Characterization of somatic mutations in sporadic uveal melanoma and uveal melanoma in patients with germline BAP1 pathogenic variants.

Genetic analyses were conducted on tumor samples from 88 patients with uveal melanoma (UM), 6 of whom carry pathogenic germline variants in BAP1. We assessed the frequency, pattern, and prognostic significance of somatic aberrations, and investigated differences between germline BAP1 variant carriers compared to sporadic cases. The frequency of the main oncogenic driver mutations was not significantly different between these groups. Patients with germline BAP1 variants did not have significantly different overall survival compared to the wildtype or somatic BAP1 mutation groups. Patients with a somatic BAP1 mutation (n = 24) had a significantly worse prognosis compared to wildtype (n = 58). All patients with stage III tumors and a somatic BAP1 mutation (n = 7) developed metastasis, however four of 28 stage I-II tumors without metastasis had somatic BAP1 mutations, with observation time >5 years. The tumor from one germline BAP1 carrier (stage IIIC) with a somatic EIF1AX splice variant, has not developed metastasis within a 22-year observation time.

Tumour immune characterisation of primary triple-negative breast cancer using automated image quantification of immunohistochemistry-stained immune cells.

The tumour immune microenvironment (TIME) in breast cancer is acknowledged with an increasing role in treatment response and prognosis. With a growing number of immune markers analysed, digital image analysis may facilitate broader TIME understanding, even in single-plex IHC data. To facilitate analyses of the latter an open-source image analysis pipeline, Tissue microarray MArker Quantification (TMArQ), was developed and applied to single-plex stainings for p53, CD3, CD4, CD8, CD20, CD68, FOXP3, and PD-L1 (SP142 antibody) in a 218-patient triple negative breast cancer (TNBC) cohort with complementary pathology scorings, clinicopathological, whole genome sequencing, and RNA-sequencing data. TMArQ's cell counts for analysed immune markers were on par with results from alternative methods and consistent with both estimates from human pathology review, different quantifications and classifications derived from RNA-sequencing as well as known prognostic patterns of immune response in TNBC. The digital cell counts demonstrated how immune markers are coexpressed in the TIME when considering TNBC molecular subtypes and DNA repair deficiency, and how combination of immune status with DNA repair deficiency status can improve the prognostic stratification in chemotherapy treated patients. These results underscore the value and potential of integrating TIME and specific tumour intrinsic alterations/phenotypes for the molecular understanding of TNBC.

Molecular patterns of resistance to immune checkpoint blockade in melanoma.

Immune checkpoint blockade (ICB) has improved outcome for patients with metastatic melanoma but not all benefit from treatment. Several immune- and tumor intrinsic features are associated with clinical response at baseline. However, we need to further understand the molecular changes occurring during development of ICB resistance. Here, we collect biopsies from a cohort of 44 patients with melanoma after progression on anti-CTLA4 or anti-PD1 monotherapy. Genetic alterations of antigen presentation and interferon gamma signaling pathways are observed in approximately 25% of ICB resistant cases. Anti-CTLA4 resistant lesions have a sustained immune response, including immune-regulatory features, as suggested by multiplex spatial and T cell receptor (TCR) clonality analyses. One anti-PD1 resistant lesion harbors a distinct immune cell niche, however, anti-PD1 resistant tumors are generally immune poor with non-expanded TCR clones. Such immune poor microenvironments are associated with melanoma cells having a de-differentiated phenotype lacking expression of MHC-I molecules. In addition, anti-PD1 resistant tumors have reduced fractions of PD1+ CD8+ T cells as compared to ICB naïve metastases. Collectively, these data show the complexity of ICB resistance and highlight differences between anti-CTLA4 and anti-PD1 resistance that may underlie differential clinical outcomes of therapy sequence and combination.

BioMEL: a translational research biobank of melanocytic lesions and melanoma.

