Low Mutational Load Research Articles

Vaccination with Designed Neopeptides Induces Intratumoral, Cross-reactive CD4+ T-cell Responses in Glioblastoma.

The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.

HL-370 Fragmentóme Profiling Reveals Distinct Molecular Subgroups of Classic Hodgkin Lymphoma

Classic Hodgkin lymphoma (cHL) is a phenotypically heterogeneous disease with enigmatic biology and pathogenesis. By leveraging ctDNA analysis, here we identify molecular groups of cHL with phenotype- and outcome-associated signatures. Identification and biological characterization of novel cHL subgroups. We sequenced 202 cHL patients targeting ~344 kb of genomic space. The length of ctDNA mapped reads was extracted, and unsupervised clustering was performed. PET/CT scan and 71 plasma cytokines were obtained for clinical correlation. Single-cell RNA sequencing was performed for 8 patients. To identify molecular subgroups within cHL, we focused on the fragmentation profile of cfDNA, a non-genetic way of detecting tumor-derived DNA in the cfDNA samples. We identified two distinct clusters, one with a fragmentation profile close to healthy (N=135), named mono-nucleosomal, and a submono-nucleosomal cluster characterized by a global shift toward shorter fragments, whose length was less than the typical wrap around the nucleosome (N=67). We explored whether cHL subtypes defined by the cfDNA fragmentation profile are clinically and biologically validated. Patients with the submono-nucleosomal fragmentome showed more frequent advanced-stage B symptoms and elevated ESR at the clinical level, higher tumor volume (TMTV and TLG) at the radiomic level, and lower PFS in terms of outcome. Tumor mutations and immune microenvironment are pathophysiologic features of cHL. The submono-nucleosomal cluster carried a higher tumor mutation burden because of heavier aberrant somatic hypermutation (ASHM). The submono-nucleosomal cluster had an immune-suppressive microenvironment enriched of T-regs and higher plasmatic levels of T-regs chemoattractants and inducers of PD1 expression (IL6, CCL2, CCL4). Conversely, mono-nucleosomal cHL had a microenvironment enriched of TFH cells and cytotoxic CD8 TEM cells and associated with cytokines and chemokines that are known for being attractants and activators of effector T cells (IL-1a, CCL21, CXCL12, IL-17A). Our observations indicate that cHL subgroups belonging to the submono-nucleosomal cluster have a more aggressive disease, a higher mutation load and more neoantigens likely due to denser ASHM, and an immune-suppressive microenvironment. Instead, cHL subgroups belonging to the mono-nucleosomal cluster have a less aggressive disease, a lower mutation load and lower neoantigens likely due to lighter ASHM, and a tumor-suppressive microenvironment.

Sexual selection for males with beneficial mutations

Sexual selection is the process by which traits providing a mating advantage are favoured. Theoretical treatments of the evolution of sex by sexual selection propose that it operates by reducing the load of deleterious mutations. Here, we postulate instead that sexual selection primarily acts through females preferentially mating with males carrying beneficial mutations. We used simulation and analytical modelling to investigate the evolutionary dynamics of beneficial mutations in the presence of sexual selection. We found that female choice for males with beneficial mutations had a much greater impact on genetic quality than choice for males with low mutational load. We also relaxed the typical assumption of a fixed mutation rate. For deleterious mutations, mutation rate should always be minimized, but when rare beneficial mutations can occur, female choice for males with those rare beneficial mutations could overcome a decline in average fitness and allow an increase in mutation rate. We propose that sexual selection for beneficial mutations could overcome the ‘two-fold cost of sex’ much more readily than choice for males with low mutational load and may therefore be a more powerful explanation for the prevalence of sexual reproduction than the existing theory. If sexual selection results in higher fitness at higher mutation rates, and if the variability produced by mutation itself promotes sexual selection, then a feedback loop between these two factors could have had a decisive role in driving adaptation.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Abstract 5764: Genomic profiling of cutaneous, acral and mucosal melanomas in Chinese populations

