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Review
Biomarkers for response to TIL therapy: a comprehensive review
  1. Víctor Albarrán Fernández1,2,
  2. Pablo Ballestín Martínez1,3,
  3. Joachim Stoltenborg Granhøj1,
  4. Troels Holz Borch1,
  5. Marco Donia1 and
  6. Inge Marie Svane1
  1. 1National Center for Cancer Immune Therapy (CCIT-DK), Department of Oncology, Copenhagen University Hospital, Herlev, Denmark
  2. 2Ramón y Cajal University Hospital, Department of Medical Oncology, Madrid, Spain
  3. 3Clínico San Carlos University Hospital, Department of Medical Oncology, Madrid, Spain
  1. Correspondence to Dr Inge Marie Svane; Inge.Marie.Svane{at}regionh.dk

Abstract

Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) has demonstrated durable clinical responses in patients with metastatic melanoma, substantiated by recent positive results of the first phase III trial on TIL therapy. Being a demanding and logistically complex treatment, extensive preclinical and clinical effort is required to optimize patient selection by identifying predictive biomarkers of response. This review aims to comprehensively summarize the current evidence regarding the potential impact of tumor-related factors (such as mutational burden, neoantigen load, immune infiltration, status of oncogenic driver genes, and epigenetic modifications), patient characteristics (including disease burden and location, baseline cytokines and lactate dehydrogenase serum levels, human leucocyte antigen haplotype, or prior exposure to immune checkpoint inhibitors and other anticancer therapies), phenotypic features of the transferred T cells (mainly the total cell count, CD8:CD4 ratio, ex vivo culture time, expression of exhaustion markers, costimulatory signals, antitumor reactivity, and scope of target tumor-associated antigens), and other treatment-related factors (such as lymphodepleting chemotherapy and postinfusion administration of interleukin-2).

  • Tumor infiltrating lymphocyte - TIL
  • Adoptive cell therapy - ACT
  • Immune Checkpoint Inhibitor
  • Biomarker
  • Solid tumor
http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jitc-2023-008640

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    Introduction

    The use of tumor-infiltrating lymphocytes (TIL) as a modality of adoptive cell therapy (ACT) has shown efficacy in advanced melanoma and promising early clinical results in some other malignancies. In contrast to single antigen-targeted ACT strategies, such as chimeric antigen receptor (CAR)-T or T-cell receptor (TCR)-engineered T cells, the antigenic heterogeneity encompassed by TIL and their intrinsic ability to traffic to the tumor site provide an exciting rationale for their application against solid tumors.1

    Essentially, candidates for this therapy undergo resection of a tumor lesion which will be mechanically fragmented or enzymatically digested. Tumor fragments are cultured in interleukin-2 (IL-2)-enriched media for 2–3 weeks, generating cultures of predominantly “young” TIL which will be further expanded in a rapid expansion protocol (REP) for 14 additional days—in the presence of IL-2, irradiated peripheral blood mononuclear cells (“feeder” cells), and anti-CD3 antibodies. Expanded TIL are harvested and administered to the patient after lymphodepleting cyclophosphamide and fludarabine-based chemotherapy (CT). Cell product infusion is usually followed by high doses of IL-2 to support the functionality of infused TIL in vivo.2 3

    Since the first trials conducted by Rosenberg and colleagues in the 1990s and the 2000s,4–7 subsequent studies have confirmed the efficacy of TIL therapy in metastatic melanoma (MM),8–11 with objective response rates (ORRs) ranging from 33% to 56%, and a significant rate of durable complete responses (CRs). A meta-analysis comprising 13 studies (410 patients) showed pooled overall ORR and CR rate estimates of 41% and 12%, respectively.12 In 2022, the first phase III trial involving TIL therapy showcased its superiority over the anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) antibody ipilimumab in patients with anti-PD-1-refractory MM (ORR 49% vs 21%).13 While evidence in other tumors is less extensive and provided by phase I/II trials, sustained responses have been reported in patients with uveal melanoma,14 non-small cell lung cancer (NSCLC),15 16 cervical cancer,17 18 head and neck squamous cell carcinoma,18 19 breast cancer,20 osteosarcoma,21 colorectal cancer,22 and cholangiocarcinoma.23 Strategies to improve TIL therapy including combinations with immune checkpoint inhibitors (ICI), selection of neoantigen-targeted TIL, and genetically engineered TIL via clustered regularly interspaced short palindromic repeat (CRISPR) technology comprise an exciting field of preclinical research.3

    However, TIL therapy faces practical obstacles that are hindering its incorporation into clinical practice, including elevated rates of toxicity, requirement of highly specialized facilities and human resources, extended manufacturing periods, and regulatory considerations.24 The challenges in its large-scale implementation underscore the relevance of identifying pertinent biomarkers for optimal patient selection. This article endeavors to provide a comprehensive review of current insights into predictors of response to TIL therapy, mainly based on published experience in cutaneous melanoma, encompassing patient and tumor characteristics alongside parameters associated with the manufacturing process and the TIL phenotype.

