function. As clear cell RCC (ccRCC) is by far the most frequent histotype of RCC, we focused on these patients, which were grouped based on the pathological WHO/ISUP grading system to low- and high-grade patients, indicative of prognosis. We also evaluated mitochondrial function in organoids derived from tumour tissue of 7 patients. RESULTS:ccRCC tumours were characterized by mutated von Hippel-Lindau gene and high expression of carbonic anhydrase IX. We found low levels of mitochondrial DNA, protein and function, together with CII function in ccRCC tumour tissue, but not in other RCC types and non-tumour tissues. Mitochondrial content increased in high-grade tumours, while the function of CII remained low. Tumour organoids from ccRCC patients recapitulated molecular characteristics of RCC tissue. CONCLUSIONS:Our findings suggest that the state of CII, epitomized by its assembly and SDHB levels, deteriorates with the progressive severity of ccRCC. These observations hold the potential for stratification of patients with worse prognosis and may guide the exploration of targeted therapeutic interventions.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors derived from neural crest cells from adrenal medullary chromaffin tissues and extra-adrenal paraganglia, respectively. Although the current treatment for PPGLs is surgery, optimal treatment options for advanced and metastatic cases have been limited. Hence, understanding the role of the immune system in PPGL tumorigenesis can provide essential knowledge for the development of better therapeutic and tumor management strategies, especially for those with advanced and metastatic PPGLs. The first part of this review outlines the fundamental principles of the immune system and tumor microenvironment, and their role in cancer immunoediting, particularly emphasizing PPGLs. We focus on how the unique pathophysiology of PPGLs, such as their high molecular, biochemical, and imaging heterogeneity and production of several oncometabolites, creates a tumor-specific microenvironment and immunologically "cold" tumors. Thereafter, we discuss recently published studies related to the reclustering of PPGLs based on their immune signature. The second part of this review discusses future perspectives in PPGL management, including immunodiagnostic and promising immunotherapeutic approaches for converting "cold" tumors into immunologically active or "hot" tumors known for their better immunotherapy response and patient outcomes. Special emphasis is placed on potent immune-related imaging strategies and immune signatures that could be used for the reclassification, prognostication, and management of these tumors to improve patient care and prognosis. Furthermore, we introduce currently available immunotherapies and their possible combinations with other available therapies as an emerging treatment for PPGLs that targets hostile tumor environments.

Despite strides in immunotherapy, glioblastoma multiforme (GBM) remains challenging due to low inherent immunogenicity and suppressive tumor microenvironment. Converting "cold" GBMs to "hot" is crucial for immune activation and improved outcomes. This study comprehensively characterized a therapeutic vaccination strategy for preclinical GBM models. The vaccine consists of Mannan-BAM-anchored irradiated whole tumor cells, Toll-like receptor ligands [lipoteichoic acid (LTA), polyinosinic-polycytidylic acid (Poly (I:C)), and resiquimod (R-848)], and anti-CD40 agonistic antibody (rWTC-MBTA). Intracranial GBM models (GL261, SB28 cells) are used to evaluate the vaccine efficacy. A substantial number of vaccinated mice exhibited complete regression of GBM tumors in a T-cell-dependent manner, with no significant toxicity. Long-term tumor-specific immune memory is confirmed upon tumor rechallenge. In the vaccine-draining lymph nodes of the SB28 model, rWTC-MBTA vaccination triggered a major rise in conventional dendritic cell type 1 (cDC1) 12 h post-treatment, followed by an increase in conventional dendritic cell type 2 (cDC2), monocyte-derived dendritic cell (moDC), and plasmacytoid dendritic cell (pDC) on Day 5 and Day 13. Enhanced cytotoxicity of CD4+ and CD8+ T cells in vaccinated mice is verified in co-culture with tumor cells. Analyses of immunosuppressive signals (T-cell exhaustion, myeloid-derived suppressor cells (MDSC), M2 macrophages) in the GBM microenvironment suggest potential combinations with other immunotherapies for enhanced efficacy. In conclusion, the authors findings demonstrate that rWTC-MBTA induces potent and long-term adaptive immune responses against GBM.

