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Immune checkpoint blockade (ICB) benefits only a small subset of patients with small cell lung cancer (SCLC), yet the mechanisms driving benefit are poorly understood. To identify predictors of clinical benefit to ICB, we performed immunogenomic profiling of tumor samples from patients with relapsed SCLC. Tumors of patients who derive clinical benefit from ICB exhibit cytotoxic T-cell infiltration, high expression of antigen processing and presentation machinery (APM) genes, and low neuroendocrine (NE) differentiation. However, elevated Notch signaling, which positively correlates with low NE differentiation, most significantly predicts clinical benefit to ICB. Activation of Notch signaling in a NE human SCLC cell line induces a low NE phenotype, marked by increased expression of APM genes, demonstrating a mechanistic link between Notch activation, low NE differentiation and increased intrinsic tumor immunity. Our findings suggest Notch signaling as a determinant of response to ICB in SCLC. Immune checkpoint blockade (ICB) benefits only a small subset of patients with small cell lung cancer (SCLC) and the mechanisms driving benefit are poorly understood. Here, the authors show that elevated Notch signaling predicts clinical benefit in ICB in relapsed SCLC.

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth.

Yardena Samuels and colleagues report the analysis of 501 melanoma exomes and the identification of RASA2 as a tumor-suppressor gene mutated in 5% of melanomas. RASA2 mutations led to increased RAS activation, and RASA2 loss was associated with shorter patient survival times. Analysis of 501 melanoma exomes identified RASA2 , encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.

Background: A consistent correlation between tumor mutation burden (TMB) and tumor immune microenvironment has not been observed in gliomas as in other cancers. Methods: Driver germline and somatic mutations, TMB, neoantigen, and immune cell signatures were analyzed using whole exome sequencing (WES) and transcriptome sequencing of tumor and WES of matched germline DNA in a cohort of 66 glioma samples (44 IDH-mutant and 22 IDH-wildtype). Results: Fourteen samples revealed a hypermutator phenotype (HMP). Eight pathogenic (P) or likely pathogenic (LP) germline variants were detected in 9 (19%) patients. Six of these 8 genes were DNA damage repair genes. P/LP germline variants were found in 22% of IDH-mutant gliomas and 12.5% of IDH-wildtype gliomas (p = 0.7). TMB was correlated with expressed neoantigen but showed an inverse correlation with immune score (R = −0.46, p = 0.03) in IDH-wildtype tumors and no correlation in IDH-mutant tumors. The Antigen Processing and Presentation (APP) score correlated with immune score and was surprisingly higher in NHMP versus HMP samples in IDH-wildtype gliomas, but higher in HMP versus NHMP in IDH-mutant gliomas. Conclusion: TMB was inversely correlated with immune score in IDH-wildtype gliomas and showed no correlation in IDH-mutant tumors. APP was correlated with immune score and may be further investigated as a biomarker for response to immunotherapy in gliomas. Studies of germline variants in a larger glioma cohort are warranted.

BackgroundTumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment.MethodsWe tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed “NeoExpand”, was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand.ResultsWe show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4+ and CD8+ TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models.ConclusionsTaken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation.Trial registration numberNCT00068003, NCT01174121, and NCT03412877.

BackgroundThe use of tumor-infiltrating T lymphocytes (TIL) that recognize cancer neoantigens has led to lasting remissions in metastatic melanoma and certain cases of metastatic epithelial cancer. For the treatment of the latter, selecting cells for therapy typically involves laborious screening of TIL for recognition of autologous tumor-specific mutations, detected through next-generation sequencing of freshly resected metastatic tumors. Our study explored the feasibility of using archived formalin-fixed, paraffin-embedded (FFPE) primary tumor samples for cancer neoantigen discovery, to potentially expedite this process and reduce the need for resections normally required for tumor sequencing.MethodWhole-exome sequencing was conducted on matched primary and metastatic colorectal cancer samples from 22 patients. The distribution of metastatic tumor mutations that were confirmed as neoantigens through cognate TIL screening was evaluated in the corresponding primary tumors. Mutations unique to primary tumors were screened for recognition by metastasis-derived TIL and circulating T lymphocytes.ResultsWe found that 25 (65.8%) of the 38 validated neoantigens identified in metastatic tumors from 18 patients with colorectal cancer were also present in matched primary tumor samples. This included all 12 neoantigens encoded by putative cancer driver genes, which are generally regarded as superior targets for adoptive cell therapy. The detection rate for other neoantigens, representing mutations without an established role in cancer biology, was 50% (13/26). Gene products encoding neoantigens detected in the primary tumors were not more likely to be clonal or broadly distributed among the analyzed metastatic lesions compared with those undetected in the primary tumors. Additionally, we found that mutations detected only in primary tumor samples did not elicit recognition by metastatic tumor-derived TIL but could elicit specific recognition by the autologous circulating memory T cells.ConclusionsOur findings indicate that primary FFPE tumor-derived screening libraries could be used to discover most neoantigens present in metastatic tumors requiring treatment. Furthermore, this approach can reveal additional neoantigens not present in resected metastatic tumors, prompting further research to understand their clinical relevance as potential therapeutic targets.