INTRODUCTION: Diagnosing invasive cutaneous melanoma (CM) can be challenging due to subjectivity in distinguishing equivocal nevi, melanoma in situ and thin CMs. The underlying molecular mechanisms of progression from nevus to melanoma must be better understood. Identifying biomarkers for treatment response, diagnostics and prognostics is crucial. Using biomedical data from biobanks and population-based healthcare data, translational research can improve patient care by implementing evidence-based findings. The BioMEL biobank is a prospective, multicentre, large-scale biomedical database on equivocal nevi and all stages of primary melanoma to metastases. Its purpose is to serve as a translational resource, enabling researchers to uncover objective molecular, genotypic, phenotypic and structural differences in nevi and all stages of melanoma. The main objective is to leverage BioMEL to significantly improve diagnostics, prognostics and therapy outcomes of patients with melanoma. METHODS AND ANALYSIS: The BioMEL biobank contains biological samples, epidemiological information and medical data from adult patients who receive routine care for melanoma. BioMEL is focused on primary and metastatic melanoma, but equivocal pigmented lesions such as clinically atypical nevi and melanoma in situ are also included. BioMEL data are gathered by questionnaires, blood sampling, tumour imaging, tissue sampling, medical records and histopathological reports. ETHICS AND DISSEMINATION: The BioMEL biobank project is approved by the national Swedish Ethical Review Authority (Dnr. 2013/101, 2013/339, 2020/00469, 2021/01432 and 2022/02421-02). The datasets generated are not publicly available due to regulations related to the ethical review authority. TRIAL REGISTRATION NUMBER: NCT05446155.

Intratumoral T-cell and B-cell receptor architecture associates with distinct immune tumor microenvironment features and clinical outcomes of anti-PD-1/L1 immunotherapy.

BACKGROUND: Effective cooperation between B-cells and T-cells within the tumor microenvironment may lead to the regression of established tumors. B-cells and T-cells can recognize tumor antigens with exquisite specificity via their receptor complexes. Nevertheless, whether a diverse intratumoral B-cells and T-cell receptor (BCR, TCR) repertoire affects the tumor immune microenvironment (TIME) and clinical outcomes in patients treated with immunotherapy is unclear. METHODS: We extracted information on BCR and TCR repertoire diversity from large clinical datasets and measured the association between immune receptor diversity features, the TIME, and clinical outcomes of patients treated with anti-PD-1/PD-L1 immunotherapy. RESULTS: In multiple tumor types, an increasingly diverse TCR repertoire was strongly associated with a highly activated TIME, while BCR diversity was more associated with antibody responses but not with the overall B-cell infiltration nor with measures related to intratumoral CD8+T cell activity. Neither TCR nor BCR diversity was independent prognostic biomarkers of survival across multiple cancer types. However, both TCR and BCR diversity improved the performance of predictive models combined with established biomarkers of response to immunotherapy. CONCLUSION: Overall, these data indicate a currently unexplored immunological role of intratumoral B-cells associated with BCR diversity and antibody responses but independent of classical anticancer T-cells intratumoral activities.

DNA promoter hypermethylation of melanocyte lineage genes determines melanoma phenotype.

Cellular stress contributes to the capacity of melanoma cells to undergo phenotype switching into highly migratory and drug-tolerant dedifferentiated states. Such dedifferentiated melanoma cell states are marked by loss of melanocyte-specific gene expression and increase of mesenchymal markers. Two crucial transcription factors, microphthalmia-associated transcription factor (MITF) and SRY-box transcription factor 10 (SOX10), important in melanoma development and progression, have been implicated in this process. In this study we describe that loss of MITF is associated with a distinct transcriptional program, MITF promoter hypermethylation, and poor patient survival in metastatic melanoma. From a comprehensive collection of melanoma cell lines, we observed that MITF-methylated cultures were subdivided in 2 distinct subtypes. Examining mRNA levels of neural crest-associated genes, we found that 1 subtype had lost the expression of several lineage genes, including SOX10. Intriguingly, SOX10 loss was associated with SOX10 gene promoter hypermethylation and distinct phenotypic and metastatic properties. Depletion of SOX10 in MITF-methylated melanoma cells using CRISPR/Cas9 supported these findings. In conclusion, this study describes the significance of melanoma state and the underlying functional properties explaining the aggressiveness of such states.

Infection is associated with increased risk of MPO- but not PR3-ANCA-associated vasculitis.