Abstract Background: Targeted therapies and immunotherapies have significantly improved the survival of patients with cutaneous melanoma. Nevertheless, patients with acral and mucosal melanomas show limited benefit. The proportion of acral and mucosal subtypes are much higher in Chinese populations than Caucasians. Therefore, it is of great interest to detail the genomic features of acral and mucosal melanomas and compare these with cutaneous melanoma. Methods: A total of 111 Chinese patients with melanoma treated in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were enrolled in this study, comprising 32 cutaneous(28.8%), 41 acral(36.9%) and 26 mucosal(23.4%) subtypes. NGS-based genomic profiling of major cancer-related genes was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens with matched blood samples. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. Results: The most commonly mutated genes of cutaneous melanoma include BRAF(48%), CDKN2A(41%), CDKN2B(41%), NRAS(22%) and PTEN(22%). In acral melanoma patients, the mutation rate of BRAF was 10% while NRAS(31%), CDKN2A(21%), EMSY(21%), KIT(18%), CCND1(18%)) and NF1(18%) were frequently mutated. Mucosal melanomas were found most commonly to have mutations in CDKN2A(36%), KIT(32%), CDKN2B(28%), PDGFRA(24%), KDR(20%) and BRAF(16%). In acral and mucosal subtypes, the overall mutation rates of BRAF were significantly lower while the relative proportions of non-V600 mutations were higher as compared to cutaneous subtype. These may explain the less significant survival benefit of BRAFi+/-MEKi in acral and mucosal melanomas. In contrast, KIT mutations including SNVs and amplification, were enriched in acral and mucosal melanomas. Acral melanomas had lower mutation load than mucosal melanomas(2.42 Muts/Mb vs 4.84 Muts/Mb, p=0.0002), while cutaneous melanomas(4.03 Muts/Mb) did not differ from other subtypes in mutation burden. Additionally, patients with TERT, APC and FAT1 gene mutations had a significantly higher TMB than patients with wild-type genes respectively, indicating more activated anti-tumor immune profiles and suggesting therapeutic benefit from immune checkpoint inhibitors. Conclusions: We conducted a direction comparison of comprehensive genomic profiles of different melanoma subtypes among Chinese patients. The genomic alterations identified in our study may provide reference to discover potential strategies for targeted therapy and immunotherapy in acral and mucosal melanomas. Citation Format: Jing Chen, Ting Ye, Li Fan, Jieying Zhang, Rubo Cao, Yanshen Liu. Genomic profiling of cutaneous, acral and mucosal melanomas in Chinese populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5764.

Phase I results of the INFORM2 combination study of nivolumab and entinostat in children and adolescents: INFORM2 NivEnt.

10034 Background: Pediatric patients with relapsed or refractory high-risk solid and CNS tumors have dismal survival. To date treatment with immune checkpoint inhibitors in this population has been disappointing. This study exploits the immune enhancing effects of entinostat on nivolumab in biomarker enriched subpopulations. The study aims to determine the pediatric recommended phase II dose (pRP2D) and to evaluate activity and safety. Methods: This is an exploratory non-randomized, open-label, multinational seamless phase I/II trial in children and adolescents with relapsed / refractory or progressive high-risk solid and CNS tumors. The phase I is divided in 2 age cohorts: 12–21 years (y) and 6–11y and follows a 3 + 3 design with two dose levels for entinostat (dose level 1: 2 mg/m2 and dose level 2: 4 mg/m2 once per week) and fixed dose nivolumab (3 mg/kg every 2 weeks). Patients entering the trial on pRP2D can seamlessly enter phase II which consists of a biomarker defined four group basket trial: high mutational load (group A), high PD-L1 mRNA expression (group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (group D). Results: The first patient was enrolled in May 2020 and at the time of the data cut (21-JAN-2022), 19 patients were treated. The median age at enrollment was 14 y. In the 12 – 21y cohort 15 patients were enrolled and four patients in the 6 – 11y cohort. The most frequent treatment-related AEs to date were thrombocytopenia in six (32%), nausea and vomiting both in four (21%), and neutropenia in three patients (16%). Five patients (26%) experienced grade 3/4 mostly reversible treatment-related AEs, e.g. neutropenia/leukopenia. No treatment related deaths were reported. In the 6 – 11y cohort dose escalation is ongoing. In the 12 – 21y cohort, one DLT (CTCAE grade 3 thrombocytopenia) was observed in six patients on dose level two, which was determined as the pRP2D of the combination. At the time of the data cut, 10 patients (six in arm D and four patients in which the biomarker group was not yet determined) had received at least one RECIST/RANO response evaluation by central review in phase II. One patient (17%) in arm D with metastatic relapsed renal cell carcinoma (RCC) harboring a typical PRCC-TFE3 fusion showed a PR after two cycles and finally achieved an ongoing CR. Extensive explorative analyses of immune signatures derived from INFORM RNA-Seq and WES data revealed that both the primary diagnosis and the current relapse samples harbored a remarkable high immune cell infiltration, especially CD8+ T-cells. Conclusions: The first and ongoing global INFORM2 trial has identified the pRP2D for the nivolumab and entinostat combination in the older age cohort with good tolerability. A patient with metastasized relapsed RCC experienced a CR. The role of immune infiltration as a potential predictive biomarker is currently being explored. Clinical trial information: NCT03838042.