Abstract Despite advances in immunotherapy, glioblastoma multiforme (GBM) remains challenging to treat due to its low inherent immunogenicity and a suppressive tumor microenvironment. Converting “cold” GBMs to “hot” tumors is crucial for effective immune activation and improved patient outcomes. We comprehensively characterized the rWTC-MBTA autologous cancer vaccine, consisting of Mannan-BAM-anchored irradiated whole tumor cells, Toll-like receptor ligands (LTA, Poly(I:C), R-848), and an anti-CD40 agonistic antibody, in preclinical GL261 and SB28 GBM models. A substantial number of vaccinated mice exhibited complete regression of GBM tumors in a T-cell-dependent manner, with no significant toxicity. Long-term tumor-specific immune memory was confirmed upon tumor rechallenge. In the vaccine-draining lymph nodes of the SB28 model, rWTC-MBTA vaccination triggered a major rise in cDC1 12 hours post-treatment, followed by an increase in cDC2, moDC, and pDC on Days 5 and 13. Enhanced cytotoxicity of CD4+ and CD8+ T cells in vaccinated mice was verified in co-culture with tumor cells. Comprehensive immunophenotyping in the GL261 and SB28 GBM microenvironments confirmed decreased regulatory T cells and increased CD4+ and CD8+ T cell infiltration in vaccinated mice. T-cell exhaustion analyses revealed an increase in the Tim3+CD8+ T cells. No significant differences were observed in the quantification of M-MDSC and PMN-MDSC between control and vaccinated mice, but PD-L1 expression significantly increased in M2 macrophages in vaccinated mice. Our findings demonstrate that rWTC-MBTA induces potent and long-term adaptive immune responses against GBM, warranting further investigation of combinations with other immunotherapies for enhanced efficacy.

European Journal of Clinical InvestigationEarly View e14162 RESEARCH LETTER Niche for polyamine inhibition in treatment of metastatic pheochromocytoma and paraganglioma Hans K. Ghayee, Corresponding Author Hans K. Ghayee [email protected] orcid.org/0000-0001-9201-3033 Department of Medicine, Division of Endocrinology, University of Florida and Malcom Randall VA Medical Center, Gainesville, Florida, USA Correspondence Hans K. Ghayee, Department of Medicine, Division of Endocrinology, University of Florida and Malcom Randall VA Medical Center, Gainesville, FL, USA. Email: [email protected]Search for more papers by this authorHeather Hatch, Heather Hatch Department of Physiological Sciences, University of Florida, Gainesville, Florida, USASearch for more papers by this authorRaymond J. Bergeron, Raymond J. Bergeron Department of Medicinal Chemistry, University of Florida, Gainesville, Florida, USASearch for more papers by this authorKaterina Hadrava Vanova, Katerina Hadrava Vanova Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USASearch for more papers by this authorKarel Pacak, Karel Pacak Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USASearch for more papers by this authorSergei Tevosian, Sergei Tevosian Department of Physiological Sciences, University of Florida, Gainesville, Florida, USASearch for more papers by this author Hans K. Ghayee, Corresponding Author Hans K. Ghayee [email protected] orcid.org/0000-0001-9201-3033 Department of Medicine, Division of Endocrinology, University of Florida and Malcom Randall VA Medical Center, Gainesville, Florida, USA Correspondence Hans K. Ghayee, Department of Medicine, Division of Endocrinology, University of Florida and Malcom Randall VA Medical Center, Gainesville, FL, USA. Email: [email protected]Search for more papers by this authorHeather Hatch, Heather Hatch