The immunotherapeutic role of indoleamine 2,3‐dioxygenase in head and neck squamous cell carcinoma: A systematic review

Abstract Background Novel cancer immunotherapy seeks to harness the body's own immune system and tip the balance in favour of antitumour activity. The intracellular enzyme indoleamine 2,3‐dioxygenase (IDO) is a critical regulator of the tumour microenvironment (TME) via tryptophan metabolism. The potential immunotherapeutic role of IDO in head and neck squamous cell carcinoma (HNSCC) requires further exploration. We aim to assess the evidence on IDO in HNSCC. Methods A systematic review of literature and clinical trials databases. Results We included 40 studies: seven involved cell lines: eight assessed tumour immunohistochemistry: ten measured IDO gene transcription: 15 reported on clinical trials. Increased cell line IDO expression was postulated to adversely affect tumour metabolism and apoptosis. Immunohistochemical IDO expression correlated with worse survival. Gene transcription studies associated IDO with positive PD‐L1 and human papillomavirus (HPV) status. Phase I/II clinical trials showed (a) overall response (34%‐55%) and disease control rates (62%‐70%) for IDO1 inhibitor in combination with a PD‐1 inhibitor, (b) similar safety profiles when both are used in combination therapy compared to each as monotherapies and (c) IDO gene expression as a predictive biomarker for response to PD‐L1 therapy. Conclusions IDO expression is increased in the TME of HNSCC, which correlates with poor prognosis. However, the exact mechanism of IDO‐driven immune modulation in the TME is an enigma. Future translational studies should map IDO activity during HNSCC treatment and elucidate its precise role in the TME, such research will underpin the development of clinical trials establishing the efficacy of IDO inhibitors in HNSCC.

T-lymphocyte-associated antigen (CTLA-4) mediated tumour regression and increased overall survival in melanoma patients, but was associated with frequent immune-related adverse events. [1][2][3][4][5] Programmed death 1 (PD-1) protein and its ligand PD-L1 was subsequently discovered 6,7 and shown to have good safety and efficacy in inducing durable tumour regression and prolonged stable disease in patients with advanced cancers including non-small cell lung cancer (NSCLC), melanoma, renal cell, ovarian, colorectal, pancreatic, gastric and breast cancer. 8 However, the vast majority of head and neck cancer patients, about 80%, remain unresponsive to immune checkpoint inhibitor therapy, highlighting the need for more effective immunotherapies and predictive biomarkers. 9 IDO1 inhibitors for melanoma, glioblastoma, NSCLC, pancreatic and breast cancer are under investigation by pharmaceutical companies and sponsors. 10 To date, IDO inhibitors for head and neck cancer have been tested in only several published clinical studies. [11][12][13][14][15][16][17][18] Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer worldwide and is diagnosed in 8000 new patients annually in the UK. 19 HNSCCs are divided into two clini- in the past four decades, that is most less than 50%. 21 The head and neck tumour microenvironment (TME) is a site of intense immunological activity, driving a recent emergence in immunotherapy being applied to HNSCC.

Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme
which plays a critical role in the immunity of the TME via tryptophan metabolism. Its activity is increased in the TME of many cancers and its expression was found to be a negative prognostic indicator in melanoma, 22 ovarian, 23

| Data sources and literature search
A systematic literature search was conducted in Ovid MEDLINE, Ovid Embase, Scopus, Web of Science, Cochrane library and Clini calTr ials.gov databases from inception until present day. The PRISMA guidelines for study selection were followed. 27 All studies that evaluated the involvement of IDO in HNSCC were systematically retrieved. The following search terms and strategy was used: ("indoleamine 2,3-dioxygenase" OR "IDO" OR "IDO1" OR "IDO-1" OR "IDO2" OR "IDO-2") AND ("squamous cell carcinoma" OR "squamous cell cancer" OR "SCC"). The titles and abstracts from the initial search results were screened independently by two authors DJL and JCKN. To ensure inclusion of all studies related to HNSCC, DJL and JCKN manually screened the studies with squamous cell carcinoma to include those involving the head and neck region.

| Study selection
In the initial screening, the following criteria were required for inclusion: (a) HNSCC from any head and neck subsite (oral, oropharynx, nasopharynx, larynx and hypopharynx), (b) study of IDO expression or activity, (c) all study types (prospective or retrospective, observational or experimental, pre-clinical or clinical), (d) published in English language only and (e) original articles and conference abstracts.