Novel insights based on the plasticity of γδ T cells in the tumor microenvironment

γδ T cells express unique T cell receptor (TCR) γ and TCR δ chains, with structural and functional heterogeneity. Taking advantage of the diverse γδ TCR repertoire or other ligand-receptor interactions, γδ T cells can recognize a broad spectrum of tumor-associated antigens (TAAs) in a major histocompatibility complex (MHC)-independent manner, thereby activating downstream pleiotropic effects. γδ T cells recruited into the tumor microenvironment can act as effector cells to mediate cancer immune surveillance. Their advantage lies in the ability to perceive tumors with a low mutation load, thus establishing the first line of defense against pathogens. Activated γδ T cells exhibit strong cytotoxic activity and cytokine secretion functions and are effective antitumor lymphocytes with simple and direct recognition modes and rapid responses. However, the clinical application of tumor-infiltrating γδ T cells has certain limitations. First, γδ T cells exposed to complicated cytokine networks are potentially affected by multiple inhibitory mechanisms. Additionally, these cells show highly flexible and dynamic plasticity and are extremely easily polarized into regulatory phenotypes. This review further emphasizes the diversified cross-talk between γδ T cells and other immune cells. Effective immunity of the body is often manifested by counterbalance under mutual restriction. Therefore, an in-depth understanding of γδ T cells that play conflicting roles in the tumor microenvironment is necessary. These cells may be a key factor ultimately mediating the deviation of the antagonistic response between tumor inhibition and tumor promotion. Finally, it retrospectively analyze the activation strategies and clinical relevance of existing γδ T cell adoptive immunotherapies. According to current challenges, there is a need to explore innovative immunotherapies, maximize the tumor-killing efficacy of γδ T cells, and attenuate or eliminate tumor immunosuppression. It is hoped that the host immune status can be accurately predicted and gradually advance γδ T cell precise individualized medicine.

Pro-mutagenic effects of the gut microbiota in a Lynch syndrome mouse model

ABSTRACT The gut microbiota strongly impacts the development of sporadic colorectal cancer (CRC), but it is largely unknown how the microbiota affects the pathogenesis of mismatch-repair-deficient CRC in the context of Lynch syndrome. In a mouse model for Lynch syndrome, we found a nearly complete loss of intestinal tumor development when animals were transferred from a conventional “open” animal facility to specific-pathogen-free (SPF) conditions. Using 16S sequencing we detected large changes in microbiota composition between the two facilities. Transcriptomic analyses of tumor-free intestinal tissues showed signs of strong intestinal inflammation in conventional mice. Whole exome sequencing of tumors developing in Msh2-Lynch mice revealed a much lower mutational load in the single SPF tumor than in tumors developing in conventional mice, suggesting reduced epithelial proliferation in SPF mice. Fecal microbiota transplantations with conventional feces altered the immune landscape and gut homeostasis, illustrated by increased gut length and elevated epithelial proliferation and migration. This was associated with drastic changes in microbiota composition, in particular increased relative abundances of different mucus-degrading taxa such as Desulfovibrio and Akkermansia, and increased bacterial-epithelial contact. Strikingly, transplantation of conventional microbiota increased microsatellite instability in untransformed intestinal epithelium of Msh2-Lynch mice, indicating that the composition of the microbiota influences the rate of mutagenesis in MSH2-deficient crypts.

Low Mutational Burden of Extranodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue in Patients with Primary Sjogren's Syndrome.