    The potential biomarkers for TIL-ACT efficacy are summarized in figure 1.

    Figure 1
    Figure 1

    Summarized representation of the potential patient-, tumor-, and treatment-related biomarkers for response to TIL therapy (in blue, purple, and green color, respectively). BRAF/MEKi, BRAF/MEK inhibitors; IL-2, interleukin-2; PD-1, programmed cell death protein 1; TIL, tumor-infiltrating lymphocyte.

    Tumor-related biomarkers

    Tumor mutational burden (TMB) and neoantigen load

    TMB is a predictor of response to ICI in melanoma25 and other tumors.26 27 In a phase I/II trial with TIL-ACT before the anti-PD-1 era, a higher TMB was correlated with longer progression-free survival (PFS) and overall survival (OS) in 27 patients with MM.28 Later, Levi et al29 reported higher TMB in responders both among anti-PD-1-naïve and -exposed patients, although other studies have found conflicting results.14 30–32

    A strong correlation between TMB and neoantigen load has been widely observed,28–31 and a high neoantigen load has been correlated with higher OS and ORR to TIL-ACT in different cohorts.28 31 Interestingly, patients with fewer than 200 predicted neoantigens seem to achieve poorer outcomes,31 suggesting low neoantigen load as a negative biomarker.

    Driver genes, genetic landscape, and epigenetic modifications

    BRAF mutation status has shown no association with TIL therapy outcomes in different clinical trials,7 13 28 29 33–35 and no significant association has been found between ORR and the status of other driver genes, such as RAS and NF1.7 28 In MM, outcomes do not seem to differ according to BRCA2 status, CDKN2A loss, IFN locus deletion, or B2M deletion.28 No differences have been found in DNA copy number gains at the HLA locus or in any other DNA regions.28

    A heightened expression of immune response-associated genes36 has been correlated with improved outcomes in MM,37 suggesting higher responsiveness to TIL-based immunotherapy. Following RNA sequencing of 25 MM samples from patients treated with TIL-ACT, upregulation of immune-associated genes, interferon (IFN)-γ signaling, and major histocompatibility complex (MHC) class I-dependent antigen presentation genes was associated with clinical benefit. Conversely, non-responders exhibited increased expression of cell cycle- and proliferation-associated genes.28 Single-cell transcriptomic signatures38 were applied to this cohort, suggesting that overall immune cell infiltration might favor TIL-ACT efficacy, although specific immune cell markers did not consistently correlate with clinical outcomes.28

    Further analysis of this cohort revealed a significant correlation between upregulation of CTLA-4 (T-cell exhaustion marker), HLA-C, and TAP2 (antigen presentation-related genes), and TIL-ACT efficacy. Other studies have found no association between immune signatures or infiltration and clinical responses,30 31 although they included heterogeneous populations with predominantly anti-PD-1-refractory patients.

    Following RNA sequencing of 64 melanoma samples, Creasy et al31 found a correlation between NGFR, PDE1C, and RTKN2 enrichment and ORR to TIL therapy. Contrarily, ELFN1 was consistently enriched in non-responders. In the same study, NGFR, PDE1C, and ELFN1 were found to be expressed in cancer-associated fibroblasts, and the latter two were expressed by endothelial cells within the tumor microenvironment (TME). The mechanisms of ELFN1 expression were studied in detail by this group, reporting a correlation between hypermethylation of the 5′-untranslated promoter region and subsequent decreased mRNA expression with clinical responses to TIL-ACT. In contrast, non-responders exhibited increased expression of glycolysis-related genes (GPI, PGAM4), which may favor TIL evasion by downregulating the expression of CXCL10, a T cell-attracting chemokine.39

    Other tumor characteristics

    In melanoma, responses to TIL therapy have been observed irrespective of mucosal (n=12) or non-mucosal histology in a phase II trial with 153 patients.34 Chandran et al14 reported an ORR of 32% from a 20-patient metastatic uveal melanoma cohort undergoing TIL-ACT, including one CR. In a phase II trial with TIL therapy lifileucel (LN-144), there was no clear correlation between PD-L1 expression and clinical responses, with similar ORR for patients with a PD-L1 Tumor Proportion Score <1% and ≥1%.34

    Patient-related biomarkers

    Baseline clinical characteristics

    Most patients have received TIL-ACT in clinical trials in which an Eastern Cooperative Oncology Group performance status (PS) of 0–1 was required or with a minority of patients with PS 2.13 30 32 34 40 41 As a result, evidence regarding potential outcomes of TIL-ACT in patients with poorer PS is lacking.

