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PURPOSE Metastatic breast cancer (mBrCa) is most often an incurable disease with only modest responses to available immunotherapies. This study investigates the immunogenicity of somatic mutations in breast cancer and explores the therapeutic efficacy in a pilot trial of mutation-reactive tumor-infiltrating lymphocytes (TILs) in patients with metastatic disease. PATIENTS AND METHODS Forty-two patients with mBrCa refractory to previous lines of treatment underwent surgical resection of a metastatic lesion(s), isolation of TIL cultures, identification of exomic nonsynonymous tumor mutations, and immunologic screening for neoantigen reactivity. Clinically eligible patients with appropriate reactivity were enrolled into one cohort of an ongoing phase II pilot trial of adoptive cell transfer of selected neoantigen-reactive TIL, with a short course of pembrolizumab (ClinicalTrials.gov identifier: NCT01174121 ). RESULTS TILs were isolated and grown in culture from the resected lesions of all 42 patients with mBrCa, and a median number of 112 (range: 6-563) nonsynonymous mutations per patient were identified. Twenty-eight of 42 (67%) patients contained TIL that recognized at least one immunogenic somatic mutation (median: 3 neoantigens per patient, range: 1-11), and 13 patients demonstrated robust reactivity appropriate for adoptive transfer. Eight patients remained clinically eligible for treatment, and six patients were enrolled on a protocol of adoptive cell transfer of enriched neoantigen-specific TIL, in combination with pembrolizumab (≤ 4 doses). Objective tumor regression was noted in three patients, including one complete response (now ongoing over 5.5 years) and two partial responses (6 and 10 months). CONCLUSION Most patients with breast cancer generated a natural immune response targeting the expressed products of their cancer mutations. Adoptive transfer of TIL is a highly personalized experimental option for patients with mBrCa shown to be capable of mediating objective responses in this pilot trial and deserves further study.

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo–expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53–reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02–restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers. See related Spotlight by Klebanoff, p. 919

Supplementary Figure from Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor–Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo–expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53–reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02–restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers.See related Spotlight by Klebanoff, p. 919

HLA typing data.

Tumor mutation profile.

Mutation expression in metastatic lesions at autopsy.

Clonal architecture of 4062 tumor.

Recognition of mRNA-encoded RBPJ mutation.

Phenotypic characterization of TIL.

Purpose: The administration of autologous tumor-infiltrating lymphocytes (TILs) can mediate durable tumor regressions in patients with melanoma likely based on the recognition of immunogenic somatic mutations expressed by the cancer. There are limited data regarding the immunogenicity of mutations in breast cancer. We sought to identify immunogenic nonsynonymous mutations in a patient with triple-negative breast cancer (TNBC) to identify and isolate mutation-reactive TILs for possible use in adoptive cell transfer.Experimental Design: A TNBC metastasis was resected for TIL generation and whole-exome sequencing. Tandem minigenes or long 25-mer peptides encoding selected mutations were electroporated or pulsed onto autologous antigen-presenting cells, and reactivity of TIL was screened by upregulation of CD137 and IFNγ ELISPOT. The nature of the T-cell response against a unique nonsynonymous mutation was characterized.Results: We identified 72 nonsynonymous mutations from the tumor of a patient with TNBC. CD4+ and HLA-DRB1*1501–restricted TILs isolated from this tumor recognized a single mutation in RBPJ (recombination signal binding protein for immunoglobulin kappa J region). Analysis of 16 metastatic sites revealed that the mutation was ubiquitously present in all samples.Conclusions: Breast cancers can express naturally processed and presented unique nonsynonymous mutations that are recognized by a patient's immune system. TILs recognizing these immunogenic mutations can be isolated from a patient's tumor, suggesting that adoptive cell transfer of mutation-reactive TILs could be a viable treatment option for patients with breast cancer. Clin Cancer Res; 23(15); 4347–53. ©2017 AACR.