Simple SummaryPatients with primary Sjogren’s syndrome (pSS) are at risk of developing extranodal marginal zone lymphoma (ENMZL) of the mucosa-associated lymphoid tissue (MALT) in the parotid glands. The genetic mechanism underlying development of MALT lymphoma in the context of pSS is unknown. The aim of our study was to define the genomic landscape of pSS-associated MALT lymphoma. For 17 localized pSS-associated MALT lymphomas, we analyzed the presence of nonsynonymous mutations, copy number alterations (CNAs) and MALT1 translocations. pSS-associated MALT lymphomas were characterized by a low mutational load (median number of nonsynonymous somatic variants per case was 7, range 2–78) and a limited number of CNAs. Unlike the recurrent genomic aberrations observed in MALT lymphoma, which were not associated with pSS, pSS-associated MALT lacked a clear lymphoma-related profile. The data suggest that localized pSS-associated MALT lymphomas are a distinct type of ENMZL, which are genomically stable and most likely depend on a stimulatory micro-environment.Patients with primary Sjogren’s syndrome (pSS) are at risk of developing extranodal marginal zone lymphoma (ENMZL) of the mucosa-associated lymphoid tissue (MALT) in the parotid glands. Unlike recurrent genomic aberrations observed in MALT lymphoma, which were not associated with pSS (non-pSS), it is unknown which somatic aberrations underlie the development of pSS-associated MALT lymphomas. Whole-exome sequencing was performed on 17 pSS-associated MALT lymphomas. In total, 222 nonsynonymous somatic variants affecting 182 genes were identified across the 17 cases. The median number of variants was seven (range 2–78), including three cases with a relatively high mutational load (≥24/case). Out of 16 recurrently mutated genes, ID3, TBL1XR1, PAX5, IGLL5 and APC are known to be associated with lymphomagenesis. A total of 18 copy number alterations were detected in eight cases. MALT1 translocations were not detected. With respect to outcome, only two cases relapsed outside of the salivary glands. Both had a high mutational load, suggesting a more advanced stage of lymphoma. The low mutational load and lack of a clear lymphoma-related mutation profile suggests that localized pSS-associated MALT lymphomas are genomically more stable than non-pSS MALT lymphomas and most likely depend on a stimulatory micro-environment.

A Network-Centric Framework for the Evaluation of Mutual Exclusivity Tests on Cancer Drivers.

One of the key concepts employed in cancer driver gene identification is that of mutual exclusivity (ME); a driver mutation is less likely to occur in case of an earlier mutation that has common functionality in the same molecular pathway. Several ME tests have been proposed recently, however the current protocols to evaluate ME tests have two main limitations. Firstly the evaluations are mostly with respect to simulated data and secondly the evaluation metrics lack a network-centric view. The latter is especially crucial as the notion of common functionality can be achieved through searching for interaction patterns in relevant networks. We propose a network-centric framework to evaluate the pairwise significances found by statistical ME tests. It has three main components. The first component consists of metrics employed in the network-centric ME evaluations. Such metrics are designed so that network knowledge and the reference set of known cancer genes are incorporated in ME evaluations under a careful definition of proper control groups. The other two components are designed as further mechanisms to avoid confounders inherent in ME detection on top of the network-centric view. To this end, our second objective is to dissect the side effects caused by mutation load artifacts where mutations driving tumor subtypes with low mutation load might be incorrectly diagnosed as mutually exclusive. Finally, as part of the third main component, the confounding issue stemming from the use of nonspecific interaction networks generated as combinations of interactions from different tissues is resolved through the creation and use of tissue-specific networks in the proposed framework. The data, the source code and useful scripts are available at: https://github.com/abu-compbio/NetCentric.