BackgroundMetastatic cholangiocarcinoma (CC), a form of gastrointestinal cancer that originates from the bile ducts, cannot be cured by currently available therapies, and is associated with dismal prognosis. In a previous case report, adoptive transfer of autologous tumor infiltrating lymphocytes (TILs), the majority of which recognized a tumor-specific point mutation, led to a profound and durable cancer regression in a patient with metastatic CC. Thus, more effective treatment for patients with this disease may be developed by using TILs that target cancer-specific mutations, but also other genetic aberrations such as gene fusions. In this context, fusions that involve fibroblast growth factor receptor 2 (FGFR2) and function as oncogenes in a subset of patients with intrahepatic CC (ICC) represent particularly attractive targets for adoptive cell therapy. However, no study to date has explored whether FGFR2 fusions can be recognized by patients’ T cells.MethodTo address whether FGFR2 fusions can be recognized by patients’ T cells, we tested TILs from four patients with FGFR2 fusion-positive ICC for recognition of peptides and minigenes that represented the breakpoint regions of these fusions, which were unique to each of the four patients.ResultsWe found that CD4+ TILs from one patient specifically recognized the breakpoint region of a unique FGFR2-TDRD1 (tudor domain-containing 1) fusion, and we isolated a T-cell receptor responsible for its recognition.ConclusionsThis finding suggests that FGFR2 fusion-reactive TILs can be isolated from some patients with metastatic ICC, and thus provides a rationale for future exploration of T cell-based therapy targeting FGFR2 fusions in patients with cancer. Furthermore, it augments the rationale for extending such efforts to other types of solid tumors hallmarked by oncogenic gene fusions.