Keypoints
• IDO is integral to TME immunity in HNSCC particularly in HPV-positive cancers.
• IDO can be used to modulate existing therapies and has applications in combinatorial immunotherapy.
• Retrospective studies have shown its presence in the TME and suggest a link to HNSCC treatment outcome.
• However, the exact mechanism of IDO-driven immune modulation in the HNSCC TME remains unclear.
• We now require prospective longitudinal studies to track IDO activity and expression throughout HNSCC treatment, thence optimise IDO-based immunotherapy.
Duplicates, correspondence, review articles and studies without data on IDO in the context of HNSCC were excluded.

| Data extraction and analysis
Following the generation of a list of articles meeting the inclusion criteria, DJL and JCKN each performed an in-depth review of the studies and extracted data for comparison. Similar studies were grouped together for qualitative analysis.

| Included studies
A total of 273 studies were identified from databases and seven studies from additional sources, and 146 were screened after removal of 134 duplicates. A total of 100 studies were excluded with reasons described in the PRISMA flow diagram in Figure 1

| Cell line studies on IDO in HNSCC
The seven studies which investigated IDO in HNSCC cell lines, each is an immortalised cell culture developed from a single human HNSCC tumour, are summarised in Table 1  Thus, changing the tumour microenvironment from immunologically "cold" (ie inactive) to "hot" (ie active/inflamed) could enhance the efficacy of other immunotherapeutic drugs in combination.

| Tumour immunohistochemistry studies on IDO in HNSCC
The majority of tumour IHC studies were performed on formalinfixed paraffin-embedded tissue blocks. The studies in this group were designed with a retrospective method of analysis; none used prospective, fresh tissue collection or analysis of IDO activity within the tissues. The tissues studied came from the lower lip, oral cavity, tongue, tonsil and larynx. The eight studies included in this group are summarised in Table 2. Anti-IDO monoclonal antibody clone 10.1 was widely used. IDO staining was most commonly seen at the invasive front of the tumour and increased IDO staining correlated with worse survival. [35][36][37] Four of the studies [35][36][37][38] were prognostic studies which reported on IDO expression and correlation with outcome.
Only two studies fulfilled all REMARK checklist criteria for prognos-

TA B L E 2 (Continued)
increase in IDO expression was seen in clinical non-responders to Nimotuzumab (anti-epidermal growth factor receptor) therapy, suggesting that IDO may be a biomarker of immune status in the TME during therapy in oral SCC patients. 38

| IDO gene transcription studies in HNSCC
We included 10 studies which investigated IDO gene transcription, summarised in Table 3 IDO gene expression as a predictive biomarker for response to PD-L1 therapy. 18 The most common treatment-related adverse event associated with the immune checkpoint trials was fatigue (22%-32%). 11,13,15 Existing clinical trials testing IDO inhibitors in HNSCC, registered on Clini calTr ials.gov without published results are summarised in Table S2.

| Summary of main results
Existing evidence suggests that IDO is integral to TME immunity

| IDO inhibitors commonly applied in preclinical and clinical studies
IDO can be expressed in tumour cells but multiple TME cell types may also express IDO including dendritic cells, macrophages, fibroblasts,

endothelial/epithelial cells and PBMCs, 50,51 though lymphoid cells (eg T cells and tumour-infiltrating lymphocytes) rarely express IDO. It is
important to distinguish between IDO protein abundance in the TME Drugs being tested in clinical trials include the non-selective IDO and TDO (tryptophan 2,3-dioxygenase) inhibitor navoximod (NLG-919), 53,54 which is approximately tenfold more selective for IDO1 than TDO2; the selective IDO1 inhibitor linrodostat (BMS-986205) 55 and IDO1 and TDO2 inhibitor PF-06840003 56 which are both more than 100-fold selective for IDO1 than TDO2. Indoximod, epacadostat and linrodostat are also being evaluated in combination drug trials given the strong rationale for the use of IDOi drugs as immunometabolic adjuvants to increase the efficacy of (chemo)radiotherapy and immunotherapies. 57 In vitro studies of IDOi drugs in cancer include those investigating indoximod 58 and linrodostat. 59 Applied as monotherapy to patient-derived colorectal cancer cell lines, indoximod exhibited rather low direct cytotoxic activity, whereas coculturing the cell lines in an allogeneic setting using naïve, "unprimed" lymphocytes from healthy volunteers generally boosted the antitumoural effect of indoximod. 58 However, this was not tested in an autologous setting using partially exhausted lymphocytes from cancer patients.
Interestingly, low IDO expressing cells responded better to indoximod monotherapy, suggesting that indoximod likely targets additional pathways, although the precise mechanism of action is yet to be elu-