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8 + and CD4 + neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature–derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

BackgroundAutologous patient T cells engineered to express antitumor T cell receptors (TCRs) and chimeric antigen receptors (CARs) have been effective for the treatment of certain cancer types,1–4 and tumor neoantigens encoded by cancer-specific mutations have emerged as major targets of CD4+ and CD8+ T cells in immune checkpoint blockade (ICB) and in adoptive cell therapy (ACT).5–9 However, only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells.10–12 Conventional approaches to isolate tumor-reactive T cells and identify their TCRs from tumors rely on T cell function and can be impaired due to T cell exhaustion and dysfunction.13 14MethodsWe performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 46,000 T cells isolated from eleven archival metastatic tumor samples whose primary cancer types included colon, rectal, breast, anal, and melanoma. From these samples, 15 CD8+ and 17 CD4+ neoantigen-reactive TCR clonotypes (NeoTCRs) were known. We then performed transcriptomic clustering of these cells and mapped known NeoTCR clonotypes onto the transcriptomic map. Subsequently we predicted NeoTCRs from prospective metastatic colon cancer samples based on their presence within clusters sharing gene expression with NeoTCR+ clusters in the archival samples.ResultsProjecting known NeoTCRs onto the TIL transcriptomic map, we observed 325 total T cells bearing these NeoTCRs, and the majority (>80%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, that included by expression of CXCL13, ENTPD1 (CD39), TOX, TIGIT, LAG3, and PDCD1 (PD-1), indicating a dysfunctional state. Reasoning that T cells sharing phenotypes with those within the NeoTCR clusters could be novel NeoTCRs, we developed gene signatures (NeoTCR4 and NeoTCR8) of CD4 and CD8 NeoTCR+ cells, respectively, and four prospective patients' TIL were analyzed by scRNA/TCR-seq and scored according to NeoTCR signatures. We expressed predicted NeoTCRs in healthy donor PBL and screened them with antigen presenting cells (APCs) expressing candidate neoantigens. 33/73 predicted NeoTCRs (including both CD4 and CD8) were reactive against patients' tumors or candidate neoantigens.ConclusionsThis study enabled successful detection of tumor-specific NeoTCRs in the sequenced TIL of 14/14 patients for whom reactivity was studied. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapeutics, and prospective NeoTCR isolation based on TIL transcriptional phenotypes will allow for rapid development of personalized immunotherapy in the form of lymphocytes expressing these tumor-specific TCRs.AcknowledgementsWe thank the Surgery Branch TIL Laboratory and clinical team for generating TIL, and patients enrolled in our clinical protocols. Support from CCR Single Cell Analysis Facility was funded by FNLCR Contract HHSN261200800001E. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We also thank NIDAP for providing additional computational support and the CCR Genomics Core for next-generation sequencing supportReferencesRobbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CCR, Levy CL, Li YF, El-Gamil M, Schwarz SL, Laurencot C, Rosenberg SA.Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011;29:917–924.Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006;314:126–129.June CH, Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med 2018;379:64–73.Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. Blood 2010;116:3875–3886.Tran E, Robbins PF, Rosenberg SA, “Final common pathway” of human cancer immunotherapy: targeting random somatic mutations. Nat Immunol 2017;18:255–262.Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, Rosenberg SA, Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 2013;19:747–752.Parkhurst MR, Robbins PF, Tran E, Prickett TD, Gartner JJ, Jia L, Ivey G, Li YF, El-Gamil M, Lalani A, Crystal JS, Sachs A, Groh E, Ray S, Ngo LT, Kivitz S, Pasetto A, Yossef R, Lowery FJ, Goff SL, Lo W, Cafri G, Deniger DC, Malekzadeh P, Ahmadzadeh M, Wunderlich JR, Somerville RPT, Rosenberg SA. Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers. Cancer Discov 2019;9:1022–1035.Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SSK, Korman AJ, Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG, Melief CJM, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 2014;515:577–581.van Rooij N, van Buuren MM, Philips D, Velds A, Toebes M, Heemskerk B, van Dijk LJA, Behjati S, Hilkmann H, el Atmioui D, Nieuwland M, Stratton MR, Kerkhoven RM, Keşmir C, Haanen JB, Kvistborg P, Schumacher TN. Tumor Exome Analysis Reveals Neoantigen-Specific T-Cell Reactivity in an Ipilimumab-Responsive Melanoma. Journal of Clinical Oncology 2013;31:e439–e442.Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, Goodall CP, Blair TC, Fox BA, McDermott JE, Chang SC, Grunkemeier G, Leidner R, Bell RB, Weinberg AD. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9:2724.Simoni Y, Becht E, Fehlings M, Loh CY, Koo SL, Teng KWW, Yeong JPS, Nahar R, Zhang T, Kared H, Duan K, Ang N, Poidinger M, Lee YY, Larbi A, Khng AJ, Tan E, Fu C, Mathew R, Teo M, Lim WT, Toh CK, Ong BH, Koh T, Hillmer AM, Takano A, Lim TKH, Tan EH, Zhai W, Tan DSW, Tan IB, Newell EW, Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–579.Scheper W, Kelderman S, Fanchi LF, Linnemann C, Bendle G, de Rooij MAJ, Hirt C, Mezzadra R, Slagter M, Dijkstra K, Kluin RJC, Snaebjornsson P, Milne K, Nelson BH, Zijlmans H, Kenter G, Voest EE, Haanen JBAG, Schumacher TN. Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. Nat Med 2019;25:89–94.Blank CU, Haining WN, Held W, Hogan PG, Kallies A, Lugli E, Lynn RC, Philip M, Rao A, Restifo NP, Schietinger A, Schumacher TN, Schwartzberg PL, Sharpe AH, Speiser DE, Wherry EJ, Youngblood BA, Zehn D. Defining “T cell exhaustion.” Nat Rev Immunol 2019;19:665–674.van der Leun AM, Thommen DS, Schumacher TN. CD8 T cell states in human cancer: insights from single-cell analysis. Nat Rev Cancer 2020;20:218–232.