Department of Physiological Sciences, University of Florida, Gainesville, Florida, USASearch for more papers by this authorRaymond J. Bergeron, Raymond J. Bergeron Department of Medicinal Chemistry, University of Florida, Gainesville, Florida, USASearch for more papers by this authorKaterina Hadrava Vanova, Katerina Hadrava Vanova Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USASearch for more papers by this authorKarel Pacak, Karel Pacak Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USASearch for more papers by this authorSergei Tevosian, Sergei Tevosian Department of Physiological Sciences, University of Florida, Gainesville, Florida, USASearch for more papers by this author First published: 21 February 2024 https://doi.org/10.1111/eci.14162Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. CONFLICT OF INTEREST STATEMENT HKG has received royalties from the University of Texas Southwestern Medical Center at Dallas. REFERENCES 1Tevosian SG, Ghayee HK. Pheochromocytomas and paragangliomas. Endocrinol Metab Clin N Am. 2019; 48(4): 727-750. 10.1016/j.ecl.2019.08.006 PubMedWeb of Science®Google Scholar 2Taieb D, Taïeb D, Wanna GB, et al. Clinical consensus guideline on the management of phaeochromocytoma and paraganglioma in patients harbouring germline SDHD pathogenic variants. Lancet Diabetes Endocrinol. 2023; 11(5): 345-361. 10.1016/S2213-8587(23)00038-4 PubMedWeb of Science®Google Scholar 3Mete O, Asa SL, Gill AJ, Kimura N, de Krijger RR, Tischler A. Overview of the 2022 WHO classification of paragangliomas and pheochromocytomas. Endocr Pathol. 2022; 33(1): 90-114. 10.1007/s12022-022-09704-6 CASPubMedWeb of Science®Google Scholar 4Fishbein L, Leshchiner I, Walter V, et al. Comprehensive molecular characterization of pheochromocytoma and paraganglioma. Cancer Cell. 2017; 31(2): 181-193. 10.1016/j.ccell.2017.01.001 CASPubMedWeb of Science®Google Scholar 5Baudin E, Goichot B, Berruti A, et al. 567O First International Randomized Study in Malignant Progressive Pheochromocytoma and Paragangliomas (FIRSTMAPPP): an academic double-blind trial investigating sunitinib. Ann Oncol. 2021; 32: S621. 10.1016/j.annonc.2021.08.702 Web of Science®Google Scholar 6Zhuang Z, Yang C, Lorenzo F, et al. Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia. N Engl J Med. 2012; 367(10): 922-930. 10.1056/NEJMoa1205119 CASPubMedWeb of Science®Google Scholar 7Pryma DA, Chin BB, Noto RB, et al. Efficacy and safety of high-specific-activity (131)I-MIBG therapy in patients with advanced pheochromocytoma or paraganglioma. J Nucl Med. 2019; 60(5): 623-630. 10.2967/jnumed.118.217463 CASPubMedWeb of Science®Google Scholar 8Rai SK, Bril F, Hatch HM, et al. Targeting pheochromocytoma/paraganglioma with polyamine inhibitors. Metabolism. 2020; 110:154297. 10.1016/j.metabol.2020.154297 CASPubMedWeb of Science®Google Scholar 9Kamihara J, Hamilton KV, Pollard JA, et al. Belzutifan, a potent HIF2alpha inhibitor, in the Pacak-Zhuang syndrome. N Engl J Med. 2021; 385(22): 2059-2065. 10.1056/NEJMoa2110051 CASPubMedWeb of Science®Google Scholar 10Voss B, Wilop S, Jonas S, et al. Tumor volume as a prognostic factor in resectable malignant melanoma. Dermatology. 2014; 228(1): 66-70. 10.1159/000356121 PubMedWeb of Science®Google Scholar 11Hahm HA, Goyal L, Supko JG, et al. Phase I study of N(1),N(11)-diethylnorspermine in patients with non-small cell lung cancer. Clin Cancer Res. 2002; 8(3): 684-690. CASPubMedWeb of Science®Google Scholar Early ViewOnline Version of Record before inclusion in an issuee14162 ReferencesRelatedInformation

Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.

