Low Somatic Mutation Research Articles

Unraveling the testicular seminoma tumor microenvironment by single-cell RNA-sequencing.

522 Background: Seminomas is a malignant germ cell tumor (GCT) affecting male adults and is characterized by low somatic mutation burden, histological homogeneity, and high survival rate. It is currently unknown what drives tumorigenesis of this curable testicular cancer. Tumor/immune microenvironment (TME) has a vital role in cancer survival and progression with implications in cell death resistance and immune escape. Herein, we aimed to unravel the transcriptomic TME landscape of seminomas and better understand seminoma pathogenesis. Methods: We performed single cell RNA-sequencing (scRNA-seq; 10X-Illumina platform) on surgically resected pure seminomas from four patients. A total of ~24,000 single cells were analyzed using Seurat/Bioconductor R packages and standard means for data visualization. Key findings were validated by in situ hybridization RNA analysis (RNAscope) on histological sections from these samples. Results: T-cells and Natural-Killer cell populations were mostly predominantly enriched in this seminoma cohort, where tumor cells consisted of a distinct subpopulation with limited presence (1%). Other distinct cell populations were also identified for additional immune cell types e.g., B-cells, macrophages/monocytes, endothelial and smooth muscle cells. ScRNA-seq revealed a unique set of signature marker genes (e.g., POU5F1, KHDC3L, DPPA5, NANOS3) highly expressed in seminoma cells which are associated with pivotal cellular processes e.g., developmental pluripotency and germ cell development. Notably, this gene set was also highly differentially expressed (DE) in The Cancer Genome Atlas (TGCA) seminomas (n=73) compared to TCGA non-seminomas (n=38) (FDR<0.01). Amongst top marker genes, KHDC3L, also higher expressed in seminomas compared to normal testis, was validated by RNA-scope. KHDC3L demonstrated the strongest positive correlation with DPPA5 (Spearman/Pearson; R2=0.8, p<0.0001), top #3 ranked DE gene as well as with other top 20 DE genes identified by scRNA-seq. Remarkably, similar strong correlations among other top DE genes were also found in the testicular TGCA cohort. Top DE genes including master transcription factors (e.g., POU5F1, NANOG, SOX4) are involved in the same protein pathway/networks likely suggesting synergic effect/involvement in the same regulatory circuitry. Gene Set Enrichment (GSEA) analyses demonstrated enrichment in immune signatures and pivotal pathways e.g., Wnt/beta catenin signaling and MYC targets. Conclusions: Our analyses revealed novel marker genes and immune signatures, uniquely expressed in seminomas which consist of a homogeneous cell population across all 4 tumors analyzed, clearly distinct from other cell types identified.

A Rare Case of CD1a +/CD207 + and S100 - Langerhans Cell Histiocytosis with Multi-System Risk Organ Involvement in Adult