    Other patient-intrinsic variables such as sex, age, or HLA-A0201 frequency do not seem to differ between responders and non-responders, according to different studies.7 10 34 35 As an exception, Besser et al33 reported female sex as an independent poor prognostic marker after a multivariable analysis in 57 patients.

    Lactate dehydrogenase (LDH) serum levels and disease burden

    LDH stands as an independent negative prognostic factor in MM.42 LDH levels strongly correlated with poorer OS in a cohort of 74 patients undergoing TIL-ACT,32 with consistent results in other studies.10 33 Initial analysis of 66 patients from the LN-144 phase II trial showed responses to TIL-ACT irrespective of LDH levels surpassing the upper limit.11 However, elevated LDH levels and disease burden were both correlated with worse outcomes after an update with 153 patients.34 Higher disease burden and LDH levels were significantly correlated with poorer responses in another 226-patient cohort.35 Noteworthy, among 88 patients with elevated LDH levels, a response rate of 45% was observed, including 15 CRs. In contrast, LDH levels did not differ between responders and non-responders in other studies,7 including the phase III trial13—although it should be noted that patients with LDH levels more than two times the upper limit of the normal range were excluded from this trial.

    Patients with baseline liver and/or brain metastases did not achieve poorer responses in the phase II trial with LN-144. In a National Cancer Institute cohort of patients with MM,43 the ORR was 33% for those with central nervous system (CNS) metastases and 49% for those without CNS involvement. Although this difference was not statistically significant, OS was shorter in patients with brain metastases untreated prior to TIL therapy, and most of them required additional brain-specific treatment after TIL-ACT.

    Influence of prior systemic therapies

    The landscape of systemic treatment for MM has undergone profound changes with the widespread availability of ICI and BRAF/MEK inhibitors (BRAF/MEKi). Currently, patients with advanced melanoma are likely to receive TIL-ACT after progression on ICI and/or targeted therapy, if BRAF V600mut.

    Several studies have failed to establish a significant correlation between response likelihood and previous exposure to anti-CTLA-4 treatment.7 9 10 32–35 41 Forget et al32 reported a non-significant decline in ORR and OS among 30 ipilimumab-exposed patients (9 of whom had also been exposed to anti-PD-1 antibodies), compared with 44 ipilimumab-naïve patients. The median duration of response (DOR) dramatically, but not significantly, decreased among responders with prior exposure to anti-CTLA-4 therapy.

    More recent studies have included patients widely treated with anti-PD-1 antibodies and BRAF/MEKi, if indicated. An analysis of 55 patients undergoing TIL-ACT across trials at our institution41 revealed significantly better PFS and DOR for all the anti-PD-1-naïve population, including those with prior exposure to anti-CTLA-4 antibodies. In a 226-patient cohort treated with TIL-ACT from 2000 to 2018, Seitter et al35 reported decreased ORR among those pretreated with anti-PD-1 or BRAF/MEKi. Melanoma-specific survival (MSS) and PFS significantly decreased in anti-PD-1-refractory patients, and an updated 150-patient analysis29 described a significantly higher ORR in the anti-PD-1-naïve compared with anti-PD-1-refractory population (55% vs 26%).

    The phase III trial comparing TIL-ACT to ipilimumab included a less heavily pretreated population, primarily consisting of patients who had progressed on anti-PD-1 antibodies either in an adjuvant or first-line setting. No substantial differences in PFS after TIL-ACT were observed between these and the previously untreated patients.13 Accounting for the BRAF V600-mutated population in Seitter et al’s cohort,35 the ORR significantly differed between BRAF/MEKi-refractory and BRAF/MEKi-naïve patients (21% and 60%, respectively). The MSS and PFS significantly decreased in the targeted therapy-resistant group. Similar findings have been reported by Levi et al.29 Notably, a higher number of previous treatment lines have shown a significant negative impact in different trials.32 41

    Baseline serum circulating factors

    Forget et al32 prospectively analyzed circulating factors in blood samples from 74 patients undergoing TIL-ACT, finding a significant association between high baseline IL-9 levels and response (serum IL-9 concentration of 5.3 pg/mL or greater predicted response in 7 out of 9 patients). In this cohort, soluble tumor necrosis factor (TNF) receptor 1 levels 21 days after TIL infusion were associated with a lack of response.

