Immune checkpoint inhibitors (ICI) can effectively restore the activity of exhausted CD8⁺ cytotoxic T cells and thereby trigger antitumor immune responses. During the last years, several ICI were approved for more than ten different tumor entities. However, still only the minority of patients shows durable responses.1 In clinical trials, the expression of programmed cell death-ligand 1 (PD-L1) on tumor and/or immune cells has been frequently used to select patient subgroups with higher chances of treatment response. However, the predictive power of PD-L1 expression is restricted and its expression varies, as its expression is for example increased after radiotherapy.2–5 Consequently, large effort has been made to identify routinely available blood and clinical markers that may predict the response to ICI therapies,6 besides and complementary to the analyzes of genomic instability.7 The latter has been assessed via tumor mutational burden (TMB)8 or mismatch repair deficiency.9 All of these methods have the potential to predict treatment responses to a certain extent, but are still too complex to be used in the clinical routine. A further challenge is imaging of the cancer treatment response to ICI. Tumor lesions can initially increase in size due to inflammatory events.10 This pseudoprogression cannot be distinguished easily from an increase in size due to tumor growth. Consequently, the classical RECIST V.1.1 criterial were modified to the iRECIST criteria, which recommend treatment beyond the first progression.11 The main problem of this procedure is that patients with real tumor progression loose time for a probably more efficient radiotherapy and/or chemotherapy.

A recent strategy to complement these approaches is to analyze changes of peripheral blood immune cells at early time points during treatment.12 The peripheral blood immune status may have a high predictive power for the treatment response of solid tumors to ICI, as several immune cells that were already proven to predict treatment responses when being present in the tumor tissue also circulate through the peripheral blood.13 Furthermore, peripheral blood can be drawn easily and be repeated without an additional risk for the patients compared with repeated biopsies of the tumor tissue. Immunophenotyping (IPT) of peripheral blood from patients with stage IV melanoma before and after treatment with ICI already identified pharmacodynamic changes in circulating CD8⁺ T cells with an exhausted-phenotype. Clinical failure of the patients was identified to be associated with an imbalance between T-cell reinvigoration and tumor burden.14 Another study identified a predictive character of proliferating (Ki-67⁺) PD1⁺CD8⁺ T cells for the response to anti-PD-1 immunotherapy in solid tumors.15 Using high-dimensional single-cell mass cytometry, Krieg and colleagues identified monocytes, expressing high levels of HLA-DR to be predictive for the treatment response to anti-PD-1 therapy.16

In the introduced preplanned biomarker analysis of the prospective ST-ICI cohort, the peripheral blood immune phenotype of patients with recurrent and/or metastatic cancer treated with ICI was prospectively monitored by multicolor flow cytometry. Our aim was to construct a predictive immune signature, based on the peripheral blood immunophenotype that identifies patients who benefit from ICI.

In recent years, ICI with or without chemotherapy were approved for multiple recurrent or metastatic cancer types. However, only approximately 20% of non-selected patients experience long-term benefit.2 24 Numerous studies suggest that TMB may predict the clinical response to ICI across multiple cancer types.25–27 However, different studies used different cut-off values based on independently companies’ database or cancer types to define low and high TMB.

The detection of PD-L1 by different methods or cut-off values could also guide anti-PD-1/PD-L1 therapy, but still harbors the risk of a false-negative patient stratification.28–30 The gene methylation status may also serve to select patients who will experience a clinical benefit from the PD-1 blockade.31 Nevertheless, there is still particular need of additional or complementary time saving, cost-effective, safe, and easy applicable tools to identify patients with cancer who could benefit from ICI.

In this context, a detailed knowledge about the immune status, both in the tumor and in the periphery, is needed to judge about the potential of cancer immunotherapy and the efficacies of immunotherapies.32 Tumor immunogenicity scores are under evaluation as predictors for responses to ICI.33 Previous studies already indicated peripheral blood immune cell subsets like PD-1⁺CD56⁺ T cells34 or CD4+ and CD8+ cells35 to be associated with favorable outcome for advanced melanoma patients treated with ipilimumab directed against cytotoxic T lymphocyte antigen-4 (CTLA-4). Now we identified a liquid immune profile-based signature to predict response of patients with metastatic cancer to anti-PD-1/PD-L1 treatment at an early time point.