Introduction: Langerhans Cell Histiocytosis (LCH) is a rare neoplastic disorder of myeloid dendritic cell precursors, leading to a spectrum of organ involvement and dysfunction. The nonspecific presentations result in missed or delayed diagnoses in adults, and data to guide the clinical care of adults with LCH is limited to case studies and series. Case Description: A 40-year-old Hispanic woman presented to the Emergency Department with ongoing symptoms of shortness of breath, muscle cramps, and intermittent chest pain for two weeks. She was found to have microcytic-hypochromic anemia requiring packed Red Blood Cells (pRBC) transfusion. A CT pulmonary angiogram showed incidental findings of splenomegaly and right axillary lymphadenopathy. A month before this presentation, she was evaluated for symptoms of polyuria, polydipsia, nocturia, and amenorrhea, ongoing for eight months. Arginine Vasopressin Deficiency (AVP-D) was diagnosed, and desmopressin was initiated. MRI Brain had revealed enhancement of the infundibular stalk. On follow-up in the clinic, an ultrasound of the right axilla showed a 2.4 cm lymph node (LN) with thickened cortex. Over the following month, she continued to require multiple pRBC transfusions. Further workup with Fluorodeoxyglucose (FDG)-Positron Emission Tomography (PET) revealed hypermetabolic splenomegaly with multifocal uptake, including the right axilla suspicious for metastatic disease. ( Figure 1-A) A biopsy of the right axillary LN ( Figure 2 - A, B) showed clusters of Langerhans cells and immunohistochemistry positive for CD1a, CD3, CD20, CD68, keratin, and CD 207 (Langerin) staining and variable positivity for S100. Next-Generation Sequencing showed BRAF V600E missense mutation and NOTCH3, DICER1, CREBBP, JAK1, and CTNNA1 as Variants of Unknown Significance (VUS). Bone marrow (BM) aspirate and flow cytometry were unremarkable. A diagnosis of Multi-System LCH with Risk Organ Involvement (MS-RO +) was made. Cytarabine therapy was started at 150 mg/m 2(days 1-5, 28-day cycle). She was hospitalized twice due to sepsis, leukocytosis, severe anemia: Hemoglobin (Hb) 3.1, and grade 4 thrombocytopenia. Cycle 2 was delayed, and the dose was reduced to 50%. Post-infusions, Hb decline necessitated multiple pRBC transfusions. Repeat FDG-PET after cycles 2 and 5 ( Figure 1-B, C) showed continued metabolic and anatomic response to therapy. A repeat brain MRI showed no change in pituitary findings. The patient continues to improve clinically with plans to complete a total of one year of chemotherapy. Discussion: LCH is defined by the presence of CD1a +/CD207 + histiocytes. Although usually positive for S-100, rare S-100 negative variants have been reported in skin-limited LCH and LC-Sarcoma. An aberrant maturation process was proposed as a possible explanation, but the exact pathology remains unknown. In children <15 years, the incidence is 2.6-8.9 cases/million/year; in adults, the incidence is less defined at 0.07 cases/million/year and is higher in Hispanics. Adult LCH typically presents after the fourth decade; two-thirds have multi-system involvement. Hematopoietic dysfunction, cytopenias, and liver involvement carry a poor prognosis. AVP-D is the most frequently associated endocrine abnormality. Whole-exome sequencing has shown very low somatic mutation frequency in LCH compared to other neoplastic disorders. BRAF, a central kinase in the RAS-RAF-MEK pathway, has a missense V600E mutation in roughly 50% of LCH, rendering it constitutionally active. Other mutations include BRAF, MAP2K1, ARAF, ERBB3, NRAS, and KRAS, but the VUS mentioned above have been reported infrequently. The LCH-III trial confirmed Vinblastine/Prednisone as the standard regimen for MS-LCH patients <18 years, with or without RO involvement. While it is effective in adults, its limited tolerance favors the use of cytarabine or cladribine. NCT02670707 is an ongoing trial comparing cytarabine monotherapy versus Vinblastine/Prednisone for frontline treatment. BRAF inhibitors are also being increasingly investigated. Prospective trials to optimize the duration of therapy (LCH-IV) and potential combination therapies are currently underway.

TSC2 inactivation, low mutation burden and high macrophage infiltration characterise hepatic angiomyolipomas.

Although TSC1 or TSC2 inactivating mutations that lead to mTORC1 hyperactivation have been reported in hepatic angiomyolipomas (hAML), the role of other somatic genetic events that may contribute to hAML development is unknown. There are also limited data regarding the tumour microenvironment (TME) of hAML. The aim of the present study was to identify other somatic events in genomic level and changes in TME that contribute to tumorigenesis in hAML. In this study, we performed exome sequencing in nine sporadic hAML tumours and deep-coverage targeted sequencing for TSC2 in three additional hAML. Immunohistochemistry and multiplex immunofluorescence were carried out for15 proteins to characterise the tumour microenvironment and assess immune cell infiltration. Inactivating somatic variants in TSC2 were identified in 10 of 12 (83%) cases, with a median allele frequency of 13.6%. Five to 18 somatic variants (median number: nine, median allele frequency 21%) not in TSC1 or TSC2 were also identified, mostly of uncertain clinical significance. Copy number changes were rare, but detection wasimpaired by low tumour purity. Immunohistochemistry demonstrated numerous CD68+ macrophages of distinct appearance from Küpffer cells. Multiplex immunofluorescence revealed low numbers of exhausted PD-1+/PD-L1+, FOXP3+ and CD8+ T cells. hAML tumours have consistent inactivating mutations in TSC2 and have a low somatic mutation rate, similar to other TSC-associated tumours. Careful histological review, standard IHC and multiplex immunofluorescence demonstrated marked infiltration by non-neoplastic inflammatory cells, mostly macrophages.

EPEN-05. TRANSCRIPTIONAL FACTOR-DIRECTED EPIGENETIC REGULATION IN CANCER NEUROSCIENCE OF ZFTA-RELA EPENDYMOMA

Abstract ZFTA-RELA (ZRFUS) ependymoma is a pediatric brain tumor with dismal 10-year progression free survival rates. Prior studies revealed low somatic mutation load in pediatric ependymoma compared to adult glioma, suggesting that developmental mechanisms play a key role during disease development. Using human tissues and mouse tumors generated from our autochthonous in utero electroporation (IUE) model, we identified a cohort of developmental transcription factors (TFs) linked to ZRFUS by investigating histone serotonylation changes with other epigenetic datasets. We further performed in vivo barcode-screening and identified the ETS variant transcription factor 5 (ETV5) is sufficient and required for disease progression. To further reveal the disease mechanism driven by ETV5, we applied transcriptomic, epigenetic, and proteomic analyses, and found ETV5 suppresses neuropeptide Y (NPY) expression in mouse ZRFUS tumors by binding with epigenetic modulator, chromobox 3 (CBX3). We further found NPY overexpression delays tumor progression and suppressed tumor-driven neuronal activity in mice. This study suggests developmental TF can shape tumor-promoting microenvironment during disease progression through epigenetic regulation.