    In a phase I/II trial conducted at our institution, an analysis of baseline cytokines revealed numerically lower mean levels of IL-6, IL-8, IL-10, and TNF in patients exhibiting longer PFS, although no significant difference was found when evaluating cytokines individually.30 Serum IL-15 and IL-7 levels, which are known to increase after lymphodepletion,44 45 did not differ among response groups in a trial conducted by Goff et al.10 This group also showed a significantly lower median baseline neutrophil-to-lymphocyte ratio in responders to TIL therapy.

    Autoimmune reactions after infusion

    Autoimmune reactions, including vitiligo and uveitis, were only observed among clinical responders in a phase I/II trial.9 A non-significant trend between patients responding to melanocyte differentiation antigen-specific TIL and autoimmune manifestations was reported by Dudley et al.6 Additional assessment of this parameter will be required in further clinical trials.

    Treatment and TIL-related biomarkers

    Before infusion: TIL procurement, cell expansion, and patient conditioning

    Prior to cell product infusion, several parameters related to the manufacturing process and patient conditioning could potentially influence the efficacy of TIL therapy.

    The location of the resected lesion appears to have minimal impact on TIL expansion. Objective responses have been observed in patients with different metastasectomy sites,5 6 21 46 47 including brain metastases.8 48 Since myelotoxic CT will be required a few weeks after surgery, anatomic sites that allow minimally invasive procedures and minimize complications (eg, subcutaneous/soft tissue nodules, or lymph nodes) are preferable,13 while those with a higher risk of bacterial contamination (eg, skin, bowel lesions, preirradiated, or ulcerated tumors) should be avoided if possible.49 Procurement from secondary lymphoid organs (eg, the spleen or bowel) might pose limitations due to bystander non-tumor-reactive lymphocytes that could preferentially expand within the culture.49 Lymph nodes or peripheral tumor areas closer to blood or lymphatic vessels are preferred, in contrast to necrotic, hemorrhagic, or adipose areas, which negatively correlate with TIL growth.50

    Regarding TIL expansion, a short ex vivo cell culture time is a robust biomarker for response to TIL-ACT. Since the initial trials, longer culture periods have been observed to hinder TIL functionality.51 52 In a study of 31 patients with MM with 15 objective responses (ORR 48.4%), the median TIL age at REP initiation was significantly shorter among responders (13.2 days vs 19.7 days; p<0.001) and <20 days in all of them.47 Similar results were obtained by Besser et al46 in 20 patients with MM. Longer telomeres in the infused TIL, which are inversely correlated with culture time,53 have been found in patients with objective responses and persistent tumor-reactive T cell clonotypes.7 54 Shen et al55 observed that TIL failed to induce telomerase activity in vivo, undergoing a rapid reduction in telomere length within days after infusion. This may lead to rapid replicative senescence for “older” TIL with shorter baseline telomeres, suggesting that “young” TIL are those able to persist and mediate antitumor effects. The expression of costimulatory molecules CD27 and CD28 seems to be higher in “young” TIL with an earlier differentiation state,54 56 supporting the concept of cell senescence as a detrimental biomarker.

    Adequate conditioning is usually considered a prerequisite for TIL-ACT. Lymphodepletion enhances the antitumor response of infused T cells through various mechanisms, including antigenic presentation induction,57 elimination of immunosuppressive CD4+ regulatory T cells (Treg) and natural killer (NK) cells,58 and reduction of TIL competition for stimulating cytokines.44 59 Intense lymphodepletion has been correlated with increased levels of proinflammatory cytokines (IL-2, IL-6, IFN-γ, TNF-α, and IL-1α)58 60 and lymphocyte homeostatic cytokines (IL-7 and IL-15),44 60 with higher serum levels in patients achieving more intense lymphodepletion. In different studies, lymphodepletion intensity has been correlated with the tumor-reactive CD8+ TIL ratio and treatment efficacy.7 58 According to these observations, high serum levels of these cytokines might be a surrogate for adequate pretreatment conditioning, although their value as predictive biomarkers has not been confirmed in clinical trials. In a randomized study of patients with MM, increased lymphodepletion intensity with total body irradiation did not improve treatment outcomes.10

    Cell product and TIL phenotype: “quantity and quality”