BackgroundImmunotherapies represent promising approaches to treat metastatic solid tumors, yet their response rates remain low.1 2 Identifying antitumor T cells, their antigens, and their cognate T cell receptors (TCRs) can provide crucial insights into the design of next-generation cellular immunotherapies. Circulating T cells from the peripheral blood (PBL) can provide a rich and non-invasive source for identifying and studying antitumor T cells as an alternative to tumor-infiltrating lymphocytes (TIL). Yet, pre-surgery antitumor T cell frequencies in PBL of patients with metastatic cancer are often low,3 4 limiting the accurate definition of their phenotypic states.MethodsWe employed single-cell phenotypic profiling of 36 vetted neoantigen-specific T cell clones from 6 metastatic epithelial cancer patients to derive the transcriptional and cell surface protein signatures of PBL-resident antitumor CD8+ T cells (NeoTCRPBL). In 4 samples, we compared T cell gene signatures of 24 paired neoantigen TCR clonotypes between the PBL and TIL compartments. We developed a NeoTCRPBL gene signature to assess its sensitivity and specificity in discovering new antitumor TCRs from PBL of prospective patients with different tumor types. Finally, we compared the frequency and avidity of antitumor TCR clonotypes between the TIL and PBL compartments in all patients.ResultsBlood-resident NeoTCRPBL T cells were clonally expanded, but low in frequency (<=0.001–0.002% per clone) necessitating their enrichment for studies. NeoTCRPBL T cells exhibited phenotypes distinct from common T cell subsets and bystander viral-reactive T cells displaying transcriptional programs of both dysfunctional and tissue-resident memory T cells (figure 1A,B). Within the same patient, intra-clonotype comparison of 24 TIL-and PBL-neoantigen-specific T cell clones suggested that relative to their TIL counterparts, circulating NeoTCRPBLT cells displayed less-dysfunctional immunotherapy-response associated progenitor phenotypic states (figure 1C).5–7 Combined analysis of 100 antitumor T cell clones revealed that circulating NeoTCRPBL T cells largely targeted the same clonal, subclonal neoantigens with comparable avidity as TIL (>79% shared), but their TCR-repertoire was only partially shared with TIL (47% shared) (figure 1F-G). Finally, prediction and testing of 64 clonally expanded NeoTCRPBL signature-enriched TCRs from prospective samples discovered 20 neoantigen-TCRs demonstrating that NeoTCRPBL signature can successfully identify antitumor TCRs from very low circulating PBL frequencies(< 0.002%) (figure 1D,E).ConclusionsCirculating antitumor T cells are low in frequency exhibiting distinct clonotypic repertoire and phenotypic states in patients with metastatic solid tumors. The NeoTCRPBL signature provides an alternative source for identifying antitumor T cells and their TCRs non-invasively from pre-surgery blood samples enabling immune monitoring and designing cancer immunotherapies.AcknowledgementsWe thank Abraham Sachs, Zhili Zheng, Zhiya Yu, and Biman C. Paria for their technical support. We thank the NIDAP for their computational support. We also thank Pia Kvitsborg, from the Netherlands Cancer Institute, for providing the photo-cleavable HLA monomers.ReferencesYarcohen, et al. Tumor mutational burden and response rate to PD-1 inhibition. NEJM 2017.Parkhurst, et al. Unique neoantigens arise from somatic mutations in patients with gastrointestinal cancers, Cancer Discovery, 2020.Gros, et al. Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nature Medicine 2016.Strønen, et al. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science, 2016.Krishna S, et al. Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science 2020.Lowery FJ, et al. Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers. Science 2022.Sade-Feldman, et al. Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 2018.Ethics ApprovalSamples were collected from patients enrolled on Surgery Branch treatment protocol NCT00068003. Apheresis samples, used to identify circulating neoantigen-reactive CD8+ T cells, were collected under NCT00001823 protocol. Metastatic tumor specimens were obtained from patients enrolled in NCT01174121 protocol. Clinical protocols were reviewed and approved by National Cancer Institute (NCI) Institutional Review Board (IRB). In accordance with the Declaration of Helsinki, informed consent was reviewed, signed, and documented.ConsentSamples were collected from patients enrolled on Surgery Branch treatment protocol NCT00068003. Apheresis samples, used to identify circulating neoantigen-reactive CD8+ T cells, were collected under NCT00001823 protocol. Metastatic tumor specimens were obtained from patients enrolled in NCT01174121 protocol. Clinical protocols were reviewed and approved by National Cancer Institute (NCI) Institutional Review Board (IRB). In accordance with the Declaration of Helsinki, informed consent was reviewed, signed, and documented.Abstract 1216 Figure 1(A) Transcriptional states of circulating CD8+ cells from six metastatic colon cancer patients’ blood samples and projection of neoantigen- and viral-reactive clones on the UMAP. (B) Schematic representing the combined TIL+ PBL neoantigen T cell phenotypic states analysis within each patient. (C) Average gene signature scores (scGSEA) scores of immunotherapy response and non-response associated gene signatures within each individual neoantigen-reactive clone compared between TIL and PBL compartment. ***P < 0.0001 by Paired T-test per each neoantigen T cell clonotype. (D) FACS-sorting enrichment gating of circulating CD8+ T cells from patient for scRNA. (E) Projection of predicted antitumor T cells from PBL based on NeoTCRPBL gene signature (left) and results of testing, 5 antitumor TCRs were discovered (right). (F) Summary of the landscape of neoantigen-reactive TCR clonotypes and their cognate neoantigens shared between TIL and PBL. (G) Functional avidity of 44 NeoTCR clones that were either found only in the PBL compartment (Blood), TIL compartment (Tumor), or shared between PBL and TIL (Shared).

Adoptive cell transfer (ACT) with neoantigen-reactive T lymphocytes can mediate cancer regression. Here we isolated unique, personalized, neoantigen-reactive T cell receptors (TCRs) from tumor-infiltrating lymphocytes of patients with metastatic gastrointestinal cancers and incorporated the TCR α and β chains into gamma retroviral vectors. We transduced autologous peripheral blood lymphocytes and adoptively transferred these cells into patients after lymphodepleting chemotherapy. In a phase 2 single-arm study, we treated seven patients with metastatic, mismatch repair-proficient colorectal cancers who had progressive disease following multiple previous therapies. The primary end point of the study was the objective response rate as measured using RECIST 1.1, and the secondary end points were safety and tolerability. There was no prespecified interim analysis defined in this study. Three patients had objective clinical responses by RECIST criteria including regressions of metastases to the liver, lungs and lymph nodes lasting 4 to 7 months. All patients received T cell populations containing ≥50% TCR-transduced cells, and all T cell populations were polyfunctional in that they secreted IFNγ, GM-CSF, IL-2 and granzyme B specifically in response to mutant peptides compared with wild-type counterparts. TCR-transduced cells were detected in the peripheral blood of five patients, including the three responders, at levels ≥10% of CD3 + cells 1 month post-ACT. In one patient who responded to therapy, ~20% of CD3 + peripheral blood lymphocytes expressed transduced TCRs more than 2 years after treatment. This study provides early results suggesting that ACT with T cells genetically modified to express personalized neoantigen-reactive TCRs can be tolerated and can mediate tumor regression in patients with metastatic colorectal cancers. ClinicalTrials.gov registration: NCT03412877 . Preliminary findings in seven patients with mismatch repair-proficient metastatic colorectal cancer who were treated in the context of a phase 2 trial show that adoptive transfer of autologous peripheral blood T lymphocytes that were retrovirally transduced with personalized neoantigen-reactive T cell receptors can be safe and induce early clinical responses.