To our best knowledge, the prospective ST-ICI cohort is the first one, which prospectively included a whole-blood multicolor flow cytometry-based approach for the identification of a detailed peripheral immune status in a clinical relevant multitype advanced cancer patient cohort treated with anti-PD-1/PD-L1 ICI. For these patients the treatment response according to RECIST V.1.1 is less appropriate for defining a prognostic signature. The main cohorts in ST-ICI are NSCLC and HNSCC. Pseudo-progression is a rare, but a frequently appearing phenomenon in PD-1 inhibitor therapy. These patients are rated as “progressive disease” according to RECIST V.1.1, even though they are probably the best responders later on. In NSCLC patients continuing immunotherapy beyond progression had significantly prolonged OS.36 The newly developed iRECIST criteria might be a method to cope with this problem, but have not been established in prospective clinical trials so far. Also, in HNSCC a secondary analysis of the CheckMate-141 trial identified responses after initial progression.37 A total of 24% of patients developed a reduction of the RECIST sum of diameters after initial progression. This highlights also in HNSCC that patients benefit from ICI even if tumor progression is identified in the first imaging assessment. Growing evidence indicates that there is a large number of patients that benefit from ICI also without developing an objective response according to RECIST V.1.1. Consequently, we decided to use the more valid endpoint OS for the development of our prognostic signature.

To avoid large volume blood sampling for preparation of peripheral blood mononuclear cells and to include granulocytes in the analyzes, the IPT was performed in fresh whole blood without preceding isolation of specific cells. Thus, whole blood assays gain importance as they are an easy approach to screen a large number of blood parameters. Within the ST-ICI cohort, 54 immune cell subsets were monitored in the patients with the need of less than 1 mL of peripheral whole blood. Our identified LIPS has the advantage of being easily integrated into clinical use for nominal expense. In a first step, 14 immune cell subsets were identified to be associated with OS. Furthermore, LASSO regression constructed the LIPS signature including neutrophils, pDCs, NKT cells (CD3⁺/CD56⁺/CD16⁺), monocytes (CD14^high), and CD8⁺ T cells (PD-1⁺). This signature includes innate and adaptive immune cells and serves as an effective tool for the identification of patients with metastatic cancer who benefit from ICI treatment. It is becoming more and more evident that multiple immune cell subsets in a concerted action may help for selecting patients that are likely to respond to ICI.38

We identified that increasing amounts of neutrophils in the peripheral blood of patients with cancer were associated with less benefit from ICI treatment. Neutrophils have been reported to support for example the development of metastasis through multiple mechanisms, such as the release of proteases that degrade antitumor factors, and leukotrienes that propagate metastasis-initiating cells. In addition, pro-tumorigenic neutrophils suppress antitumor T cell responses.39 Decreased migration of neutrophils to tumor areas or the inhibition of granulocyte colony stimulating factor to decrease the amount of neutrophils has already shown efficacy in preclinical models.40 Recent findings suggest that neutrophil antagonism will improve the efficacy of ICI therapy in the future.12

Regarding innate immune cells not of the granulocytic, but of the monocytic compartment, increased amounts of CD14 high expressing monocytes in the peripheral blood are associated with improved prognosis. This is in accordance with the findings of Krieg et al who identified CD14⁺/HLA-DR⁺ monocytes as a strong predictor of PFS and OS in response to anti-PD-1 immunotherapy in melanoma patients16. We here show the importance of CD14 high expressing monocytes in the peripheral blood for non-melanoma solid tumor patients.

Further, we found that increased amounts of plasmacytoid dendritic cells (pDCs) are beneficial for the PFS and OS. pDCs have a central role in activating host innate and adaptive immune responses and they are known as the major interferon type I-producing cells. Thereby, pDCs activate many other cell types, such as monocytes, NK cells, and T cells which are known to be also crucial for antitumor immune responses.41 High levels of circulating pDCs were already found to be predict a favorable outcome in patients with breast cancer42 and for patients with pancreatic adenocarcinoma.43

NKT cells are suggested to induce a cross talk of pDCs with conventional DCs that result in induction of memory CD8⁺ T cells.44 We identified increased numbers of NKT cells in patients of the ST-ICI cohort to be favorable for PFS and OS. Just recently, it was shown for patients with NSCLC that increased amounts of peripheral NK cells correlate with responses to anti-PD-1 treatment.45 The role for NKT cells in this scenario has not been investigated before, but it is becoming more and more evident that NKT cells are important in antitumor immunity as they, for example, reinvigorate exhausted immune cells in the tumor microenvironment.46