OBJECTIVES: To determine whether development of ANCA-associated vasculitis (AAV) shows a relationship with a prior infection and if prior infection affects disease characteristics and outcome. METHODS: All incident cases of AAV diagnosed in a defined region of Sweden from 2000 through 2016 were identified. For each case, 10 individuals from the general population, matched for age, sex and area of residence, were selected. Infections occurring in AAV patients and controls prior to the date of AAV diagnosis (index date for respective controls) were identified using an administrative database. Conditional logistic regression models were used to calculate odds ratios (OR) of developing AAV. Occurrence, clinical characteristics and outcome of AAV were analysed with respect to prior infection. RESULTS: Two-hundred and seventy patients with AAV (48% female) and 2687 controls were included. Prior to diagnosis/index date, 146 (54%) AAV patients had been diagnosed with infection vs 1282 (48%) controls, with OR for AAV 1.57 (95% CI 1.18, 2.19) in those with infections of the upper respiratory tract and 1.68 (1.02, 2.77) in those with pneumonia. Difference from controls was significant in patients with MPO-ANCA 1.99 (95% CI 1.25, 3.1) but not in those with PR3-ANCA 1.0 (0.61, 1.52). Patients with prior infection showed higher disease activity at AAV diagnosis. No differences in disease characteristics, comorbidities or outcome in those with and without prior infections were observed. CONCLUSIONS: Respiratory tract infections are positively associated with development of MPO- but not PR3-ANCA vasculitis. Prior infection is associated with higher disease activity at AAV diagnosis.

Neoantigen-reactive CD8+ T cells affect clinical outcome of adoptive cell therapy with tumor-infiltrating lymphocytes in melanoma.

BACKGROUNDNeoantigen-driven recognition and T cell-mediated killing contribute to tumor clearance following adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs). Yet how diversity, frequency, and persistence of expanded neoepitope-specific CD8+ T cells derived from TIL infusion products affect patient outcome is not fully determined.METHODSUsing barcoded pMHC multimers, we provide a comprehensive mapping of CD8+ T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic melanoma patients who received ACT.RESULTSWe identified 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observed neoepitope-specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we found that the frequency of neoepitope-specific CD8+ T cells in TIL infusion products correlated with increased survival and that neoepitope-specific CD8+ T cells shared with the infusion product in posttreatment blood samples were unique to responders of TIL-ACT. Finally, we found that a transcriptional signature for lymphocyte activity within the tumor microenvironment was associated with a higher frequency of neoepitope-specific CD8+ T cells in the infusion product.CONCLUSIONSThese data support previous case studies of neoepitope-specific CD8+ T cells in melanoma and indicate that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8+ T cells.FUNDINGNEYE Foundation; European Research Council; Lundbeck Foundation Fellowship; Carlsberg Foundation.

B Cells and Tertiary Lymphoid Structures: Friends or Foes in Cancer Immunotherapy?

Tumor cells pose a challenge to the adaptive immune system, and its key cell types, T and B cells, have frequently been associated with an improved prognosis. The success of immune checkpoint blockade has confirmed the relevance of T cells. However, the role of B cells is increasingly recognized, and highlighted in this review. Recent data suggest that tumors contain a diverse set of B cells reflecting different developmental states and exerting functions such as antigen presentation, antibody production, and regulatory effects. Further, B cells are frequently located in tertiary lymphoid structures (TLS), which are immune cell niches that sustain an immune response at sites of chronic inflammation. TLSs in tumors display substantial heterogeneity, ranging from cell aggregates to mature structures with an active germinal center. Recent studies have provided insights into initiation, cellular and spatial composition, and function of TLS in a variety of cancer types; however, several critical issues still need to be resolved. Currently, initial reports are discerning the role of TLSs in immunotherapy, with the majority of studies observing TLSs to confer favorable patient outcome. Finally, TLS induction in tumors is evaluated, with the therapeutic aim to reactivate the host immune response.

Transcriptomic signatures of tumors undergoing T cell attack.