TMOD-14. CONJOINED CLASS I AND II EPITOPES ENHANCE NEOANTIGEN-TARGETED ACTIVE IMMUNOTHERAPY

Abstract INTRODUCTION Cancer vaccines involve the administration of tumor-associated antigens into the host to generate anti-tumor T cell responses. Glioblastoma (GBM) is a disease with poor prognosis. GBM has a limited number of immunotherapeutic targets due to low mutational load, and is also highly heterogeneous; targeting a single antigen leads to antigen escape and tumor growth. METHODS VMDK mice were subcutaneously implanted with 750,000 SMA560 cells and on days 1 and 8 post implantation, mice were treated with 15 nmol of the universal helper epitope, P30, conjoined to the MHCI-restricted neoepitopes Odc1MHCI-P30 or Topbp1MHCI-P30. Human CD27 (hCD27) transgenic mice were intracranially implanted with CT2A-Odc1, followed by anti-CD27 and 15 nmol of Odc1MHCI-P30. B16.OVA or B16.F10 tumor cells were intracranially implanted in hCD27 mice and received SIINFEKL-P30 or Trp2-P30 conjoined peptides. Tumor growth, survival, or IFNγ secretion of splenic or tumor-infiltrating cells was assessed. RESULTS Unlike Odc1MHCI mixed with P30, conjoined Odc1MHCI-P30 had equivalent immunogenicity and anti-tumor efficacy to that observed with native long Odc1 peptide. Native long peptide of Topbp1 did not elicit an antitumor response, yet Topbp1MHCI-P30 caused an increase in numbers of IFNγ-secreting splenocytes and a decrease in tumor growth and similar to that seen with Odc1MHCI-P30 . Anti-CD27 treatment significantly increased numbers of IFNγ secreting splenocytes in Odc1MHCI-P30 vaccinated hCD27 mice, and the use of anti-CD27 with Odc1MHCI-P30 achieved long-term survival in 90% of tumor bearing hCD27 mice. Anti-CD27 synergized with SIINFEKL-P30 and Trp2-P30 to significantly improve survival after administration of these peptides. CONCLUSIONS Our work shows that poorly immunogenic neoantigens can be conjoined to P30 and used to generate an anti-tumor response in mouse models of GBM, and anti-tumor responses of conjoined peptides can be enhanced with anti-CD27 treatment. Together, these data demonstrate the efficacy of neoantigen vaccines for the treatment of heterogeneous GBM.

Low Mutational Burden of Extra Nodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue in Patients with Primary Sjogren's Syndrome

Background: Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of the exocrine glands. Patients with pSS are at increased risk of developing extra nodal marginal zone lymphoma of the mucosa-associated lymphoid tissue (MALT) type, predominantly in the salivary glands. The presence of cryoglobulinemia, low C4 levels and salivary gland enlargement, are important risk factors for lymphoma development in pSS. Although lymphoma associated mutations have been described in circulating B-cells of patients with pSS associated cryoglobulinemia, it is unknown which somatic aberrations underlie the development of pSS-associated MALT lymphomas. The aim of the current study was to define the genomic landscape of pSS salivary gland MALT lymphomas. Identification of specific recurrent mutations or copy number aberrations and defining the affected pathways would help to understand the mechanisms by which intra-epithelial B-cells undergo malignant transformation.Methods: Whole exome sequencing was performed on 14 fresh frozen and 3 paraffin embedded MALT tissue samples of pSS patients at the University Medical Center Groningen (UMCG). Matched peripheral white blood cell samples were available for 12 patients and were used as germline controls. Fluorescence in situ hybridization was performed for the detection of MALT1 translocations.Results: Presence of a clonal B-cell population, indicative of a MALT lymphoma was confirmed by IgH PCR (BIOMED-2) for all patients. Whole exome sequencing resulted in a median target coverage of 130x, ranging from 84-163. More than 90% of the target regions had a coverage of >50x. In total we identified 232 somatic mutations in 184 genes. Three cases had a relatively high mutational load (>25 / case), while the median number of mutations in the remaining 14 cases was 7. Across the 17 cases there were 18 recurrently mutated genes. PRKD1, associated with malignant epithelial tumor of the salivary glands, was the most frequently mutated gene (six cases). Besides PRKD1, five additional recurrently mutated genes are also involved in epithelial surface and/or extracellular matrix (MAMDC4, COL14A1, CAMSAP3, TMEM2 and MUC4). Five genes, all with mutations in two cases, were shown to be associated with lymphomagenesis (ID3, TBL1XR1, PAX5, IGLL5, APC). A total of 18 copy number alterations (CNAs) were detected in 8 of the 14 evaluable cases, with gains of part of chromosomes 2 and 19, and loss of chromosome 6 each being detected in two cases. With respect to outcome, only 2 cases with high mutational load relapsed outside of the salivary glands, suggesting a more advanced stage of lymphoma.Conclusion: The low mutational load and lack of a clear lymphoma related gene signature suggests that localized pSS MALT lymphomas are genetically stable and most likely depend on the inflammatory state of the micro-environment. Only those cases characterized by higher mutational load showed a relapse-remitting disease, as is typical for indolent lymphoma. These observations could be translated to the clinical setting and potentially have value as biomarker for identification of patients with a more aggressive disease. Confirmation in larger cohorts are required to confirm this potential association. This study also paves way for treatment strategies targeting the micro-environment. DisclosuresNo relevant conflicts of interest to declare.