| Other combination immunotherapeutic strategies
Apart from immune checkpoint blockade alone, other combination immunotherapeutic strategies have been studied. Rational combinations have been tested based on the knowledge that activating STING in the TME of mice stimulated protective antitumour immunity; however, preliminary outcomes from a clinical trial reveal little benefit of STING agonist monotherapy. 60 To overcome this therapy resistance, Lemos and colleagues showed that in mice bearing established Lewis lung carcinoma (LLC) tumours, intratumoural treatment with STING agonist, synthetic cyclic diadenyl monophosphate (CDA) and co-treatment with selective COX2 inhibitor celecoxib eliminated the primary tumour burden, prevented metastases and induced durable protective antitumour immunity. 61 Co-treatment with IDOi drugs indoximod, navoximod and linrodostat also enhanced antitumour responses to CDA, especially in co-treatment with linrodostat which induced rapid tumour regression and increased survival, however, did not eliminate the primary tumours.
Interestingly, inhibiting COX2 also significantly reduced IDO activity, which may contribute to greater antitumour activity elicited by celecoxib in combination with CDA. Another strategy to enhance antitumour immunity is to deliver recombinant enzymes that act downstream of IDO to reduce the level of immune-suppressive Trp catabolites in the TME. patients is yet to be proven. Although speculative at present, combining IDOi drugs with radiotherapy treatment may modulate the TME in favour of antitumour activity and help overcome treatment resistance in cases where the disease is less radiosensitive, for instance in HPVnegative HNSCCs where radiotherapy controls less than 50% of disease cases that have concurrent nodal metastases. The hypothesised role of IDO in the immune microenvironment is summarised in Figure 2.

| Strengths, limitations and potential bias of evidence
This narrative systematic review synthesises the existing evidence on studies involving IDO in HNSCC. Due to the heterogeneity among the studies, a meta-analysis was not possible and a qualitative analysis was therefore performed on the groupings of study types. Although existing HNSCC cell line studies showed IDO involvement in certain cell lines (SCC4, SCC15) and influenced by F I G U R E 2 IDO immune microenvironment hypothesis. (Chemo)radiotherapy and CDA treatments activate STING to incite antitumour immunity but also boost immune regulation to enhance therapy resistance. Multiple STING-responsive pathways involving chronic inflammation and tumour progression and associated with immune checkpoints (PD-1/L, CTLA-4, IDO) result in therapy resistance. Blocking these pathways modulate the TME in favour of antitumour immunity. Immune, inflammatory and metabolic biomarkers in blood reflect changes in the TME caused by treatments and therapy resistance. Abbreviations: CDA, cyclic diadenyl monophosphate; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand 1; CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; IDO, indoleamine 2,3-dioxygenase; IFN-1, interferon-1; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells F I G U R E 3 Expression of IDO1 in HNSCC based on HPV status. Box and whisker plots of IDO1 expression in HPV-positive, HPV-negative and normal adjacent tissue generated from TCGA data. This comparison shows significant differences in IDO1 expression when comparing: HPV-positive vs normal tissue (P = .00002), HPV-positive vs HPVnegative (P = .00112) and HPV-negative vs normal tissue (P < .00001) STING activation, conclusions cannot be drawn from these studies on the influence of the whole immune system or local TME dendritic

| Implications for future clinical practice and research
Although there exists a spectrum of immune cell infiltrates in shows that HPV-positive HNSCCs have significantly higher IDO1 expression when compared to HPV-negative HNSCCs (P = .001) and adjacent normal tissue (P < .001) (Figure 3) 68 although the IDO expression is also elevated in HPV-negative HNSCC when compared to normal tissue. Furthermore, studies in various solid cancers investigating the differences between primary tumour and corresponding lymph node metastases have shown that strong tumoural IDO expression is associated with metastatic disease. [69][70][71] It has been observed in both colorectal and breast cancer that IDO expression pattern is consistent between the primary tumour and metastatic sites. 72,73 This suggests IDO expression as a modulator of cancer inflammation and immune evasion in both primary and metastatic tumour progression. 51 As more evidence mounts in favour of the involvement of IDO in the TME of HNSCC and a better understanding of its mechanistic role in HNSCC immune modu- Improved understanding of the mechanistic role of IDO in modulating local TME immunity will inform the targeting of the IDO pathway to optimise HNSCC therapy. The ability to measure IDO activity in the peripheral blood of cancer patients 76 allows researchers to prospectively map and characterise potential groups of patients who may benefit from modified treatment doses (eg radiotherapy) based on IDO immune status. The correlation of IDO activity and expression at the tumour, draining lymph nodes, and in peripheral blood can potentially lead to less invasive sentinel lymph node and liquid biopsies to inform stratified, personalised HNSCC immune-based therapy.

| CON CLUS IONS
Current evidence shows the presence of IDO in the TME and suggests a link to prognosis and prediction of HNSCC treatment outcome. However, the exact mechanism of immune modulation by the IDO pathway in the TME of HNSCC remains unclear. Future translational studies need to prospectively map the activity and expression of IDO throughout HNSCC treatment to achieve a mechanistic understanding of its involvement in TME immunity and to inform the design of precision, stratified immunotherapeutic approaches involving IDO.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.