Adoptive cell therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from rare mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. Here, we describe a library of T cell receptors (TCRs) that can target TP53 mutations shared among 7.3% of patients with solid cancers. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner both in vitro and in vivo. Patients with chemorefractory epithelial cancers treated with ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) naturally reactive with mutant p53 experienced limited clinical responses (2 PRs/12 patients), and we detected low frequencies, exhausted phenotypes, and poor persistence of the infused mutant p53-reactive TILs. Alternatively, we treated one patient with a chemorefractory breast cancer with ACT by transducing autologous peripheral blood lymphocytes with an HLA-A*02-restricted anti-p53R175H TCR. The infused cells exhibited an improved immunophenotype and prolonged persistence compared to the TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these data demonstrate the feasibility of off-the-shelf TCR-engineered cell therapies targeting shared p53 neoantigens to treat human cancers.

Tumor neoepitopes presented by major histocompatibility complex (MHC) class I are recognized by tumor-infiltrating lymphocytes (TIL) and are targeted by adoptive T-cell therapies. Identifying which mutant neoepitopes from tumor cells are capable of recognition by T cells can assist in the development of tumor-specific, cell-based therapies and can shed light on antitumor responses. Here, we generate a ranking algorithm for class I candidate neoepitopes by using next-generation sequencing data and a dataset of 185 neoepitopes that are recognized by HLA class I–restricted TIL from individuals with metastatic cancer. Random forest model analysis showed that the inclusion of multiple factors impacting epitope presentation and recognition increased output sensitivity and specificity compared to the use of predicted HLA binding alone. The ranking score output provides a set of class I candidate neoantigens that may serve as therapeutic targets and provides a tool to facilitate in vitro and in vivo studies aimed at the development of more effective immunotherapies. Robbins and colleagues develop and test a machine learning neoantigen ranking model using experimentally validated neoantigens from human tumors, providing a resource of targetable neoantigens for future immunotherapies.

