Therapeutic approaches targeting immunosuppressive features of the tumor microenvironment (TME) or cancer cells may reinforce antitumor immune responses in both solid and hematological malignancies. Up-regulation of inhibitory checkpoints on the surface of innate and acquired immune cells is considered as one of the important immunosuppressive features in most types of cancers (1). After the approval of cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) inhibitor ipilimumab in 2011, immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment (2). Under physiological conditions, immune checkpoint proteins (ICPs) that involve in various inhibitory and stimulatory pathways regulate the homeostasis between pro-inflammatory and anti-inflammatory signals while in cancer, the malignant cells promote an immunosuppressive tumor microenvironment to diminish anti-tumor immune response for immune evasion. Currently, clinically approved ICIs target either CTLA-4 or PD-1 signaling pathways while others targeting TIM3 and LAG-3 are currently under investigation (3, 4). CTLA-4 and PD-1 pathways differ from each other according to the periods of the immune response they involve (5). CTLA-4 expression is restricted to T cells, inhibiting cellular activation at the initial stages, mainly in lymph nodes while PD-1, expressed on T cells, B cells and myeloid cells regulates activated T cells in peripheral tissues. In summary, activation of both ICPs exerts similar effects on T-cell activity but their signaling mechanisms as well as the location where cellular interactions take place differ.

Immune checkpoint blockade (ICB) refers to usage of monoclonal antibodies which target immune checkpoint molecules or their ligands on the surface of immune and/or tumor cells which act by hampering the inhibitory mechanisms on T cells in addition to activating cells taking part in innate and adaptive immunity to exert an effective anti-tumor response (6, 7). This immunotherapeutic approach was first approved by FDA for the treatment of melanoma and non-small cell lung cancer (NSCLC), and its beneficial effects in treatment of hematological malignancies have also been proven (8–10). Historically, the promising effects of immunotherapy in the field of hematological malignancies have been initially recognized after success of stem cell transplantation (SCT) as well as various mAbs against tumor surface proteins. Accordingly, accumulating data have demonstrated that due to some genetic factors, the physiologic functions of inhibitory checkpoints are dysregulated in several hematologic diseases such as myelodysplastic syndrome (MDS), lymphoid neoplasms and acute myeloid leukemia (AML) (11). ICB therapies in hematological malignancies have primarily started with anti-CTLA-4 (Ipilimumab) and anti PD-1/PD-L1 antibodies (Pembrolizumab, Nivolumab, Atezolizumab, Avelumab, and Durvalumab) and there are numerous clinical trials evaluating their therapeutic benefits alone or in combination with other treatments (Table S1); however, they have shown clinical benefits mostly in certain types of hematological malignancies. Moreover, blockade of other inhibitory checkpoints such as LAG-3, TIM-3, and TIGIT or some innate checkpoints on NK cells and macrophage with therapeutic agents are under clinical evaluation as a potential approach to overcome acquired resistance (11–13). Some ongoing studies are investigating efficacy of ICB in association with other immunotherapies such as CAR-T cell therapy, with promising outcomes (14, 15).

Despite favorable clinical benefits of these immunotherapies in most types of hematologic malignancies, ICB is used only in the treatment of certain tumor types which have high infiltration of immune cells. Furthermore, due to some primary or acquired resistance mechanisms, checkpoint blockade therapies (CBTs) on their own are not able to achieve long-term disease control in leukemia, and thus, certain patients diagnosed with leukemia may not respond well to this treatment or benefit from it only during the initial stage. On the other hand, as inhibitory checkpoints play role in maintaining the balance of immune function, patients treated with these agents may suffer from a series of side effects called immune-related adverse events (irAEs) (16). Due to high cost of CBT, its use only in certain leukemia patients, and its potential toxicities or adverse effects, there is an unmet need for assessment of laboratory biomarkers to identify and monitor patient populations who are more likely respond to this type of therapy and management of side effects. Despite availability of several potential biomarkers and laboratory approaches, further studies are required since still there is no consensus. In solid tumors, checkpoints or their ligands’ expression levels in the tumor tissue as well as the serum levels of their soluble forms are usually evaluated, however in case of hematologic malignancies, it is more complex and identification of reliable predictive biomarkers is more difficult (17). Here, we aimed to review recent information on the predictive and prognostic value of laboratory methods and biomarkers for clinical follow-up of patients with leukemia or lymphoma who were treated with ICBs, particularly with anti-CTLA-4 and anti-PD-1/PD-L1 antibodies, the most widely used ICIs.