Clonal haematopoiesis of indeterminate potential in patients with venous thromboembolism.

Over the last decade, the concept of Clonal haematopoiesis of undetermined potential (CHIP) has emerged. Low frequency somatic mutations in hematopoietic cells can occur with age and might allow formation of clones in individuals with no characterized haematological pathology. These CHIP mutations are associated with an increased risk of cancer or atherothrombosis, and their prevalence are more and more studied in pathologies with an inflammatory component. In our study, we analysed, by next generation sequencing, the prevalence of CHIP mutation in 94 patients with deep venous thrombosis (DVT), distinguishing two clinical phenotypes: provoked distal and non-provoked proximal DVTs. We show that there is no difference in CHIP prevalence between these two groups, nor with a matched-aged control group. The number of mutation per patients and the affected genes remain also the same between the three groups. Consequently and despite the relative small number of patients in each cohort, it seems that CHIP is not a strong concern in venous thromboembolism.

A gut microbiota rheostat to forecast response to PDL1-VEGF blockade in mesothelioma.

e20534 Background: Targeting of the immune inhibitory PD1-PDL1 axis has proven clinically effective in mesothelioma, despite a low somatic mutation burden, and high rate of CDKN2A deletion. Tumour responses to anti PD1 or PDL1 immune checkpoint inhibition are heterogeneous, and the factors underpinning sensitivity remain poorly understood. We therefore addressed this knowledge gap through multi-omic interrogation of tumours from patients enrolled into arm 4 of the Mesothelioma Stratified Therapy umbrella trial (NCT03654833, MiST4), a multi-centre single arm phase IIA trial of atezolizumab and bevacizumab in patients with relapsed mesothelioma. Methods: Next generation sequencing of whole exomes (mesotheliomas and matched germline DNA), transcriptomes, and 16sRNA to profile gut microbiota were undertaken. Spatial phenotyping of the immune landscape employed multiplex immunofluorescence analysis using a 19x depth 4 panel detection. Tumour proportion score (TPS) for PDL1 was assessed using the 22C3 clone, and BAP1, p16ink4a assessed by immunohistochemistry. The MIST4 cohort was dichotomised by best tumour response (50:50) into those patients exhibiting any tumour reduction (R) versus those without (NR). Machine learning (boosting and bagging) was employed to decipher correlates of response. Results: Tumour responses correlated with progression-free survival (PFS, p = 0.0003). Neither PDL1 TPS or CDKN2A expression were predictive. The NR group exhibited a greater degree of genomic instability with higher somatic copy number burden (p = 0.02), homologous recombination deficiency (HRD, p = 0.03), and uniparental disomy (UPD, p = 0.01). Notably the burden of nonsynonymous mutations and neoantigens did not differ significantly between groups. The NR group was transcriptionally enriched for epithelial mesenchymal transition (p < 0.05). Conversely, 16s RNA sequencing revealed higher gut microbial diversity in the R group compared with NR (Shannon index p = 0.009) with R-group enrichment of the type 2 enterotype (provotella 33% vs 9%). R-group enriched genera comprised prevotella, butyricicoccus, bilophilla, Eubacterium ventriosum, whereas the NR group was enriched for erysipelatoclostidium. The log ratio of genera, ie. Log[GR/GNR] was 2-log higher for the R group (p < 0.0001) vs NR group, and was highly predictive of response (with an area under the receiver operator curve of 0.99). Log[GR/GNR] positively correlated with tumour CD8 T cell infiltration (r = 0.6, p = 0.05) and PFS (p = 0.04), but negatively with CD68 monocyte infiltration (p = 0.05), UPD (p = 0.008) and HRD (p = 0.05). Conclusions: We propose a model in which interacting tumour intrinsic and extrinsic factors correlate with response to PDL1-VEGF inhibition in patients with mesothelioma. Gut microbiota composition represents a new, potentially modifiable target with potential to improve immunotherapy outcomes in patients with mesothelioma. Clinical trial information: NCT03654833 .

A naturally arising broad and potent CD4-binding site antibody with low somatic mutation.