Background Adoptive cell therapies (ACT) directed against the products of somatic mutations in cancer cells can lead to long lasting clinical responses. We focused on ACT against shared p53 mutations to be used to potentially treat a broad range of patients with common cancers. We have built a library of anti-mutant p53 T cell receptors (TCRs) to be used for the treatment of patients with epithelial cancers in the autologous setting and as ‘off-the-shelf’ reagents for patients sharing the same p53 mutation and HLA. Methods Tumor infiltrating lymphocytes (TILs) were screened for recognition of p53 mutations and were expanded as previously described.1 For treatment of patient 4349 with metastatic breast cancer, the patient‘s peripheral blood T cells were retrovirally engineered to express the allogeneic anti-p53 R175H TCR. Results We identified TILs recognizing ‘hotspot’ p53 mutations, such as R175H, Y220C, and R273C as well as less frequent but recurrent mutations, such as L111R, C135Y, and Q331H (table 1). First, we adoptively transferred TILs that included T cells reactive to a p53 mutation in an autologous manner for the treatment of patients with metastatic epithelial cancers (n=12). Except for the two patients who exhibited an objective response (RECIST), most of the patients did not respond to the therapy, possibly due to low frequencies of anti-mutant p53 cells in the infusion product, exhausted phenotype, and/or poor persistence (table 2). To overcome these barriers to TIL treatment, we retrovirally transduced autologous peripheral blood T cells to express an allogeneic anti-mutant p53 TCR. We engineered the HLA-A*02:01-restricted anti-p53 R175H TCR into patient 4349’s lymphocytes (transduction efficiency of 64%) and saw less expression of exhaustion markers relative to the TIL infusion products (table 2). This patient with metastatic breast cancer was refractory to the six prior chemotherapy regimens. After the transfer of 5.3e10 cells, the patient experienced an objective partial response, showing regression by 55% of skin and mediastinal lesions for 7 months. The persistence of the infused T cells was higher than the other patients who received the TIL treatment (table 2). Conclusions The library of anti-mutant p53 TCRs we have generated can potentially be used to treat ~6% of all cancer patients. We are pursuing the adoptive transfer of TILs against mutant p53 naturally occurring in the tumor or TCR-engineered cells using ‘off-the-shelf’ receptors against mutant p53. Ethics Approval This study was approved by the Institutional Review Board (IRB) of the NCI, and the approval numbers are as follows:Protocol 10-C-0166 (TIL treatment); Protocol 18-C-0049 (allogeneic TCR engineered T cell therapy) Reference Lo, W., et al., Immunologic Recognition of a Shared p53 Mutated Neoantigen in a Patient with Metastatic Colorectal Cancer. Cancer Immunol Res, 2019. 7(4): p. 534–543.

The adoptive transfer of tumor-infiltrating lymphocytes (TILs) expanded in vitro can mediate regressions of established tumors, yet only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells. This complexity underpins the need to phenotypically characterize T cells infiltrating the tumor microenvironment. In this study, we performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 45,000 T cells isolated from ten archival metastatic tumor samples whose primary histologies included colon, rectal, breast, and melanoma. By transcriptomic clustering of the T cells and mapping TCR identity data onto these blueprints, we observed a diversity of T cell phenotypes spanning from memory-like to dysfunctional effector-like, with clonal expansion occurring more widely in the effector-like cells. Through sequencing of peripheral blood lymphocytes (PBL) obtained at the time of tumor resection, we found T cell clusters that were either tumor enriched or distinctly PBL enriched, potentially representing recent tumor immigrants. Prior knowledge of 30 CD4 and CD8 tumor neoantigen-reactive TCRs (NeoTCRs) from eight patients allowed us to project these NeoTCRs onto the TIL transcriptomic map where we observed 380 total T cells bearing these NeoTCRs. The majority (>85%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, marked by expression of CXCL13, ENTPD1 (CD39), TOX, and PDCD1 (PD-1), indicating a dysfunctional state. We reasoned that other dominant T cell clonotypes within the NeoTCR clusters could contain additional novel NeoTCR clonotypes. We expressed these predicted NeoTCRs in donor PBL and screened them in vitro with antigen presenting cells (APCs) pulsed with peptides or tandem minigenes encoded by patient-specific tumor mutations, yielding 24 new NeoTCR clones (251 cells), which included neoantigen reactivities undetected in the same patients' fragment TIL screens. Using a NeoTCR signature score derived from the archival samples, three prospective patients' TIL were analyzed by scRNA/TCRseq and clusters scored according to their NeoTCR signature. TCRs reconstructed from these clusters and tested against patients' tumor-specific reagents yielded novel CD4 and CD8 NeoTCRs. Altogether, this study enabled successful enrichment and detection of tumor-specific NeoTCRs in the sequenced TIL of 12/12 patients for whom reactivity was either previously assessed or prospectively identified. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapy, as prospective NeoTCR isolation based on transcriptional phenotype of TIL will allow for rapid therapeutic development in the form of T cells expressing these tumor-specific TCRs. Citation Format: Frank J. Lowery, Sri Krishna, Rami Yossef, Praveen D. Chatani, Neilesh B. Parikh, Yong-Chen Lu, Nikolaos Zacharakis, Noam Levin, Ken-ichi Hanada, James C. Yang, Paul F. Robbins, Maria R. Parkhurst, Steven A. Rosenberg. Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer [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 127.