    A high amount of total infused TIL and CD8+ TIL correlates with responses to TIL therapy across different tumor subtypes.8 12 21 32 46 47 61 Among 78 patients from the phase III trial,13 the median number of infused cells was 44.3×109 in responders versus 36.0×109 in non-responders (p=0.013), and the median CD8+ TIL count was also significantly higher in the first group (p<0.01).62 A prospective study of 74 patients suggested that the predictive value of this variable may be stronger in anti-CTLA-4-naïve patients.32

    A higher CD8:CD4 ratio correlates with better antitumor activity in vitro52 and in vivo.47 61 However, transferred CD4+ T cells also exhibit antitumor effects,63 64 mediating neoantigen-specific effector responses,65 and durable responses have been reported with predominantly CD4-dense products.61 66 Interestingly, a randomized trial evaluating CD8-enriched versus unselected TIL in patients with MM showed no significantly, but numerically inferior, response rates with CD8-selected than unselected young TIL.67

    As expected, several studies have found a correlation between clinical responses and the amount of infused tumor-reactive CD8+ cells.8 9 18 Trials with antigen-specific clonally expanded CD8+ TIL have been ineffective,68 69 highlighting the importance of targeting a diverse antigenic spectrum. A translational study of TIL infusion products from patients with melanoma using single-cell RNA and TCR sequencing revealed a correlation between responses and higher clonality of the TCR repertoire in the transferred cells.70 Reflecting the predictive value of neoantigen load, the abundance of neoepitope-specific CD8+ cells in the infusion product and post-treatment blood samples has been correlated with responses in melanoma71 72 and NSCLC.15

    Variations in the TCR heterodimeric structure entail biologic features with a potential impact on antitumor activity. In contrast to the predominant αβ T cells, γδ T cells recognize tumor-associated antigens without MHC restriction, being considered a bridge between innate and adaptive immunity.73 However, γδ T cells seem to display dual antitumor and protumor roles, being able to induce tumor progression by activating the signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways.74 Accordingly, a higher percentage of infused γδ T cells may worsen TIL-ACT outcomes,9 while TCR-Vβ gene signals seem to favor TIL growth.75

    Expression of exhaustion markers is another relevant phenotypic feature. Cell exhaustion protects T cells from overstimulation and hinders the efficacy of immunotherapy.76 77 High expression of exhaustion markers (programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT)) has been detected in TIL from ovarian cancer,78 human papillomavirus (HPV)-associated cancer,18 and NSCLC,15 hypothesizing that an exhausted phenotype might contribute to lower TIL reactivity in non-melanoma tumors. Among patients with osteosarcoma, a higher percentage of CD8+PD-1+ TIL was found in non-responders.21 However, in patients with MM, Lauss et al28 found a trend toward upregulation of exhaustion markers in responders, and the expression of PD-1, LAG-3, and TIM-3 has been shown to identify the autologous repertoire of CD8+ tumor-reactive T cells,79 thus the role of exhaustion markers in melanoma remains unclear.

    In contrast to other exhaustion molecules, B- and T-lymphocyte attenuator (BTLA) seems to activate the Akt/PI3K pathway in differentiated T cells, thus protecting them from senescence.80 A correlation between higher percentages of CD8+BTLA+ TIL and clinical responses has been observed in MM.61 62 The predictive value of BTLA expression, similar to the total cell count and the CD8:CD4 ratio, might be stronger in anti-CTLA-4-naïve patients.32

    Some studies have outlined the relevance of other immunosuppressive molecules on infused TIL, mainly the transcription factor Forkhead box P3 (FoxP3). FoxP3 contributes to the activation of Treg,81 82 which mediate immune self-tolerance and suppress the effector activity of CD8+ cells.83 CD4+FoxP3+ TIL from melanoma are unable to produce IL-2 and IFN-γ on ex vivo stimulation as their FoxP3− counterparts,84 suggesting that FoxP3 expression identifies Treg in the TME. A higher percentage of infused CD4+FoxP3+ TIL has been correlated with worse outcomes in melanoma7 85 and osteosarcoma.21

    The expression of costimulatory molecules also seems to modulate antitumor reactivity. The TNF receptor-family member CD137 (4-1BB) induces antiapoptotic signals (Bcl-2 and Bcl-xL) in CD8+ TIL,86 thereby promoting their effector functions. CD137 costimulation might be particularly relevant after the naturally occurring loss of CD27/CD28 during REP, and higher levels of CD137 in post-REP CD8+ TIL have been correlated with increased cytotoxicity against melanoma cells.86 In ovarian cancer, CD137 expression is higher in TIL than in circulating T cells, hence identifying naturally tumor-reactive T cells, and has been correlated with better TIL efficacy.87