Cancer immunotherapy has shown remarkable clinical progress in recent years. Although age is one of the biggest leading risk factors for cancer development and older adults represent a majority of cancer patients, only a few new cancer immunotherapeutic interventions have been preclinically tested in aged animals. Thus, the lack of preclinical studies focused on age-dependent effect during cancer immunotherapy could lead to different therapeutic outcomes in young and aged animals and future modifications of human clinical trials. Here, we compare the efficacy of previously developed and tested intratumoral immunotherapy, based on the combination of polysaccharide mannan, toll-like receptor ligands, and anti-CD40 antibody (MBTA immunotherapy), in young (6 weeks) and aged (71 weeks) mice bearing experimental pheochromocytoma (PHEO). The presented results point out that despite faster growth of PHEO in aged mice MBTA intratumoral immunotherapy is effective approach without age dependence and could be one of the possible therapeutic interventions to enhance immune response to pheochromocytoma and perhaps other tumor types in aged and young hosts.

Abstract Metformin, a lipophilic biguanide inhibiting hepatic gluconeogenesis and improving peripheral utilization of glucose, is a well-established medication for the management of type 2 diabetes. Furthermore, metformin has been shown to have pleiotropic effects targeting oxidative phosphorylation via complex I inhibition. Thus, it regulates the energy supply to cells from mitochondrial complex I respiration along with reduced glycolytic metabolism. Several pre-clinical in vitro studies on cell lines derived from pheochromocytoma (PHEO) have suggested anti-proliferative potential of metformin which hints that metformin could be used as a potent candidate for PHEO anti-cancer therapy. However, these results have yet to be confirmed in vivo. Murine PHEO MPC cells and human progenitor hPheo1 cells were treated with metformin in vitro to study the effect on cell proliferation and ATP production. Mice were injected with MTT cells subcutaneously and when the tumors reached the average volume of 120.2±48.1 mm3, they were randomized into 3 groups (n=5) treated with vehicle, 125 mg/kg metformin i.p. daily (6 times a week), or the drug dissolved into drinking water (5 mg/ml). Tumor progression and survival data were collected. Metformin effectively decreased PHEO cell proliferation and overall ATP production in dose-dependent manner in vitro. Tumor progression was similar in both metformin-treated groups and control group with no significant effect on the growth or mice survival. Even though the metformin showed antiproliferative effects in both murine and human derived cells in vitro, we did not observe tumor growth limitation in our model of murine pheochromocytoma in vivo. However, further studies including newer models and variable treatment conditions including timing, are needed to clarify the lack of efficacy in vivo. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Metastatic pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors associated with poor prognosis and limited therapeutic options. Recent advances in oncology-related immunotherapy, specifically in targeting of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathways, have identified a new treatment potential in a variety of tumors, including advanced and rare tumors. Only a fraction of patients being treated by immune checkpoint inhibitors have shown to benefit from it, displaying a need for strategies which identify patients who may most likely show a favorable response. Building on recent, promising outcomes in a clinical study of metastatic PPGL using pembrolizumab, a humanized IgG4κ monoclonal antibody targeting the PD-1/PD-L1 pathway, we examined PD-L1 and PD-L2 expression in relation to oncogenic drivers in our PPGL patient cohort to explore whether expression can predict metastatic potential and/or be considered a predictive marker for targeted therapy. We evaluated RNA expression in the NIH cohort of 48 patients with known genetic predisposition (sporadic; pseudohypoxia: SDHB, VHL, EPAS1, EGLN1; kinase signaling: RET, NF1) and 6 normal medulla samples (NAM). For comparison, 72 PPGL samples from The Cancer Genome Atlas (TCGA) were used for analysis of gene expression based on the variant status (pseudohypoxia: SDHB, VHL, EPAS1, EGLN1; kinase signaling: NF1, RET). Expression of PD-L1 was elevated in the PPGL cohort compared to normal adrenal medulla, aligning with the TCGA analysis, whereas PD-L2 was not elevated. However, expression of PD-L1 was lower in the pseudohypoxia cluster compared to the sporadic and the kinase signaling subtype cluster, suggesting that sporadic and kinase signaling cluster PPGLs could benefit from PD-1/PD-L1 therapy more than the pseudohypoxia cluster. Within the pseudohypoxia cluster, expression of PD-L1 was significantly lower in both SDHB- and non-SDHB-mutated tumors compared to sporadic tumors. PD-L1 and PD-L2 expression was not affected by the metastatic status. We conclude that PD-L1 and PD-L2 expression in our cohort of PPGL tumors was not linked to metastatic behavior, however, the presence of PPGL driver mutation could be a predictive marker for PD-L1-targeted therapy and an important feature for further clinical studies in patients with PPGL.