The induction of broadly neutralizing antibodies (bNAbs) is a potential strategy for a vaccine against HIV-1. However, most bNAbs exhibit features such as unusually high somatic hypermutation, including insertions and deletions, which make their induction challenging. VRC01-class bNAbs not only exhibit extraordinary breadth and potency but also rank among the most highly somatically mutated bNAbs. Here, we describe a VRC01-class antibody isolated from a viremic controller, BG24, that is much less mutated than most relatives of its class while achieving comparable breadth and potency. A 3.8-Å x-ray crystal structure of a BG24-BG505 Env trimer complex revealed conserved contacts at the gp120 interface characteristic of the VRC01-class Abs, despite lacking common CDR3 sequence motifs. The existence of moderately mutated CD4-binding site (CD4bs) bNAbs such as BG24 provides a simpler blueprint for CD4bs antibody induction by a vaccine, raising the prospect that such an induction might be feasible with a germline-targeting approach.

Abstract 46: Independent clonal origins of synchronous primary tumors in multifocal ileal neuroendocrine tumor patients

Abstract Background: Small intestinal neuroendocrine tumors (SI-NETs) are the most common neoplasms of the small bowel, accounting for 40% of all small intestinal malignancies. Previous high-throughput sequencing studies have shown that SI-NETs have a low somatic mutation rate. Loss of heterozygosity (LOH) at chromosome 18 is the most frequent genomic alteration identified, occurring in ~60% of tumors, and the only recurrent mutations reported to date are loss-of-function mutations in CDKN1B in ~10% of tumors. Most previous studies have focused on sequencing a single primary tumor from each SI-NET patient, even though up to 50% of the patients are diagnosed with multiple synchronous tumors. Thus, the mechanisms by which multifocal SI-NETs arise remain elusive. A better understanding of their tumorigenesis is essential for the optimal treatment of the patients. Materials and Methods: We performed a comprehensive genomic profiling of 75 de-identified synchronous primary tumors, 15 metastases, and 18 normal ileal mucosa and/or whole blood specimens from 13 patients with multifocal ileal NETs using whole genome sequencing. Results: We observed lack of shared somatic variation among the multifocal ileal NET patients and between the synchronous primary tumors within each patient, indicating that these tumors develop independently. Although recurrent copy-number alterations were identified, additional chromosome mapping revealed that tumors from the same patient can gain or lose different parental alleles. In addition to the previously reported CDKN1B loss-of-function mutations, we identified potential driver gene alterations in TNRC6B in a subset of ileal NETs. We also observed two different metastatic dissemination patterns among patients with multiple metastases. Metastases were either clonal or independent, originating from one or several primary tumors. Conclusions: Our study demonstrates notable genomic diversity among multifocal ileal NETs, which corroborates previous literature suggesting that these tumors originate independently. We identified potential loss-of-function mutations in TNRC6B, a candidate tumor suppressor gene in a small subset of ileal NETs. Identification of different metastatic dissemination patterns has an important clinical implication, highlighting the need to carefully remove all synchronous primary tumors of the entire jejunum and ileum at the time of surgery. Lastly, our results indicate that the tumorigenesis of multifocal ileal NETs is not exclusively driven by genomic alterations, suggesting that SI-NETs could be also driven by epigenetic mechanisms, or arise from morphologically normal small intestine as a result of field cancerization. Also, the tumor microenvironment in the ileum may play a role in the growth and development of SI-NETs. Citation Format: NETTA MÄKINEN, Meng Zhou, Zhouwei Zhang, Yosuke Kasai, Elizabeth Perez, Grace E. Kim, Chrissie Thirlwell, Eric Nakakura, Matthew Meyerson. Independent clonal origins of synchronous primary tumors in multifocal ileal neuroendocrine tumor patients [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 46.

Tumor-infiltrating lymphocytes-based subtypes and genomic characteristics of EBV-associated lymphoepithelioma-like carcinoma.

Tumor-infiltrating lymphocytes (TILs) offer a key for morphological diagnosis of lymphoepithelioma-like carcinoma (LELC) and are the foundation of oncoimmunology. To date, no reports have found a specific risk stratification value of TILs and related it to genomic variation in LELC. Based on the stromal TILs (str-TILs) ratio, we classified 105 Epstein-Barr virus (EBV)-associated LELC cases into two subtypes: patients with ≥60% str-TILs area ratio in tumor were classified as subtype I, and otherwise as subtype II. Subtype I patients had significantly better progression-free survival (PFS) and overall survival (OS). We also explored the genomic characteristics of EBV-associated LELC within different involved organs. We performed whole-exome sequencing for 51 patients with enough tissue and analyzed the genomic characteristics of EBV-associated LELC. Overall, EBV-associated LELCs were characterized by a low somatic mutation rate and copy number variations; the enriched genetic lesions affected RTK-RAS, PI3K, and cell cycle pathways. Moreover, EBV-associated LELCs from different organs were more similar to each other genetically as compared with other traditional carcinomas of the same sites-as evidenced by unsupervised clustering based on the quantitative data from both mutation signature and chromosomal aneuploidies. Notably, EBV-associated LELC patients with oncogenic driver alterations showed a worse prognosis compared with patients without such alterations. © 2022 The Pathological Society of Great Britain and Ireland.