    As mentioned above, CD27 and CD28 are costimulatory molecules preferentially expressed in young TIL and correlate with clinical responses in melanoma.7 62 Nevertheless, their predictive value remains unclear, since other studies have reported better results for CD27− TIL with an effector phenotype (Teff), in contrast to CD27+ TIL with a memory (TM) phenotype.61

    The presence of memory-progenitor TIL with a stem-like phenotype (CD39−CD69−) has been correlated with positive clinical outcomes, in opposition to terminally differentiated CD39+ TIL, probably due to better self-renewal and persistence in vivo.88 However, most antitumor neoantigen-reactive TIL were found in the differentiated CD39+ state in this study, and among patients from the phase III trial,62 a higher proportion of CD8+CD39+ TIL was observed in responders; hence, the value of CD39/CD69 expression remains confusing.

    Postinfusion: role of IL-2 and TIL durability

    Intravenous IL-2 is generally administered to sustain infused TIL functionality in vivo. Although responses to low-dose (LD)8 89 or decrescendo regimes9 of IL-2 have been observed, high doses (HDs) of 600 000–720 000 IU/kg every 8 hours have been used by most studies, including the phase III trial13 and are currently the standard approach. In the abovementioned meta-analysis, the HD IL-2 group had better outcomes,12 which is supported by clinical evidence suggesting that LD IL-2 facilitates CD4+FoxP3+ Treg reconstitution.90 91 A higher number of IL-2 doses may also favor Treg reconstitution after lymphodepletion,85 and some trials have reported fewer IL-2 doses in responders,6 7 although other studies have not confirmed these results.46

    The persistence of infused TIL in the peripheral blood has a clear impact on clinical outcomes. Tumor-reactive CD8+ TIL detection 1 month after infusion has been correlated with durable responses in melanoma.7 In the prospective study of patients with MM by Forget et al,32 high levels of UL16 binding protein 1, an activating natural killer group 2D (NKG2D) ligand of cytotoxic CD8+ T and NK cells expressed on tumor cells, were detected among responders 3 months after TIL-ACT. Interestingly, short durability may explain the lack of response in some patients with high counts of tumor-reactive CD8+ TIL,9 suggesting that TIL persistence in the peripheral blood is a powerful predictor of long-term benefit. A positive correlation between persistent tumor-reactive TIL and clinical outcomes has also been found in patients with NSCLC15 and HPV-associated tumors.18

    Evidence for each potential biomarker has been synthesized in table 1.

    View this table:
    Table 1

    Evidence regarding potential biomarkers for tumor-infiltrating lymphocytes (TIL) therapy

    Discussion

    Considerable advances in understanding the biologic basis of TIL-ACT have been made in the last decade, although there is still a long way to go before feasible clinical biomarkers can reliably predict clinical responses. In this review, we have searched for contrasted or potential biomarkers for TIL therapy that could be validated by further prospective studies, sorting them into three categories: variables related to tumor biology, patient clinical features, and treatment or cell product characteristics. It should be noted that as the available information is mainly derived from published studies on cutaneous melanoma, extrapolation to other cancers is currently limited.

    Within tumor characteristics, high TMB and neoantigen load stand as relevant biomarkers across different trials, even after progression on ICI, suggesting distinct mechanisms of resistance to different immunotherapies. Interestingly, a low neoantigenic load could be considered as a negative biomarker for TIL-ACT. Tumor immune infiltration and favorable transcriptomic signatures have also been correlated with clinical responses, probably reflecting enhanced tumor-reactive TIL production due to high tumor antigenicity.

    To date, clinical evidence suggests that baseline patient characteristics do not significantly impact responses. However, patients with a PS ≥2 are under-represented, and those with significant cardiovascular or respiratory comorbidities have been excluded from clinical trials because of their inability to receive HD IL-213, hence little is known about TIL therapy outcomes in this more vulnerable population. TIL-ACT entails a complex and time-consuming process, as well as acute and potentially severe toxicities, challenges that are greater to overcome in clinically deteriorated patients.

    LDH levels and a greater disease burden have been associated with poorer outcomes. We hypothesize that this correlation may be explained by the prognostic role of LDH, and patients with elevated LDH levels should still be considered for TIL-ACT, as durable responses have been reported in this population.