Purpose: The administration of autologous tumor-infiltrating lymphocytes (TILs) can mediate durable tumor regressions in patients with melanoma likely based on the recognition of immunogenic somatic mutations expressed by the cancer. There are limited data regarding the immunogenicity of mutations in breast cancer. We sought to identify immunogenic nonsynonymous mutations in a patient with triple-negative breast cancer (TNBC) to identify and isolate mutation-reactive TILs for possible use in adoptive cell transfer. Experimental Design: A TNBC metastasis was resected for TIL generation and whole-exome sequencing. Tandem minigenes or long 25-mer peptides encoding selected mutations were electroporated or pulsed onto autologous antigen-presenting cells, and reactivity of TIL was screened by upregulation of CD137 and IFNγ ELISPOT. The nature of the T-cell response against a unique nonsynonymous mutation was characterized. Results: We identified 72 nonsynonymous mutations from the tumor of a patient with TNBC. CD4+ and HLA-DRB1*1501–restricted TILs isolated from this tumor recognized a single mutation in RBPJ (recombination signal binding protein for immunoglobulin kappa J region). Analysis of 16 metastatic sites revealed that the mutation was ubiquitously present in all samples. Conclusions: Breast cancers can express naturally processed and presented unique nonsynonymous mutations that are recognized by a patient's immune system. TILs recognizing these immunogenic mutations can be isolated from a patient's tumor, suggesting that adoptive cell transfer of mutation-reactive TILs could be a viable treatment option for patients with breast cancer. Clin Cancer Res; 23(15); 4347–53. ©2017 AACR.

Summary T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.

Background & Translational Relevance: Adoptive transfer of tumor infiltrating lymphocytes (TIL) can effect long-term durable regression in patients with metastatic melanoma but has not been widely tested in common epithelial cancers. When examining the TIL of successfully treated patients with melanoma, a heterogeneous T cell population can be identified with reactivity against melanoma differentiation antigens, cancer germline antigens, and personalized non-synonymous somatic mutations. Common epithelial cancers, including breast cancer, express far fewer somatic mutations than melanoma, however, a patient with metastatic cholangiocarcinoma was treated with autologous CD4+ TIL enriched for neoantigen specificity and has experienced an ongoing partial response (>2 years). It is known that the presence of TIL on pathologic examination of triple-negative breast cancers is a positive prognostic marker for disease-free survival and overall survival. The identification of enriched populations of neoantigen-specific TIL could form the basis for personalized cell therapy for patients with metastatic breast cancer. This pilot study investigates the ability to grow TIL from breast cancer metastases, to identify personalized non-synonymous somatic mutations and potential neoantigens, and to adoptively transfer TIL into patients with breast cancer. Methods: Eligible patients were evaluated and treated under IRB-approved protocols for tissue procurement, genomic testing, and adoptive cell transfer. Portions of resected tumors were placed in culture under standard TIL conditions. DNA was extracted from tumor and matched normal peripheral blood samples for whole exome sequencing (WES). Non-synonymous somatic mutations were identified and tested for potential immunogenicity. Results: Nine patients have undergone surgical resection in this ongoing pilot study, and TIL was successfully grown from the tumors of all patients. All were primarily CD3+ (median 79%) with a small population of natural killer cells. Of the CD3+ cells, 7 of 9 patients had a predominantly CD4+ population (median CD4:CD8 ratio 2.2, range 0.4-5.8). With the exception of a single patient with inflammatory breast cancer, tumor purity allowed for WES of the tumors of eight patients, and non-synonymous somatic mutations were identified as potential neoantigens (median 96.5, range 71-148). Autologous T cell reactivity has been identified against tumor-specific mutations in 4 of 6 patients studied. The TIL of one patient demonstrated in vitro reactivity to a mutated form of RBPJ, a DNA-binding protein involved in Notch1 signaling. In addition, specimens obtained from this patient at autopsy contained the specific RBPJ mutation (RBPJ c.A611T) in every sampled tumor (n=16). Other patient-specific neoantigens identified by autologous reactivity include SLC3A2, KIAA0368, and a mutated TCRBV domain. Conclusions: Tumor-infiltrating lymphocytes derived from metastatic breast cancer can react to tumor-specific non-synonymous somatic mutations in vitro. TIL grown from breast cancers are predominantly CD4+ and can form the basis of an adoptive cell transfer experimental approach to patients with metastatic breast cancer. Citation Format: Goff SL, Zacharakis N, Assadipour Y, Prickett T, Gartner J, Somerville R, Black MA, Xu H, Chinnasamy H, Kriley I, Lu L, Statler M, Wunderlich J, Robbins PF, Rosenberg SA, Feldman SA. Recognition of autologous neoantigens by tumor infiltrating lymphocytes derived from breast cancer metastases [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-02.