PURPOSE:To understand prognostic immune cell infiltration signatures in neuroendocrine neoplasms (NENs), particularly pheochromocytoma and paraganglioma (PCPG), we analyzed tumor transcriptomic data from The Cancer Genome Atlas (TCGA) and other published tumor transcriptomic data of NENs.METHODS:We used CIBERSORT to infer immune cell infiltrations from bulk tumor transcriptomic data from PCPGs, in comparison to gastroenteropancreatic neuroendocrine tumors (GEPNETs) and small cell lung carcinomas (SCLCs). PCPG immune signature was validated with NanoString immune panel in an independent cohort. Unsupervised clustering of the immune infiltration scores from CIBERSORT was used to find immune clusters. A prognostic immune score model for PCPGs and the other NENs were calculated as a linear combination of the estimated infiltration of activated CD8+/CD4+ T cells, activated NK cells, and M0 and M2 macrophages.RESULTS:In PCPGs, we found five dominant immune clusters, associated with M2 macrophages, monocytes, activated NK cells, M0 macrophages and regulatory T cells, and CD8+/CD4+ T cells respectively. Non-metastatic tumors were associated with activated NK cells and metastatic tumors were associated with M0 macrophages and regulatory T cells. In GEPNETs and SCLCs, M0 macrophages and regulatory T cells were associated with unfavorable outcomes and features, such as metastasis and high-grade tumors. The prognostic immune score model for PCPGs and the NENs could predict non-aggressive and non-metastatic diseases. In PCPGs, the immune score was also an independent predictor of metastasis-free survival in a multivariate Cox regression analysis.CONCLUSION:The transcriptomic immune signature in PCPG correlates with clinical features like metastasis and prognosis.

Gliomas are the most common central nervous tumors in children and adolescents. However, spinal cord low-grade gliomas (sLGGs) are rare, with scarce information on tumor genomics and epigenomics. To define the molecular landscape of sLGGs, we integrated clinical data, histology, and multi-level genetic and epigenetic analyses on a consecutive cohort of 26 pediatric patients. Driver molecular alteration was found in 92% of patients (24/26). A novel variant of KIAA1549:BRAF fusion (ex10:ex9) was identified using RNA-seq in four cases. Importantly, only one-third of oncogenic drivers could be revealed using standard diagnostic methods, and two-thirds of pediatric patients with sLGGs required extensive molecular examination. The majority (23/24) of detected alterations were potentially druggable targets. Four patients in our cohort received targeted therapy with MEK or NTRK inhibitors. Three of those exhibited clinical improvement (two with trametinib, one with larotrectinib), and two patients achieved partial response. Methylation profiling was implemented to further refine the diagnosis and revealed intertumoral heterogeneity in sLGGs. Although 55% of tumors clustered with pilocytic astrocytoma, other rare entities were identified in this patient population. In particular, diffuse leptomeningeal glioneuronal tumors (n = 3) and high-grade astrocytoma with piloid features (n = 1) and pleomorphic xanthoastrocytoma (n = 1) were present. A proportion of tumors (14%) had no match with the current version of the classifier. Complex molecular genetic sLGGs characterization was invaluable to refine diagnosis, which has proven to be essential in such a rare tumor entity. Moreover, identifying a high proportion of drugable targets in sLGGs opened an opportunity for new treatment modalities.