BACKGROUND: Studying tumor cell-T cell interactions in the tumor microenvironment (TME) can elucidate tumor immune escape mechanisms and help predict responses to cancer immunotherapy. METHODS: We selected 14 pairs of highly tumor-reactive tumor-infiltrating lymphocytes (TILs) and autologous short-term cultured cell lines, covering four distinct tumor types, and co-cultured TILs and tumors at sub-lethal ratios in vitro to mimic the interactions occurring in the TME. We extracted gene signatures associated with a tumor-directed T cell attack based on transcriptomic data of tumor cells. RESULTS: An autologous T cell attack induced pronounced transcriptomic changes in the attacked tumor cells, partially independent of IFN-γ signaling. Transcriptomic changes were mostly independent of the tumor histological type and allowed identifying common gene expression changes, including a shared gene set of 55 transcripts influenced by T cell recognition (Tumors undergoing T cell attack, or TuTack, focused gene set). TuTack scores, calculated from tumor biopsies, predicted the clinical outcome after anti-PD-1/anti-PD-L1 therapy in multiple tumor histologies. Notably, the TuTack scores did not correlate to the tumor mutational burden, indicating that these two biomarkers measure distinct biological phenomena. CONCLUSIONS: The TuTack scores measure the effects on tumor cells of an anti-tumor immune response and represent a comprehensive method to identify immunologically responsive tumors. Our findings suggest that TuTack may allow patient selection in immunotherapy clinical trials and warrant its application in multimodal biomarker strategies.

Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens.

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.

Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

Clinical efficacy of T-cell therapy after short-term BRAF-inhibitor priming in patients with checkpoint inhibitor-resistant metastatic melanoma.

PURPOSE: Despite impressive response rates following adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, improvement is needed to increase the efficacy and broaden the applicability of this treatment. We evaluated the use of vemurafenib, a small-molecule BRAF inhibitor with immunomodulatory properties, as priming before TIL harvest and adoptive T cell therapy in a phase I/II clinical trial. METHODS: 12 patients were treated with vemurafenib for 7 days before tumor excision and during the following weeks until TIL infusion. TILs were grown from tumor fragments, expanded in vitro and reinfused to the patient preceded by a lymphodepleting chemotherapy regimen and followed by interleukin-2 infusion. Extensive immune monitoring, tumor profiling and T cell receptor sequencing were performed. RESULTS: No unexpected toxicity was observed, and treatment was well tolerated. Of 12 patients, 1 achieved a complete response, 8 achieved partial response and 3 achieved stable disease. A PR and the CR are ongoing for 23 and 43 months, respectively. In vitro anti-tumor reactivity was found in TILs from 10 patients, including all patients achieving objective response. Serum and tumor biomarker analyses indicate that baseline cytokine levels and the number of T cell clones may predict response to TIL therapy. Further, TCR sequencing suggested skewing of TCR repertoire during in vitro expansion, promoting certain low frequency clonotypes. CONCLUSIONS: Priming with vemurafenib before infusion of TILs was safe and feasible, and induced objective clinical responses in this cohort of patients with checkpoint inhibitor-resistant metastatic melanoma. In this trial, vemurafenib treatment seemed to decrease attrition and could be considered to bridge the waiting time while TILs are prepared.

BRN2 is a non-canonical melanoma tumor-suppressor.

While the major drivers of melanoma initiation, including activation of NRAS/BRAF and loss of PTEN or CDKN2A, have been identified, the role of key transcription factors that impose altered transcriptional states in response to deregulated signaling is not well understood. The POU domain transcription factor BRN2 is a key regulator of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, in a BrafV600E PtenF/+ context, we show that BRN2 haplo-insufficiency promotes melanoma initiation and metastasis. However, metastatic colonization is less efficient in the absence of Brn2. Mechanistically, BRN2 directly induces PTEN expression and in consequence represses PI3K signaling. Moreover, MITF, a BRN2 target, represses PTEN transcription. Collectively, our results suggest that on a PTEN heterozygous background somatic deletion of one BRN2 allele and temporal regulation of the other allele elicits melanoma initiation and progression.

PTENP1-AS contributes to BRAF inhibitor resistance and is associated with adverse clinical outcome in stage III melanoma.

BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.

Tumour gene expression signature in primary melanoma predicts long-term outcomes.

Adjuvant systemic therapies are now routinely used following resection of stage III melanoma, however accurate prognostic information is needed to better stratify patients. We use differential expression analyses of primary tumours from 204 RNA-sequenced melanomas within a large adjuvant trial, identifying a 121 metastasis-associated gene signature. This signature strongly associated with progression-free (HR = 1.63, p = 5.24 × 10-5) and overall survival (HR = 1.61, p = 1.67 × 10-4), was validated in 175 regional lymph nodes metastasis as well as two externally ascertained datasets. The machine learning classification models trained using the signature genes performed significantly better in predicting metastases than models trained with clinical covariates (pAUROC = 7.03 × 10-4), or published prognostic signatures (pAUROC < 0.05). The signature score negatively correlated with measures of immune cell infiltration (ρ = -0.75, p < 2.2 × 10-16), with a higher score representing reduced lymphocyte infiltration and a higher 5-year risk of death in stage II melanoma. Our expression signature identifies melanoma patients at higher risk of metastases and warrants further evaluation in adjuvant clinical trials.