Introduction: Adoptive transfer of tumor infiltrating lymphocytes (TIL) can mediate long-term durable regression in patients with metastatic melanoma, a type of cancer which is characterized by a high number of mutated genes and pronounced lymphocytic infiltrate. In contrast, metastatic breast cancer expresses far fewer somatic mutations and may contain lower levels of TIL. This pilot study investigated the ability to identify immunogenic non-synonymous somatic mutations which can serve as targets for adoptively transferred autologous T cells into patients with metastatic breast cancer, refractory to other treatments. Methods: WES and RNAseq were performed on metastatic lesions resected in the Surgery branch, NCI, NIH from patients with metastatic breast cancer. Non-synonymous somatic mutations were identified and, in parallel, TIL from the same lesions were grown ex vivo in the presence of IL-2. The somatic mutations were tested for recognition by the autologous TIL using previously described functional assays, involving tandem mini-genes (TMG), long peptides or predicted minimal peptide approaches. For some patients, the mutation-reactive T cells were sorted by FACS and the respective TCR was identified by single cell sequencing. Results: Metastatic tumor lesions from 37 patients with breast cancer were studied and all of them were found to contain and express non-synonymous somatic mutations (range: 4-1788, median: 102). TIL were successfully grown ex vivo from the tumors of all patients. Following functional assays (IFN-γ secretion and 4-1BB/OX-40 upregulation), it was found that autologous TIL recognized at least one (range: 1-11, median: 3) mutated product in 25 of 37 patients (68%) tested. Seventy-five percent of those were recognized by CD4+ and 25% by CD8+ T cells. All identified immunogenic tumor mutations (n=88) were unique and not shared by other patients. Sixty-four unique TCR pairs were isolated from the reactive cells and verified to recognize 30 immunogenic tumor mutations (range 1-7 TCRs/neoantigen). Conclusions: Patients with metastatic breast cancer contain a substantial number of non-synonymous somatic mutations and mutation-reactive TIL were found in the majority of those patients. The immunogenicity of tumor-specific mutations in metastatic breast cancer can be the platform for a highly personalized adoptive T cell transfer of selected TIL or mutation-reactive TCR transduced T cells targeting those tumor mutated genes. Citation Format: Nikolaos Zacharakis, Peter Kim, Paul Robbins, Jared Gartner, Todd Prickett, Victoria Hill, Biman Paria, Steven Feldman, Stephanie Goff, Steven Rosenberg. Tumor infiltrating lymphocytes (TIL) recognize a unique and diverse number of non-synonymous somatic mutations in patients with metastatic breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1068.

Immunotherapy using either checkpoint blockade or the adoptive transfer of antitumor lymphocytes has shown effectiveness in treating cancers with high levels of somatic mutations—such as melanoma, smoking-induced lung cancers and bladder cancer—with little effect in other common epithelial cancers that have lower mutation rates, such as those arising in the gastrointestinal tract, breast and ovary1–7. Adoptive transfer of autologous lymphocytes that specifically target proteins encoded by somatically mutated genes has mediated substantial objective clinical regressions in patients with metastatic bile duct, colon and cervical cancers8–11. We present a patient with chemorefractory hormone receptor (HR)-positive metastatic breast cancer who was treated with tumor-infiltrating lymphocytes (TILs) reactive against mutant versions of four proteins—SLC3A2, KIAA0368, CADPS2 and CTSB. Adoptive transfer of these mutant-protein-specific TILs in conjunction with interleukin (IL)-2 and checkpoint blockade mediated the complete durable regression of metastatic breast cancer, which is now ongoing for > 22 months, and it represents a new immunotherapy approach for the treatment of these patients.

Several high tibial osteotomy (HTO) surgical techniques for the treatment of medial osteoarthritis of the varus knee have been reported. Their main objectives are the achievement of the precise correction that is necessary for the lower limb mechanical axis realignment and the alleviation of the medial joint space. Early or late recurrence of the varus deformity must be avoided and various methods of fixation have been proposed to obtain this. We present a method of single level oblique HTO with no bone removal and with blade plate fixation for the treatment of medial osteoarthritis of the knee. One hundred seventeen patients (136 knees) were operated using this technique during a 12.2-year period. In 4.5 years follow-up there were 73.8% satisfactory results while in 8.4 years follow-up there were 61.1% satisfactory results. There were 11 complications: five patients with deep vein thrombosis, four with superficial wound infection and two with temporary peroneal nerve dysfunction. This study presents the operative technique in details, evaluates the results and assesses the advantages and disadvantages of the method in relation to other techniques described in the literature.