BACKGROUND:Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors with frequent mutations in genes linked to the tricarboxylic acid cycle. However, no pathogenic variant has been found to date in succinyl-CoA ligase (SUCL), an enzyme that provides substrate for succinate dehydrogenase (SDH; mitochondrial complex II [CII]), a known tumor suppressor in PPGL. METHODS:A cohort of 352 patients with apparently sporadic PPGL underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health, including the SUCLG2 subunit of SUCL. Gene deletion, succinate levels, and protein levels were assessed in tumors where possible. To confirm the possible mechanism, we used a progenitor cell line, hPheo1, derived from a human pheochromocytoma, and ablated and re-expressed SUCLG2. RESULTS:We describe 8 germline variants in the guanosine triphosphate-binding domain of SUCLG2 in 15 patients (15 of 352, 4.3%) with apparently sporadic PPGL. Analysis of SUCLG2-mutated tumors and SUCLG2-deficient hPheo1 cells revealed absence of SUCLG2 protein, decrease in the level of the SDHB subunit of SDH, and faulty assembly of the complex II, resulting in aberrant respiration and elevated succinate accumulation. CONCLUSIONS:Our study suggests SUCLG2 as a novel candidate gene in the genetic landscape of PPGL. Large-scale sequencing may uncover additional cases harboring SUCLG2 variants and provide more detailed information about their prevalence and penetrance.

Abstract INTRODUCTION: Paediatric high-grade gliomas (HGG) are characterised by the aggressive biological behaviour with dismal prognosis of long-term survival 10-15%. Current molecular-biological diagnostic approaches allow for more precise characterization and determination of new unique subgroups of HGG. Our aim was to identify novel and rare HGG subgroups within our institution cohort. PATIENTS AND METHODS: Our reference centre patients′ cohort consisted of 97 clinically annotated patients with HGG diagnosed between 2000 and 2021. Sanger sequencing was used for screening of the most common HGG-related oncogenic drivers; furthermore we employed whole genome methylation array (Illumina Infinium MethylationEPIC BeadChip) and for selected samples RNA sequencing and expression profiling. RESULTS: Based on H3 status and previous radiotherapy we separated our HGG cases into the RIG, H3mut and H3wt groups. In contrast to H3mut(n=35) and RIG(n=11) that were uniformly fatal, H3wt group contained a proportion of long-term survivors. In the H3wt group we found patients carrying driver mutations in IDH1/2 (n=2) and BRAFV600E (7). Five young patients (under 3) consisted of 3 infant hemispheric gliomas (with NTRK and ROS1 fusions), one gliomatosis cerebri and one brainstem anaplastic astrocytoma with MYB/QKI fusion. We also identified a rare EWSR1-PATZ1 gene fusion in one patient. Importantly, long-term survivors recruited from these subgroups. On the contrary, four cases of MYCN GBM with poor prognosis presented in various locations: one disseminated, one gliomatosis cerebri and two with hemispheric tumour. We identified one patient with “hypermutated” glioblastoma and used targeted therapy with Nivolumab. In three samples of our patients with thalamic glioblastomas, we detected “loss of H3K27-trimethylation” caused by EZHIP overexpression. These tumours proved to be very aggressive with early metastatic recurrence and dismal prognosis. SUMMARY: Detailed characterization of H3 wild-type HGG is very important for further understanding of their biological behaviour, diagnostics, prognostication and identification of therapeutic targets.