The TFAM-to-mtDNA ratio defines inner-cellular nucleoid populations with distinct activity levels.

In human cells, generally a single mitochondrial DNA (mtDNA) is compacted into a nucleoprotein complex denoted the nucleoid. Each cell contains hundreds of nucleoids, which tend to cluster into small groups. It is unknown whether all nucleoids are equally involved in mtDNA replication and transcription or whether distinct nucleoid subpopulations exist. Here, we use multi-color STED super-resolution microscopy to determine the activity of individual nucleoids in primary human cells. We demonstrate that only a minority of all nucleoids are active. Active nucleoids are physically larger and tend to be involved in both replication and transcription. Inactivity correlates with a high ratio of the mitochondrial transcription factor A (TFAM) to the mtDNA of the individual nucleoid, suggesting that TFAM-induced nucleoid compaction regulates nucleoid replication and transcription activity invivo. We propose that the stable population of highly compacted inactive nucleoids represents a storage pool of mtDNAs with a lower mutational load.

Comprehensive of N1-Methyladenosine Modifications Patterns and Immunological Characteristics in Ovarian Cancer.

Backgroundrecently, many researches have concentrated on the relevance between N1-methyladenosine (m1A) methylation modifications and tumor progression and prognosis. However, it remains unknown whether m1A modification has an effect in the prognosis of ovarian cancer (OC) and its immune infiltration.MethodsBased on 10 m1A modulators, we comprehensively assessed m1A modification patterns in 474 OC patients and linked them to TME immune infiltration characteristics. m1Ascore computed with principal component analysis algorithm was applied to quantify m1A modification pattern in OC patients. m1A regulators protein and mRNA expression were respectively obtained by HPA website and RT-PCR in clinical OC and normal samples.ResultsWe finally identified three different m1A modification patterns. The immune infiltration features of these m1A modification patterns correspond to three tumor immune phenotypes, including immune-desert, immune-inflamed and immune-excluded phenotypes. The results demonstrate individual tumor m1A modification patterns can predict patient survival, stage and grade. The m1Ascore was calculated to quantify individual OC patient’s m1A modification pattern. A high m1Ascore is usually accompanied by a better survival advantage and a lower mutational load. Research on m1Ascore in the treatment of OC patients showed that patients with high m1Ascore showed marked therapeutic benefits and clinical outcomes in terms of chemotherapy and immunotherapy. Lastly, we obtained four small molecule drugs that may potentially ameliorate prognosis.ConclusionThis research demonstrates that m1A methylation modification makes an essential function in the prognosis of OC and in shaping the immune microenvironment. Comprehensive evaluation of m1A modifications improves our knowledge of immune infiltration profile and provides a more efficient individualized immunotherapy strategy for OC patients.

Landscape of Immune-Related Markers and Potential Therapeutic Targets in Soft Tissue Sarcoma.

Simple SummaryDespite being a group of rare diseases of mesenchymal origin, soft tissue sarcomas are heterogenous and display varying clinical behavior, and depending on the subtype, intermediate- and high-grade sarcomas have significant metastatic potential, making it difficult to establish a standardized therapy. Our work, as well as studies by others, emphasizes the high potential of immunotherapy for the treatment of sarcoma. The aim of this study was to determine whether specific genomic alterations, as well as the expression of infiltrating cytotoxic and suppressive cell type markers identified by next-generation sequencing (NGS), warrant further consideration of immunotherapy agents for treating certain soft tissue sarcoma subtypes. Altogether, our data provide a better understanding of the immune composition of different sarcoma subtypes to better identify novel therapy targets.Soft tissue sarcomas, depending on the subtype and grade, frequently recur and become metastatic after localized treatment. There is now great interest in applying immunotherapy to sarcomas to immuno-profile the different subtypes and immune monitor for prognosis. Our group previously showed that key immunotherapy target genes are present in sarcomas. Here, we extend our findings by demonstrating that sarcomas with a relatively high mutational load are likely to be more sensitive to immunotherapy compared to sarcomas with a lower mutation load. We also show that sarcomas with a higher mutation load are associated with the expression of key immune-related genes. We found that CD8+ T cells are present in sarcoma subtypes and that PD-L2 is highly expressed. These findings further define potential mechanisms behind the immunotherapy response of specific sarcoma subtypes and can be used to develop more optimal treatments in the future.

Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors

BackgroundAtypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as...

Combining Algorithms to Find Signatures That Predict Risk in Early-Stage Stomach Cancer.

This study applied two mathematical algorithms, lattice up-stream targeting (LUST) and D -basis, to the identification of prognostic signatures from cancer gene expression data. The LUST algorithm looks for metagenes, which are sets of genes that are either overexpressed or underexpressed in the same patients. Whereas LUST runs unsupervised by clinical data, the D -basis algorithm uses implications and association rules to relate gene expression to clinical outcomes. The D -basis selects a small subset of the metagene (a signature) to predict survival. The two algorithms, LUST and D-basis, were combined and applied to mRNA expression and clinical data from The Cancer Genome Atlas (TCGA) for 203 stage 1 and 2 stomach cancer patients. Two small (four-gene) signatures effectively predict survival in early-stage stomach cancer patients. These signatures could be used as a guide for treatment. The first signature (DU4) consists of genes that are underexpressed on the long-survival/low-risk group: FLRT2, KCNB1, MYOC, and TNXB. The second signature consists of genes that are overexpressed on the short-survival/high-risk group: ASB5, SFRP1, SMYD1, and TACR2. Another nine-gene signature (REC9) predicts recurrence: BNC2, CCDC8, DPYSL3, MOXD1, MXRA8, PRELP, SCARF2, TAGLN, and ZNF423. Each patient is assigned a score that is a linear combination of the expression levels for the genes in the signature. Scores below a selected threshold predict low-risk/long survival, whereas high scores indicate a high risk of short survival. The metagenes associate with TCGA cluster C1. Both our signatures and cluster C1 identify tumors that are genomically silent, and have a low mutation load or mutation count. Furthermore, our signatures identify tumors that are predominantly in the WHO classification of poorly cohesive and the Lauren class of diffuse samples, which have a poor prognosis.

Overcoming Leukemia-Induced Immune Suppression in BCR-ABL+ Acute Lymphoblastic Leukemia

BCR-ABL+ acute lymphoblastic leukemia patients have transient responses to current therapies. However, the fusion of BCR to ABL generates a potential leukemia-specific antigen that could be a target for immunotherapy. Herein we demonstrate that the immune system can limit BCR-ABL+ leukemia progression although ultimately this immune response fails. To address how BCR-ABL+ leukemia escapes immune surveillance, we developed a peptide: MHC-II tetramer that labels endogenous BCR-ABL-specific CD4+ T cells. Naïve mice have a small population of BCR-ABL-specific T cells that proliferate modestly upon immunization with the BCR-ABL fusion peptide (referred to as BAp hereafter). In response to BCR-ABL+ leukemia, BCR-ABL specific T cells proliferated and converted into regulatory T cells (Treg cells), a process that was dependent on MHC-II antigen presentation by leukemic cells. Treg cells were critical for leukemia progression in C57Bl/6 mice, as transient Treg cell ablation led to extended survival of leukemic mice. Leukemic B cells produced significantly more TGb1, IL10 and PDL1 than control non-transformed B cells. We are currently extending these studies to determine if leukemia-specific production of TGFb, IL10, or PDL1 plays a critical role in driving tolerance induction. Thus, BCR-ABL+ leukemia actively suppresses the anti-leukemia immune responses by converting cross-reactive leukemia-specific T cells into Treg cells. To determine if we could reverse tolerance we examined whether checkpoint blockade or therapeutic vaccination with BAp peptide could improve survival in mice with pre-established BCR-ABL+ leukemia. Consistent with the low mutation load in our leukemia model, we found that checkpoint blockade with anti-PDL1 and CTLA-4 alone had only modest effects on survival. In contrast, robust heterologous vaccination using LCMV, Listeria, and VSV infection plus a peptide derived from the BCR-ABL fusion (BAp), a key driver mutation, resulted in a small population of mice that survived long-term. Checkpoint blockade strongly synergized with heterologous vaccination to enhance overall survival in mice with leukemia. Enhanced survival did not correlate with numbers of BAp:I-Ab-specific T cells, but rather with increased expression of IL10, IL17, and GrzmB and decreased expression of PD1 on these cells. Our findings demonstrate that vaccination to key driver mutations cooperates with checkpoint blockade and allows for immune control of cancers with low non-synonymous mutation loads. We are currently examining whether we can further improve survival via combination chemotherapy (using Nilotinib which typically only induces transient remission) and immunotherapy (using checkpoint blockade with anti-PD1 or anti-PDL1). DisclosuresFarrar:Merck: Research Funding.