Background: Adoptive transfer of tumor infiltrating lymphocytes (TIL) can effect long-term durable regression in patients with metastatic melanoma but has not been widely tested in common epithelial cancers. Similar to metastatic melanoma, recent studies on epithelial GI cancers report that tumor regressions can be mediated by adoptively transferred TIL recognizing non-synonymous somatic mutations. Breast cancer metastatic tumors are infiltrated by TIL and reactivity against autologous somatic mutations can be identified. Methods: TIL were grown from a metastatic soft tissue tumor of a patient with hormone and chemotherapy-refractory ER+, HER2- breast cancer. DNA was extracted from tumor and matched normal peripheral blood samples for whole exome sequencing (WES) and RNAseq. Non-synonymous somatic mutations were identified and tested for potential recognition by autologous TIL using previously described tandem mini-gene and long peptide approaches. Recognition was assessed by IFN-γ release on ELISPOT and/or CD137 (4-1BB) upregulation with appropriate controls. Mutation reactive TIL were rapidly expanded in culture and transferred back to the patient. Adoptive cell transfer was preceded by a lymphodepleting preparative regimen and one dose of pembrolizumab. Cells were supported post-transfer with intravenous IL-2 administration, and pembrolizumab was administered for three additional doses. Deep sequencing of TCR rearrangement sequences was performed on the TIL and the pre- and post-infusion peripheral blood. Results: The resected subcutaneous tumor no longer expressed ER and was negative for PD-L1 staining. WES/RNASeq identified 96 non-synonymous mutations for testing. Multiple CD4+ clones specifically recognizing a mutation expressed in the solute carrier SLC3A2 (p.K94T) and a single CD8+ clone recognizing a mutation in the proteasome-associated protein KIAA0368 (p.S186F) were identified with no recognition of wild-type peptides. These eight T cell clones constituted 23% of the infusion bag TIL. Nine months after transfer of 82x109 cells, the patient has an ongoing partial response, with target lesions down 96% from baseline, including multiple hepatic metastases and disabling brachial plexus adenopathy. Seven of the eight T cell clones with known reactivity persisted in the peripheral blood comprising 2.4% of all CDR3 sequences at 7 months. Only two of these TCR clones were detectable, with frequencies totaling 0.005%, in the pre-treatment circulation. Conclusions: Tumor-infiltrating lymphocytes derived from a patient with metastatic breast cancer recognized tumor-specific non-synonymous somatic mutations, expanded in vitro and were used for adoptive cell transfer. TIL, co-administered with IL-2 and a short course of pembrolizumab, persisted in vivo and mediated regression of soft tissue, nodal and visceral metastases of breast cancer. Citation Format: Nikolaos Zacharakis, Katarzyna Trebska-McGowan, Robert Somerville, Yong-Chen Lu, Anna Pasetto, Mary Black, Harshini Chinnasamy, Hui Xu, Jared J. Gartner, Todd D. Prickett, Paul F. Robbins, Steven A. Rosenberg, Stephanie L. Goff, Steven A. Feldman. Regression of metastatic breast cancer after adoptive cell transfer of tumor infiltrating lymphocytes and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2017-4982

Systemic sclerosis (SSc) is a chronic autoimmune disease, characterized by immune system dysregulation, uncontrolled deposition of collagen and microvascular damage in the skin and several internal organs. T cells have a key role in the initiation and propagation of SSc. Our laboratory has previously identified the presence of high proportions of identical α- and β-chains TCR transcripts in skin biopsies from patients with SSc of recent onset, demonstrating clonal expansion of T cells in response to one or more antigens. In order to identify these antigens, pairs of those in vivo clonally expanded α- and β-chain TCR transcripts from two patients with SSc (SSc-21 and SSc-22) were expressed in mutant TCR negative T cells, J.RT3-T3.5, generating a total of 52 T cell lines, including 10 controls. An intracellular Ca2+ mobilization assay was employed to examine whether these transduced Jurkat T cell lines recognize putative SSc antigens presented by autologous EBV-transformed B cell lines. The self-antigen, DNA topoisomerase I and CMV and parvovirus antigens were tested. Statistically significant intracellular Ca2+ mobilization was observed in response to 3 DNA topoisomerase I and 2 CMV peptides by 5 T cell lines expressing clonally expanded α- and β-chain TCR transcripts from patient SSc-21 and SSc-22. These results demonstrate that clonally expanded T cells recognizing DNA topoisomerase I or CMV antigens are present in skin biopsies of recent onset of patients with SSc.