Genomic characteristics of invasive mucinous adenocarcinoma of the lung with multiple pulmonary sites of involvement

Invasive mucinous adenocarcinoma (IMA) of the lung frequently presents with diffuse pneumonic-type features or multifocal lesions, which are regarded as a pattern of intrapulmonary metastases. However, the genomics of multifocal IMAs have not been well studied. We performed whole exome sequencing on samples taken from 2 to 5 regions in seven patients with synchronous multifocal IMAs of the lung (24 regions total). Early initiating driver events, such as KRAS, NKX2-1, TP53, or ARID1A mutations, are clonal mutations and were present in all multifocal IMAs in each patient. The tumor mutational burden of multifocal IMAs was low (mean: 1.13/mega base), but further analyses suggested intra-tumor heterogeneity. The mutational signature analysis found that IMAs were predominantly associated with endogenous mutational process (signature 1), APOBEC activity (signatures 2 and 13), and defective DNA mismatch repair (signature 6), but not related to smoking signature. IMAs synchronously located in the bilateral lower lobes of two patients with background usual interstitial pneumonia had different mutation types, suggesting that they were double primaries. In conclusion, genomic evidence found in this study indicated the clonal intrapulmonary spread of diffuse pneumonic-type or multifocal IMAs, although they can occur in multicentric origins in the background of usual interstitial pneumonia. IMAs exhibited a heterogeneous genomic landscape despite the low somatic mutation burden. Further studies are warranted to determine the clinical significance of the genomic characteristics of IMAs in expanded cohorts.

The lncRNA Signatures of Genome Instability to Predict Survival in Patients with Renal Cancer.

Long noncoding RNAs (lncRNAs) exert an increasingly important effect on genome instability and the prognosis of cancer patients. The present research established a computational framework originating from the mutation assumption combining lncRNA expression profile and somatic mutation profile in the genome of renal cancer to assess the effect of lncRNAs on the gene instability of renal cancer. A total of 45 differentially expressed lncRNAs were evaluated to be genome-instability-associated from the high and low cumulative somatic mutations groups. Then we established a prognosis model based on three genome-instability-associated lncRNAs (AC156455.1, AC016405.3, and LINC01234)-GlncScore. The GlncScore was then verified in testing cohort and the total TCGA renal cancer cohort. The GlncScore was evaluated to have an accurate prediction for the survival of patients. Furthermore, GlncScore was associated with somatic mutation patterns, indicating its capacity of reflecting genome instability in renal cancer. In conclusion, this study evaluated the effect of lncRNAs on genome instability of renal cancer and provided new hidden cancer biomarkers related to genome instability in renal cancer.

Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer

BackgroundEnhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity—however, most predicted enhancer regions remain to be functionally tested.MethodsWe analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions.ResultsWe report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity.ConclusionsOur results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

P53 is functionally inhibited in clear cell renal cell carcinoma (ccRCC): a mechanistic and correlative investigation into genetic and molecular characteristics

PurposeAlthough p53 is rarely mutated in ccRCC, its overexpression has been linked to poor prognosis. The current study sought to elucidate the unique role of p53 in ccRCC with genomic, proteomic, and functional analyses.Materials and methodsData from the Cancer Genome Atlas (TCGA) were evaluated for genomic and proteomic characteristics of p53; a tissue micro array (TMA) study was carried out to evaluate the association of p53 and phosphorylated p53 (pp53) with clinical outcome. Mechanistic in vitro experiments were performed to confirm a pro-apoptotic loss of p53 in ccRCC and p53 isoforms as well as posttranslational modifications of p53 where assessed to provide possible reasons for a functional inhibition of p53 in ccRCC.ResultsA low somatic mutation rate of p53 could be confirmed. Although mRNA levels were correlated with poor prognosis and clinicopathological features, there was no monotonous association of mRNA levels with survival outcome. Higher p53 protein levels could be confirmed as poor prognostic features. In vitro, irradiation of ccRCC cell lines markedly induced levels of p53 and of activated (phosphorylated) p53. However, irradiated ccRCC cells demonstrated similar proliferation, migration, and p53 transcriptional activity like non-irradiated controls indicating a functional inhibition of p53. p53 isoforms and could not be correlated with clinical outcome of ccRCC patients.Conclusionsp53 is rarely mutated but the wildtype p53 is functionally inhibited in ccRCC. To investigate mechanisms that underlie functional inhibition of p53 may provide attractive therapeutic targets in ccRCC.