Exhausted immune cells fail to contribute to antitumor immunity. Increased numbers of PD-L1⁺ NK cells in the peripheral blood, for example, are associated with poor response to anti-PD-1 treatment of patients with NSCLC.47 However, intratumoral CD8⁺ T cells which are positive for PD-1⁺ were shown to be predictive for response and survival on anti-PD-1 antibody therapy, as shown in a small cohort of patients with NSCLC.48 Studies in chronic myeloid leukemia patients just recently revealed that differences in PD-1. Expression on CD8⁺ T-cells might predict the disease course.49 Our immune monitoring suggests that increased amounts of circulating PD-1⁺ CD8⁺ T cells at early time points of ICI therapy predict a poorer OS and PFS in patients with metastatic solid tumors treated with ICI. This might indicate that in these cases the activated CD8⁺ T cells cannot enter properly into the tumor and exert their antitumor activity.

The ST-ICI cohort composes a clinically highly relevant group of advanced stage patients with solid tumors, that includes some confounding factors such as the treatment with different ICI, different tumor types, brain metastases, the number of previous treatments and PD-L1 expression. La and colleagues identified a median OS of NSCLC patients of 9.9 months in the second line ICI treatment, and a median OS of HNSCC patients of 7.4 months in real-world cohorts.50 This is in line with our findings with a median OS of 8.7 months in a cohort consisting mainly of NSCLC and HNSCC. In the ST-ICI cohort, 32 patients had 0–1 previous treatments, while 57 patients had ≥2 of previous treatments. Patients with less (0–1) of previous treatments did not show significantly prolonged OS (HR=0.60 (0.34–1.08), median OS(0–1)=12.46 (9.76-NE) months, median OS(≥2)=9.44 (7.43–14.5) months), but a prolonged PFS (HR=0.49 (0.30–0.82), median PFS(0–1)=7.79 (4.01–15.88) months, median PFS(≥2)=3.22 (2.93–5.79) months). Furthermore, number of previous treatments (0–1 vs ≥2) was significantly associated with PFS in the univariate analysis, however, it was not significantly associated with PFS in the multivariate analysis. The number of previous treatments was also not significantly associated with OS in the univariate and multivariate analysis. Taken together, this factor did not impact our LIPS signature as predictor for ICI. The multivariable Cox regression and subgroup analyzes suggest that LIPS is an independent predictive factor for patients with advanced cancer treated with ICI targeting the PD-1/PD-L1 axis. The subgroup analyzes suggest that this immune signature can reliably predict OS and PFS benefit for patients with cancer treated with ICI in any subgroups, except for brain metastases. This may be a consequence of a continuous administration of glucocorticoids in these patients. Although patients with brain metastases can benefit from ICI treatment,51 the knowledge about the immune microenvironment at this tumor location side is scarce,52 53 and future trials will have to particularly focus on this subgroup of the ST-ICI study population. In present clinical practices, PD-L1 expression is an essential biomarker as drug approvals mostly depend on its expression. However, our subgroup analyzes revealed that the LIPS predicts survival benefits independent from PD-L1 expression. Notably, LIPS identified more than 82% of all patients with advanced cancer, who benefit from anti-PD-1/PD-L1 antibodies.

It has been recognized that a non-invasive and early detection of multiple parameters including peripheral immune features are promising to predict the clinical response of patients with cancer to ICI. At the same time, the dynamics of the responses should be considered.54 As the NKT cells and neutrophils as central part of our identified LIPS can still predict OS and PFS even after the first administration of the ICI (online supplemental sFigures 9–10), our analyzes and results demonstrate that easy applicable immune phenotyping of peripheral blood of patients with metastasized cancer allows an accurate and early forecasting of OS and PFS in patients with metastatic cancer.

Our prospective analyzes of the immune status of patients of the ST-ICI cohort demonstrated that LIPS can predict which patients with cancer with advanced disease will benefit from ICI treatment and therefore could guide clinical decisions in the future. This newly identified LIPS is a low-cost, safe, easy and broadly applicable and effective early predictor for OS and PFS in patients with recurrent and/or metastatic cancer treated with ICI.