    Noteworthy, this review examines studies spanning a wide period during which new treatments have become available. While anti-CTLA-4 antibodies do not appear to drive a significant impact on TIL-ACT outcomes, anti-PD-1-naïve patients seem to achieve the highest benefit from TIL therapy. However, durable responses have also been observed in the anti-PD-1-refractory population, suggesting that TIL-ACT and ICI resistance mechanisms may be only partially shared. Given the toxicity and complexity of TIL-based ACT, it can be hardly justified to position this therapy in the first-line treatment setting of MM, in which a significant proportion of patients may achieve long-term benefit with ICI.92 Therefore, most candidates for TIL-ACT will now be ICI-refractory, highlighting the importance of understanding how tumor “immunoediting” by exposure and acquired resistance to ICI modulates TIL activity.

    Circulating factors could realistically be implemented in a daily workflow if validated as biomarkers. IL-9 is the only baseline circulating factor prospectively associated with TIL-ACT efficacy. As a member of the IL-2 receptor family, IL-9 has been proven to amplify the cytotoxic effect of CD8+ T cells and appears to have a powerful antitumorigenic role in melanoma,93 which could explain its potential as a biomarker for TIL therapy. However, validation of these findings in large cohorts and a deeper knowledge of the inter-relationship between different cytokines are required to assess their predictive value. In addition, it is poorly understood how these biomarkers vary across different tumor subtypes and how they are modulated by previous anticancer treatments or lymphodepleting chemotherapy.

    Short pre-REP culture times and high absolute cell counts are unequivocal predictors of response to TIL-ACT. Unfortunately, both come into conflict since longer culture times are generally required to achieve higher cell amounts, and little is known about how to achieve the optimal balance between these two parameters, or how to optimize TIL proliferation in vitro. This outlines the relevance of a high TIL baseline count, which is not feasible for all patients.

    TIL antitumor reactivity is another relevant feature that correlates to clinical responses. The measurement of reactivity in the infusion product is too late for TIL selection and intuitively, baseline TIL reactivity might be tested to selectively expand only tumor-reactive T cells. However, this strategy may lead to significant prolongation of culture time, favoring cell senescence and exhaustion, and has not been widely used. Although not randomized, a clinical trial by Besser et al66 compared minimally cultured “young” TIL to selected tumor-reactive TIL according to their IFN-γ production, with better outcomes for the first group. The need for minimally cultured “young” TIL impedes pre-REP cell selection, often leading to the expansion of bystander non-reactive lymphocytes, thus compromising treatment efficacy. In addition, reactivity is usually tested against autologous cell lines in vitro, but the success of cell line establishment is influenced by several features and variable across tumor types.94 The alternative use of allogeneic tumor cell lines for reactivity testing, although more consistent and homogeneous, increases the risk of not identifying neoantigen-specific tumor-reactive clones during pre-REP cell selection. Testing TIL reactivity at an early stage against autologous tumor digest would avoid the need to use autologous or allogeneic cell lines and may serve as a putative biomarker. However, the lack of standardized potency assays is a major obstacle in TIL therapy regulation and remains an unresolved issue in the field.

    The negative value of cell exhaustion in the transferred TIL might be particularly relevant in non-melanoma tumors, such as ovarian cancer, and novel strategies should be designed to overcome this problem. At our institution, a phase I/II trial combining TIL-ACT with PD-1 and CTLA-4 blockade (nivolumab plus ipilimumab) showed clinical responses across different tumor subtypes.40 A phase I trial combining ipilimumab, nivolumab, and TIL-ACT with LD IL-2 in ovarian cancer showed modest results,95 and another phase I trial combining TIL therapy with nivolumab and anti-LAG-3 relatlimab is currently ongoing (NCT04611126). Interestingly, TIL gene editing to knock out PD-1 has proven to be feasible through CRISPR/Cas9 technology in MM.96 However, a better understanding of cell exhaustion is required, as we know that at least one exhaustion marker (BTLA) has activating functions and seems to correlate with responses, and similar results have been suggested for PD-1 and TIM-3 by some studies.61

    High CD8:CD4 ratios are generally considered a positive biomarker; nevertheless, CD4-dense products can lead to clinical responses, and poor responses have been observed in patients receiving predominantly CD8+ TIL. This may be explained by the influence of other variables, including TIL durability in vivo. Besides, the concept of CD4+ TIL as a homogeneous group is probably simplistic, since now we know that some CD4+ cells exhibit antitumor effector functions, and others (as CD4+FoxP3+ TIL) display a Treg phenotype that favors immune tolerance. Therefore, a deeper understanding of the CD4+ TIL subsets is probably required before the CD8:CD4 ratio can be considered as a conclusive biomarker.