Abstract SY17-02: Multiantigen vaccines for colon cancer prevention

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US when men and women are combined. While overall CRC incidence has declined over the years, there has been an increase in CRC diagnosis in young and middle-aged adults. The US Preventive Services Task Force has recently issued draft recommendations for CRC screening to begin at 45 years of age instead of the previously recommended age of 50 for asymptomatic healthy individuals, who are at average risk of CRC, i.e. without a family history of hereditary CRC syndromes such as Lynch syndrome and familial adenomatous polyposis, or with no prior diagnosis of CRC, adenomatous polyps, inflammatory bowel disease. While the evidence supports that CRC-screening and early detection confers a substantial benefit, additional CRC prevention strategies can greatly help reduce the cancer burden in the US and around the world. A number of previous studies have shown that the use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancers, including CRC. However, their extended use can be associated with serious side effects, including gastrointestinal bleeding. Safer and more efficacious preventive approaches are needed for durable long-term CRC prevention. The majority of newly diagnosed CRC are non-familial (sporadic) and non-hypermutated/microsatellite-stable (MSS). Compared to CRC with high microsatellite instability, MSS CRC and adenomas have significantly lower somatic mutation frequencies, thus lower neoantigen load. Furthermore, MSS CRC have generally lower levels of tumor-infiltrating lymphocytes (TILs), higher density of myeloid derived suppressor cells (MDSC), and downregulation of HLA molecules. These are characteristic of immunologically “cold” tumors, making it highly challenging to immunologically control tumor growth. If cancer vaccines can successfully prime the immune system, in particular before the development of invasive cancer, vaccine-induced adaptive antitumor immunity may render “cold” tumors “hot” and achieve a favorable tumor control. One of the key questions, however, is what tumor antigens can be targeted by vaccines if tumors have lower levels of neoantigens that are known to elicit robust antitumor immune responses. Another approach for cancer vaccine-mediated immune priming is to target tumor associated antigens (TAAs). Unlike tumor-specific neoantigens, TAAs are self-antigens that are overexpressed in tumors, but may also be expressed in normal cells at various levels. Therefore, it is highly critical to identify T cell epitopes that break tolerance and help elicit effective antitumor immunity. Clinically beneficial antitumor immunity is characterized by high-density infiltrates of effector CD8+ cytotoxic T lymphocytes and effector memory T cells and the low abundance of immunosuppressive elements (e.g. regulatory T cells, MDSC, and M2-polarized tumor-associated macrophages) in the tumor microenvironment. Disis and colleagues from University of Washington have previously established a systematic and stepwise approach to identify Th1-selective epitopes for efficacious cancer vaccines while eliminating Th2-epitopes. They have shown cancer vaccines targeting TAAs thus developed could elicit robust Th1 immune response associated with antitumor effects. The NCI PREVENT Cancer Preclinical Drug Development Program (PREVENT) is a peer-reviewed agent development program focused on preclinical development of innovative interventions, including cancer vaccines and drugs, for precision cancer prevention-interception towards clinical applications (https://prevention.cancer.gov/major-programs/prevent-cancer-preclinical). PREVENT and Disis team collaborated on the development of a multi-antigen multi-peptide colon cancer vaccine, named Colovac, for immunoprevention of CRC. It consists of highly immunogenic Th1-promoting epitopes from three TAAs that are selected through CRC genomic database and literature search. TAAs selected are overexpressed in CRC, known to play a role in CRC oncogenesis, and associated with poor disease outcomes. This presentation will cover the background of Colovac development, preclinical antitumor efficacy data on Colovac used alone or in combination with naproxen, characterization of immune correlates of protection, and current ongoing development efforts toward clinical trials. Citation Format: Shizuko Sei. Multiantigen vaccines for colon cancer prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY17-02.

Lower Somatic Mutation Levels in the λ Light-Chain Repertoires with Chronic HBV Infection

To investigate the characteristics of the immunoglobulin light-chain repertoires with chronic HBV infection, the high-throughput sequencing and IMGT/HighV-QUEST were adapted to analyze the κ (IgK) and λ (IgL) light-chain repertoires from the inactive HBV carriers (IHB) and the healthy adults (HH). The comparative analysis revealed high similarity between the κ light-chain repertoires of the HBV carriers and the healthy adults. Nevertheless, the proportion of IGLV genes with ≥90% identity as the germline genes was higher in the IgL light-chain repertoire of the IHB library compared with that of HH library (74.6% vs. 69.1%). Besides, the frequency of amino acid mutations in the CDR1 regions was significantly lower in the IgL light-chain repertoire of the IHB library than that of the HH library (65.52% vs. 56.0%). These results suggested the lower somatic mutation level in the IgL repertoire of IHB library, which might indicate the biased selection of IGLV genes in the IgL repertoire with chronic HBV infection. These findings might lead to a better understanding of the characteristics of the light-chain repertoires of HBV chronically infected individuals.