Infections Are Associated With Increased Risk of Giant Cell Arteritis: A Population-based Case-control Study from Southern Sweden.

OBJECTIVE: To investigate the association between infections and the subsequent development of giant cell arteritis (GCA) in a large population-based cohort from a defined geographic area in Sweden. METHODS: Patients diagnosed with biopsy-confirmed GCA between 2000 and 2016 were identified through the database of the Department of Pathology in Skåne, the southernmost region of Sweden. For each GCA case, 10 controls matched for age, sex, and area of residence were randomly selected from the general population. Using the Skåne Healthcare Register, we identified all infection events prior to patients' date of GCA diagnosis and controls' index date. With infection as exposure, a conditional logistic regression model was employed to estimate the OR for developing GCA. The types of infections contracted nearest in time to the GCA diagnosis/index date were identified. RESULTS: A total of 1005 patients with biopsy-confirmed GCA (71% female) and 10,050 controls were included in the analysis. Infections were more common among patients subsequently diagnosed with GCA compared to controls (51% vs 41%, OR 1.78, 95% CI 1.53-2.07). Acute upper respiratory tract infection (OR 1.77, 95% CI 1.47-2.14), influenza and pneumonia (OR 1.72, 95 % CI 1.35-2.19), and unspecified infections (OR 5.35, 95 % CI 3.46-8.28) were associated with GCA. Neither skin nor gastrointestinal infections showed a correlation. CONCLUSION: Infections, especially those of the respiratory tract, were associated with subsequent development of biopsy-confirmed GCA. Our findings support the hypothesis that a range of infections may trigger GCA.

Qualitative Analysis of Tumor-Infiltrating Lymphocytes across Human Tumor Types Reveals a Higher Proportion of Bystander CD8+ T Cells in Non-Melanoma Cancers Compared to Melanoma.

Background: Human intratumoral T cell infiltrates can be defined by quantitative or qualitative features, such as their ability to recognize autologous tumor antigens. In this study, we reproduced the tumor-T cell interactions of individual patients to determine and compared the qualitative characteristics of intratumoral T cell infiltrates across multiple tumor types. Methods: We employed 187 pairs of unselected tumor-infiltrating lymphocytes (TILs) and autologous tumor cells from patients with melanoma, renal-, ovarian-cancer or sarcoma, and single-cell RNA sequencing data from a pooled cohort of 93 patients with melanoma or epithelial cancers. Measures of TIL quality including the proportion of tumor-reactive CD8+ and CD4+ TILs, and TIL response polyfunctionality were determined. Results: Tumor-specific CD8+ and CD4+ TIL responses were detected in over half of the patients in vitro, and greater CD8+ TIL responses were observed in melanoma, regardless of previous anti-PD-1 treatment, compared to renal cancer, ovarian cancer and sarcoma. The proportion of tumor-reactive CD4+ TILs was on average lower and the differences less pronounced across tumor types. Overall, the proportion of tumor-reactive TILs in vitro was remarkably low, implying a high fraction of TILs to be bystanders, and highly variable within the same tumor type. In situ analyses, based on eight single-cell RNA-sequencing datasets encompassing melanoma and five epithelial cancers types, corroborated the results obtained in vitro. Strikingly, no strong correlation between the proportion of CD8+ and CD4+ tumor-reactive TILs was detected, suggesting the accumulation of these responses in the tumor microenvironment to follow non-overlapping biological pathways. Additionally, no strong correlation between TIL responses and tumor mutational burden (TMB) in melanoma was observed, indicating that TMB was not a major driving force of response. No substantial differences in polyfunctionality across tumor types were observed. Conclusions: These analyses shed light on the functional features defining the quality of TIL infiltrates in cancer. A significant proportion of TILs across tumor types, especially non-melanoma, are bystander T cells. These results highlight the need to develop strategies focused on the tumor-reactive TIL subpopulation.