    Evidence regarding the value of costimulatory molecules remains inconclusive. While the expression of CD27/CD28 has been proposed as a predictor of efficacy, a high proportion of CD27− TIL with a Teff phenotype has also been correlated with responses. It has been hypothesized that transferred TIL may be modulated by the host “artificial” conditions after lymphodepletion, including a weaker competition for lymphocyte homeostatic cytokines. Some studies have shown that transferred Teff TIL exhibit improved migratory properties similar to those of TM.97 Others have found that in lymphodepleted hosts, TIL seem to have increased phenotypic plasticity, with TM TIL rapidly losing (and Teff TIL gaining) CD27 expression.98 This dynamic behavior in vivo might explain the contradictory results regarding its influence on TIL activity. Similarly, while CD137/4-1BB expression seems to correlate with efficacy, it also might trigger IL-13 secretion and limit Th1-mediated inflammation, thus favoring Treg reconstitution.99

    Another open question is how both stem-like and differentiated TIL contribute to the antitumor response. While stem-like cells have a better capacity for proliferation and self-renewal, terminally differentiated TIL are more prone to enter senescence and get exhausted, but simultaneously, differentiation is required for antigenic recognition and antitumor reactivity. This might explain why different studies have reported conflicting results regarding stem-like markers, suggesting that differentiated cytotoxic TIL and a subset of durable progenitor-like cells have complementary functions, both being probably required for long-term responses.

    It is relevant to mention that, while TIL reactivity and phenotype-related parameters may predict clinical outcomes, they cannot be considered conventional predictive biomarkers to guide patient selection strategies, such as patient- or tumor-related characteristics. In contrast, their understanding provides exciting potential for TIL product optimization throughout the manufacturing process.

    The mechanisms underlying TIL persistence and survival, alongside their interactions with host blood cells after recovery from lymphodepletion, are largely unknown. Postinfusion IL-2 is meant to facilitate TIL functionality in vivo, which makes the apparent correlation between fewer IL-2 doses and clinical responses an intriguing observation. It has been hypothesized that increased cytokine production by tumor-reactive TIL with high proinflammatory activity may limit the tolerance to IL-2, leading to the administration of fewer doses in responders.6 The combination of TIL-ACT with intratumoral administration of an oncolytic virus encoding TNF-α and IL-2 (TILT-123) has been proposed as a novel strategy to deliver IL-2 directly into the tumor site, thus reducing its systemic toxicity, and is being evaluated in a first-in-human trial at our institution.100

    In conclusion, some biomarkers have shown a strong correlation with TIL therapy efficacy, including high TMB and tumor neoantigenic load, short pre-REP culture of “young” TIL, high count of total and CD8+ infused cells, wide target antigenic scope, and long persistence of tumor-reactive TIL clonotypes. High CD8:CD4 ratios and downregulation of T cell exhaustion are potential biomarkers for efficacy, although antitumor activity may be also driven by certain CD4+ TIL subsets and enhanced by specific exhaustion markers. Further research is required to better understand the biologic functions of costimulatory receptors, the optimal balance between stem-like and differentiated TIL subpopulations, and how the interaction between the tumor and its immune microenvironment is modulated by prior exposure to systemic therapies, lymphodepleting CT, and administration of IL-2, among other factors.

    Ethics statements

    Patient consent for publication

    Ethics approval

    Not applicable.

    Acknowledgments

    image created with BioRender.com.

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    Footnotes

    • Twitter @vic_albarran, @Pballestin, @doniamarco

    • VAF and PBM contributed equally.

    • Contributors VAF and PBM: writing and original draft preparation; JSG, THB, and MD: review and editing of the manuscript; IMS: review and editing of the manuscript and expert supervision. All the authors read and approved the final version of the manuscript. VAF and PBM contributed equally to this work (coauthors).

    • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

    • Competing interests JSG declares speakers’ fees from BMS. MD declares advisory fees from Achilles Therapeutics and proprietary data access for Bristol-Myers Squibb and Genentech. IMS declares honoraria for speaker, consultancy or advisory role for Roche, Novartis, MSD, BMS, TILT bio, Pierre Fabre, cofounder, stake/shareholder, and clinical advisor for IO Biotech. The CCIT-DK has received research grants from Adaptimmune, Lytix Biopharma, Enara Bio, TILT Biotherapeutics, Asgard Therapeutics, and BMS. The other authors declare no relevant competing interests.

    • Provenance and peer review Commissioned; externally peer reviewed.