064 Genome analysis reveals UV signature mutations in sun-exposed skin tumors in tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a tumor syndrome caused by pathogenic variants in TSC1 or TSC2. Individuals with TSC develop skin tumors with second-hit somatic mutations in TSC1 or TSC2 in fibroblast-like tumor cells. These somatic mutations in facial angiofibromas, but not fibrous tumors in non-sun-exposed areas, are commonly UV signature mutations, prompting recommendations to limit UV exposure to reduce the severity of TSC skin tumors. In order to investigate whether UV light induces additional genetic alterations that promote TSC tumor pathogenesis, we performed whole genome sequencing of DNA from patient peripheral blood and fibroblast-like cells grown from TSC skin tumors. Unique dual-indexed PCR-free libraries were sequenced as pools on an Illumina NovaSeq 6000 generating 2x150 read pairs with a Picard mean coverage of ∼38X for blood samples and ∼63X for tumor samples. TSC1 and TSC2 germline mutations were identified in 39 out of 48 patients (5 TSC1, 34 TSC2 including 12 mosaic). TSC1 and TSC2 somatic mutations were found in 27 out of 45 tumor samples (2 TSC1, 25 TSC2), including copy neutral LOH, point mutations, large deletions, and intra- and interchromosomal gene fusions. The mutation rate per Mb was higher in sun-exposed (median 5.7/Mb, IQR 4.1-11.8) than non-sun-exposed locations (median 1.7/Mb, IQR 1.5-2.0). C>T mutations and a UV signature were more frequent throughout the genome in sun-exposed than other sites, but there were no recurrent mutations in known driver genes. In summary, TSC skin tumors have relatively low somatic mutation rates that are increased in sun-exposed sites; however, somatic alteration of TSC1 or TSC2, whether random or UV-induced, combined with a TSC1/TSC2 germline mutation, appears to be sufficient to drive tumorigenesis.

Human Lanosterol 14-Alpha Demethylase (CYP51A1) Is a Putative Target for Natural Flavonoid Luteolin 7,3'-Disulfate.

Widespread pathologies such as atherosclerosis, metabolic syndrome and cancer are associated with dysregulation of sterol biosynthesis and metabolism. Cholesterol modulates the signaling pathways of neoplastic transformation and tumor progression. Lanosterol 14-alpha demethylase (cytochrome P450(51), CYP51A1) catalyzes one of the key steps in cholesterol biosynthesis. The fairly low somatic mutation frequency of CYP51A1, its druggability, as well as the possibility of interfering with cholesterol metabolism in cancer cells collectively suggest the clinical importance of CYP51A1. Here, we show that the natural flavonoid, luteolin 7,3′-disulfate, inhibits CYP51A1 activity. We also screened baicalein and luteolin, known to have antitumor activities and low toxicity, for their ability to interact with CYP51A1. The Kd values were estimated using both a surface plasmon resonance optical biosensor and spectral titration assays. Unexpectedly, in the enzymatic activity assays, only the water-soluble form of luteolin—luteolin 7,3′-disulfate—showed the ability to potently inhibit CYP51A1. Based on molecular docking, luteolin 7,3′-disulfate binding suggests blocking of the substrate access channel. However, an alternative site on the proximal surface where the redox partner binds cannot be excluded. Overall, flavonoids have the potential to inhibit the activity of human CYP51A1 and should be further explored for their cholesterol-lowering and anti-cancer activity.

Advances in Detecting Low Prevalence Somatic TERT Promoter Mutations in Papillary Thyroid Carcinoma.

BackgroundTwo recurrent TERT (telomerase reverse transcriptase) promoter mutations, C228T and C250T, have been reported in thyroid carcinomas and were correlated with high-risk clinicopathological features and a worse prognosis. Although far more frequent in the poorly differentiated and undifferentiated thyroid cancer, the TERT promoter mutations play a significant role on PTC recurrence and disease-specific mortality. However, the prevalence varies considerably through studies and it is uncertain if these differences are due to population variation or the methodology used to detect TERT mutations. In this study we aim to compare three different strategies to detect TERT promoter mutations in PTC.MethodsDNA was isolated from formalin-fixed paraffin-embedded (FFPE) specimens from 89 PTC and 40 paired lymph node metastases. The prevalence of the hot spot TERT C228T and C250T mutations was assessed in FFPE samples using TaqMan SNP genotyping assays. Random samples were tested by Sanger Sequencing and droplet digital PCR (ddPCR).ResultsIn general, 16 out of 89 (18%) PTC samples and 14 out of 40 (35%) lymph node metastases harbored TERT promoter mutations by TaqMan assay. Sanger sequencing, performed in random selected samples, failed to detect TERT mutations in four samples that were positive by TaqMan SNP genotyping assay. Remarkably, ddPCR assay allowed detection of TERT promoter mutations in six samples that harbor very low mutant allele frequency (≤ 2%) and were negative by both genotype assay and Sanger Sequencing.ConclusionThis study observed a good concordance among the methodologies used to detect TERT promoter mutations when a high percentage of mutated alleles was present. Sanger analysis demonstrated a limit of detection for mutated alleles. Therefore, the prevalence of TERT promoter mutations in PTC may be higher than previously reported, since most studies have conventionally used Sanger sequencing. The efficient characterization of genetic alterations that are used as preoperative or postoperative diagnostic, risk stratification of the patient and individualized treatment decisions, mainly in highly heterogeneous tumors, require highly sensitive and specific approaches.