We describe three distinct types of congenital hip disease in adults. The first type is dysplasia, in which the femoral head is contained within the original true acetabulum. The second type is low dislocation, in which the femoral head articulates with a false acetabulum, the inferior lip of which contacts or overlaps the superior lip of the true acetabulum, giving the appearance of two overlapping acetabula. The third type is high dislocation, in which the femoral head has migrated superoposteriorly and there is no contact between the true and the false acetabulum. We describe and classify the acetabular abnormalities and deficiencies found with these three types. If the anterior, posterior, and superior aspects of the acetabular component cannot be covered during a total hip arthroplasty because of a deficient acetabulum in an adult who has congenital hip disease, we advocate an acetabuloplasty technique (which we have named a cotyloplasty) that involves medial advancement of the acetabular floor by the creation of a controlled comminuted fracture of its medial wall, autogenous bone-grafting, and the implantation of a small acetabular component with cement. This procedure was performed in sixty-six patients (eighty-six hips). Forty-nine of the hips had a high dislocation, thirty-one had a low dislocation, and six were dysplastic. Two to fifteen years (mean, seven years) after the operation, the clinical and radiographic results were satisfactory. Only two acetabular components needed to be revised for aseptic loosening, at 5.3 and 7.5 years postoperatively. Moreover, the cumulative success rate for the acetabular components was 100 per cent at five years and 93.2 per cent at ten years.

Systemic Sclerosis (SSc) is a chronic autoimmune disease characterized by excessive deposition of collagen in the skin and many internal organs, production of autoantibodies and microvascular alterations. Previous studies in our laboratory have demonstrated the presence of high proportions of identical β-chains TCR transcripts in skin biopsies from patients with SSc of recent onset, suggestive of clonal expansions of T cells. To identify the antigen(s) recognized by clonally expanded T cells in SSc biopsies, we examine the clonality of the α-chain TCR transcripts in patients with SSc. Sequence analysis of α-chain TCR transcripts following non-palindromic PCR/Vα specific PCR and cloning revealed the presence of clonally expanded α-chain TCR transcripts in skin biopsies from four patients with SSc. Substantial proportion of identical α-chain TCR transcripts were found in these skin biopsies accounting from 16% to 32% of the transcripts sequenced. These clonal expansions were confirmed with Vα-specific PCR followed by cloning and sequencing. Identical α-chain TCR transcripts, ranging from 68% to 100%, were identified in individual Vα families. In one of these patients an identical T cell clone was clonally expanded in both peripheral blood and skin. These T cells have undergone clonal expansion in response to, as yet unidentified, specific antigen(s).

The long term outcome of 84 Charnley low friction arthroplasties performed between January 1973 and December 1984 on 69 patients (84 hips) with osteoarthritis was assessed. The patients were 55 years old or younger (mean, 46 years) at the time of surgery. The probability of survival was 89.8% (range, 85.9%-93.7%) after 10 years with 69 hips at risk and 73.3% (range, 65.5%-81.1%) after 18 years with 33.5 hips at risk. Of the 84 hips in the study, 24 (28.6%) failed. Of the hips that survived, clinical results were good and excellent with Charnley scores of four or more for pain and function in all hips. Of the hips that survived, 93.3% achieved good or excellent results for movement.

In a 12- to 18-year radiographic follow-up study of 95 Charnley low-friction total hip arthroplasties (THA), there was a statistically significant correlation between location of the center of rotation of the total hip prosthesis (notably the element horizontal distance cup to tear-drop) and long-term, unfavorable, radiographic signs, such as acetabular and stem demarcation, cup wear, cup migration, subsidence of the stem, and calcar resorption. Small differences, as little as 2 mm, in the placement of the cup in relation to anatomic landmarks were responsible for these findings. A logistical regression analysis showed that the horizontal distance cup to teardrop was the most significant parameter influencing long-term unfavorable radiographic signs of a THA in comparison with the parameters of age, gender, diagnosis, and body-weight.