Background Metastatic pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors associated with poor prognosis and limited therapeutic options. Recent advances in oncology-related immunotherapy, specifically targeting of the programmed death 1 (PD-1)/-ligand 1 (PD-L1) pathways, have uncovered new treatment potential for a variety of tumors. The expression of PD-L1 and PD-L2 was recently found to be present in 18% and 16% of PPGL, respectively, but only PD-L2 expression correlated with malignancy, hypoxia markers, and shorter survival.1 However, PD-L1 was suggested to be a malignant proliferation biomarker for PPGLs in another study.2 Given the promising outcomes of a clinical study in 9 cases of PPGL using pembrolizumab, a humanized IgG4κ monoclonal antibody that targets the PD-1/PD-L1 pathway,3 we examined the PD-Ls expression in our representative PPGL cohort to explore if PD-Ls expression can predict malignancy and/or be a predictive marker for PD-Ls targeted therapy in PPGL. Methods The Cancer Genome Atlas (TCGA) provided 173 patient samples to allow for observation of gene expression across four PPGL driver mutation groups (Cluster I: SDHB, VHL; Cluster II: NF1, RET) and NAM samples. Tumor RNA from the 48-patient cohort (sporadic; Cluster I: SDHB, VHL, EPAS1; Cluster II: RET, NF1) was evaluated to validate the results. Results Expression of PD-L1, but not PD-L2, was elevated in our PPGL cohort, which aligns with TCGA analysis. Expression of PD-L1 was decreased in Cluster I PPGLs but not in Cluster II, suggesting that sporadic and Cluster II PPGLs could benefit from PD-1/PD-L1 targeted therapy more than Cluster I PPGL tumors. Within Cluster I, expression of PD-L1 was significantly lower in SDHB- and VHL-mutated tumors compared to sporadic tumors. Expression of PD-L2 did not differ between PPGL clusters. Metastatic PPGLs had significantly elevated Ki-67 levels, however PD-Ls expression was not affected by malignancy status. Conclusions We conclude that PD-Ls expression in our cohort of PPGL tumors was not linked to malignancy, however, driver mutation analysis could be a selective marker for PD-Ls-targeted therapy. References Pinato DJ, Black JR, Trousil S, Dina RE, Trivedi P, Mauri FA, Sharma R. Programmed cell death ligands expression in phaeochromocytomas and paragangliomas: Relationship with the hypoxic response, immune evasion and malignant behavior. Oncoimmunology. 2017;6(11):e1358332 Guo D, Zhao X, Wang A, Xie Q, Xu X, Sun J. PD-L1 expression and association with malignant behavior in pheochromocytomas/paragangliomas. Hum. Pathol. 2019; 86:155–162. Naing A, Gainor JF, Gelderblom H, Forde PM, Butler MO, Lin CC, Sharma S, Ochoa de Olza M, Varga A, Taylor M, Schellens JHM, Wu H, Sun H, Silva AP, Faris J, Mataraza J, Cameron S, Bauer TM. A first-in-human phase 1 dose escalation study of spartalizumab (PDR001), an anti-PD-1 antibody, in patients with advanced solid tumors. J Immunother Cancer. 2020; 8(1):e000530. Ethics Approval The study protocol was approved by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Institutional Review Board (NIH Protocol 00-CH-0093). Consent All patients provided written informed consent approved by the National Institutes of Health ethics committee.

Abstract Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors derived from neural crest cells that are frequently linked to mutations including those in Krebs cycle enzymes, particularly succinate dehydrogenase (SDH). Succinyl-CoA ligase (SUCL) catalyzes reversible conversion of succinyl-CoA to succinate providing the substrate for SDH. While mitochondrial diseases were documented for the mutations in SUCL subunits G1 and A2, the association of GDP/GTP-specific subunit SUCLG2 mutations with specific pathologies including cancer have not been reported. In our study, 352 patients with apparently sporadic PPGLs underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health. Additionally, human pheochromocytoma (hPheo1) cells were used for gene manipulation to produce SUCLG2 knock-out (KO). Tumor tissues and hPheo1 SUCLG2 KO cells were used for further analysis focusing on mechanism of germline variants effect on mitochondrial functions. We detected eight germline SUCLG2 mutations in 15 patients which represents 4.3% of the cohort. Germline variants together with LOH led to decreased levels of SDH subunit B resulting in aberrant respiration and accumulation of succinate, well recognized oncometabolite. Manipulation of SUCLG2 in hPheo1 cells confirmed decrease in SDHB leading to faulty assembly of mitochondrial complex II and alteration of its respiration and activity. In summary, our study identified an association between SUCLG2 and PPGL. Larger scale sequencing and uncovering additional cases bearing SUCLG2 variants will further clarify the relationship between SUCLG2 and SDHx, particularly SDHB, as well as their role in disease etiology.