Distinct Signatures of Genomic Copy Number Variants Define Subgroups of Merkel Cell Carcinoma Tumors.

Simple SummaryCancer results from genetic changes in cells. These changes are often mutations that alter the DNA sequence of critical genes. However, duplications and deletions in cancer-related genes can also contribute to malignant transformation. In this study we use Nanostring technology to assess DNA copy number changes in samples of Merkel cell carcinoma (MCC), a rare and aggressive neuroendocrine skin tumor. We were able to identify recurrent amplifications and deletions in cancer-related genes. We also found that MCC tumors grouped into three distinct copy number variant profiles. The first group consisted of tumors with multiple deletions. The second group contained tumors with low levels of genomic structural alterations. The last group comprised tumors containing multiple amplifications. Our study suggests that most MCC tumors are associated with deletions in cancer-related genes or are lacking in copy number changes, whereas a small percentage of tumors are associated with genomic amplifications.Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated Merkel cell polyomavirus DNA (virus-positive MCC, VP-MCC) and carry a low somatic mutation burden whereas virus-negative MCC (VN-MCC) possess numerous ultraviolet-signature mutations. In contrast to viral oncogenes and sequence mutations, little is known about genomic structural variants in MCC. To identify copy number variants in commonly altered genes, we analyzed genomic DNA from 31 tumor samples using the Nanostring nCounter copy number cancer panel. Unsupervised clustering revealed three tumor groups with distinct genomic structural variant signatures. The first cluster was characterized by multiple recurrent deletions in genes such as RB1 and WT1. The second cluster contained eight VP-MCC and displayed very few structural variations. The final cluster contained one VP-MCC and four VN-MCC with predominantly genomic amplifications in genes like MDM4, SKP2, and KIT and deletions in TP53. Overall, VN-MCC contained more structure variation than VP-MCC but did not cluster separately from VP-MCC. The observation that most MCC tumors demonstrate a deletion-dominated structural group signature, independent of virus status, suggests a shared pathophysiology among most VP-MCC and VN-MCC tumors.

Deep sequencing reveals the genomic characteristics of lung adenocarcinoma presenting as ground-glass nodules (GGNs).

BackgroundThe concept of multi-step progression from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma (ADC) has been proposed, and ground-glass nodules (GGNs) may play a critical role during the early lung tumorigenesis. We present the first comprehensive description of the genomic architecture of GGNs to unravel the genetic basis of GGN.MethodsWe investigated 30 GGN-like lungs ADC by performing >1,000× whole-exome sequencing (WES) and characterized the genomic variations and evaluate the relationship between the clinicopathologic and molecular characteristics in this disease.ResultsDespite the low somatic mutation burden, GGNs exhibited high intratumor heterogeneity (ITH) characterized by the proportion of subclonal mutations. Different mutagenesis shaped the genomes of GGN during cancer evolution and were mostly featured by molecular clock-like signatures that occur in clonal mutations and defective DNA mismatch signatures that occur in subclonal mutations. Moreover, 10.7–67.1% clonal mutations occurred after whole-genome doubling (WGD), indicating that WGD could be a frequent truncal event in GGNs. Samples with WGD showed higher genomic instability but lower ITH. These GGNs were characterized by recurrent focal copy-number changes that are highly associated with tumorigenesis, with only two genes (EGFR and RBM10) that were recurrently mutated. Additionally, GGNs with different pathological subtypes or computed tomography (CT) features exhibited distinct genetic characteristics. Lepidic predominant or pure GGNs in CT images carried a lower mutation burden and had a relatively stable genome than nonlepidic or mixed GGNs. GGNs with RBM10 mutations tended to accompany a pathologically lepidic pattern, indicating RBM10 may drive the distinct subtype of lung cancer with better prognosis.ConclusionsThese findings facilitated interpreting the genomic characteristics of GGNs, provided insight into the early stages of lung cancer evolution, and possessed potential clinical significance.