Deep Learning Algorithms Support Distinction of PV, PMF, and ET Based on Clinical and Genetic Markers

Introduction: The WHO classification (2016) defines myeloproliferative neoplasms (MPN) according to cytomorphology, bone marrow biopsy, grading of fibrosis, blood counts and several molecular markers. Polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET) are separated from each other. However, overlaps and borderline findings occur. Entities also can transform into another subtype of MPN or even AML.Aim: To separate and stratify PV, PMF, and ET using blood counts, cytogenetics, standard molecular markers (JAK2, CALR, MPL) and a panel of further 15 genes all investigated by NGS supported by deep learning algorithms.Methods and Results: As a training cohort we used well characterized samples (n=243, all BCR-ABL1 negative) diagnosed with either PMF, PV, or ET based on morphology and JAK2, MPL, CALR mutation status following WHO criteria: 65 PMF, 70 PV, and 108 ET. Mutation status of another 15 recurrently mutated genes was available in all cases (ASXL1, CBL, DNMT3A, EZH2, IDH1, IDH2, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1). Further, in 207/243 cytogenetics was available. Based on these 243 cases an algorithm was trained using platelet and white blood cell counts, hemoglobin levels, mutation status of 18 genes, cytogenetics (normal or aberrant), and low versus high JAK2 mutational load (<35/≥35%). We used Support Vector Machines with class-probabilities output. Individual models were built for PMF, PV, and ET with 3-fold cross-validation, resulting in training errors of 8%, 9%, and 10%, respectively. Afterwards, the trained models were used to predict the most probable diagnosis of patients from a test cohort (n=183) retrieving the class probabilities for each patient (Fig. A). Open-set recognition was applied to correctly handle patients that were characterized by patterns that did not match any of the trained patterns. The latter patients received the class label “unknown”. A probability of ≥60% in one of the classes was the class defining argument. The test cases were classified following the WHO criteria: 24 PMF, 3 PV, 41 ET. However, if sufficient clinical parameters were missing cases were classified as “MPN” only if JAK2, CALR, or MPL were mutated (n=61). Cases without such a mutation were classified as “suspected MPN” (n=54).Overall, we reached an accuracy of 96%, showing only 7 cases with severe clinical discordant information (Fig. B). In total, 25 cases were classified as PMF, 16 as PV, 48 as ET, and 94 as “unknown” by clinical parameters and deep learning, respectively. For all patients the probabilities of classification to all four classes are given in Figure A. 88% of ET cases were classified correctly, with only 5 cases assigned to the “unknown” labeled group. 100% of PV patients were classified correctly without any misclassification at all. Classification of PMF cases was more challenging with a number of not assigned cases. However, the classification of the large group of 61 MPN patients not further specified by WHO criteria and morphology resulted in 25 assigned cases: 19 PMF, 3 PV, and 3 ET, illustrating the power of deep learning to support clinicians by making the diagnosis based on the genetic background of the patient only if some clinical parameters are missing. The last group of cases, where no valid diagnosis of a MPN was possible (cases without a MPN defining mutation), were - as expected - grouped mainly to the “unknown” class in 76% (41/54) of cases.Conclusions: Use of WHO criteria to classify MPN leads to the diagnosis of PV, PMF, or ET. Borderline cases can be categorized by adding more molecular markers. Deep learning algorithms can support decision making with a high accuracy even if clinical data is insufficient. In cases with incomplete data set or borderline features the application of such algorithms might assist diagnostic procedures in the near future. [Display omitted] DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Radiation-induced neoantigens broaden the immunotherapeutic window of cancers with low mutational loads

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use KrasG12D x p53-/- sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8+ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.