As mentioned above, regarding the physiologic role of ICIs in maintaining the immune system in balance, their blockade might result in several side effects and toxicities and/or provoke autoimmune reactions in both solid and hematologic malignancies. They include a wide spectrum of adverse effects including dermatologic, gastrointestinal, and hepatic disorders, iritis, hypophysitis, pneumonitis and endocrine events. These treatment-related complications which are usually less frequent in PD-1/PDL1 blocking compared to CTLA-4, are managed based on their grade. Even though it has been reported that some endocrine complications have resulted in permanent organ dysfunction necessitating hormone replacement therapy, other irAEs have typically been reversible and manageable (18, 19). Here, we describe some more common immune-related adverse events that may occur following CBT in hematological malignancies.

Previous studies have shown that blockade of PD-1/PD-L1 pathway is usually well-tolerated and associated with less adverse effects. Several studies have investigated adverse events with anti-PD-1 therapies during treatment of hematologic malignancies and have shown that most patients (approximately 78%) experience minor adverse events in the form of rash and thrombocytopenia, followed by fatigue, diarrhea, nausea, pruritus, and pyrexia. Approximately 22% of patients experienced Grade 3 or higher adverse events such as lymphopenia, stomatitis, increased lipase, MDS and pancreatitis. Such irAEs were reported in the treatment of diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL) with pembrolizumab, where the most common side effect was neutropenia, followed by fatigue, diarrhea, and upper respiratory infections. Other studies indicated that anti-PD-1 therapy is associated with pancreatitis, lymph node pain, pneumonitis, and MDS (20–24). Furthermore, some of these studies have demonstrated Grade 3-4 neutropenia and thrombocytopenia (19% and 8%, respectively) following administration of pidilizumab (an anti-PD-1 antibody) after ASCT in patients with DLBCL, primary mediastinal B-cell lymphoma (PMBCL), and transformed indolent NHL, which are attributable to autoimmune etiology, but further studies are required to confirm this finding (20).

During treatment of hematological malignancies with anti-CTLA-4 antibodies (most commonly Ipilimumab), the most common Grade 3 adverse events were diarrhea and fatigue. Furthermore, AST elevation and thrombocytopenia have been also reported less frequently. Additionally, some other trials have evaluated efficacy and safety of ipilimumab in patients who had failed allo-SCT. Interestingly, administration of ipilimumab in hematopoietic malignancy after HSCT demonstrated a similar profile of toxicities (22). Davids et al. have investigated adverse events after use of ipilimumab in relapsed hematologic malignancies (including AML, lymphomas, myeloma, myelodysplastic syndrome, and myeloproliferative syndromes) following HSCT. In this study, patients received deferent doses of induction therapy with Ipilimumab (3 or 10 mg per kilogram of body weight every 3 weeks for a total of 4 doses). Of 28 evaluable patients, one case of acute GVHD of the gut and 3 cases of chronic GVHD of the liver were reported (25).

Leukemia develops in bone marrow and its surrounding microenvironment, which is a heterogenous environment with stromal cells, blood vessels, immune cells, and extracellular matrix (ECM) (26). It is easier to obtain a sample compared to solid tumors, however its heterogeneity, the need for expertise and the cost makes it challenging for clinical laboratories. Advanced technical expertise required for immunological assays that is centralized to advanced laboratories can also be considered as a drawback for hospital-based clinical laboratories.

There are three main mechanisms of resistance developed against ICI therapies; primary, adaptative and secondary or acquired resistance (26). Based on this classification, “primary” mechanism is referred to pre-existing resistance to immunotherapy and usually contains patients who do not respond at all to immunotherapeutic treatments, however, appearance of resistance as a Darwinian mechanism of adaptation is considered as “adaptive”. Finally, “acquired” (also called “secondary”) means appearance of resistance after immunotherapy instigation after a transient period of disease control. To understand and predict these mechanisms, there is a need for defining the “immune contexture” by analyzing the spatial localization, density, and functional orientation of immune cell populations in bone marrow and surrounding tissues (26). Routine immunophenotyping for leukemias and other hematological malignancies is an essential part of the diagnostic work-up. There are also some biomarkers that have been suggested for use in different types of resistance mechanisms such as myeloid-derived suppressor cells (MDSCs), tumor associated macrophages (TAMs) and tumor infiltrating cells (TILs) for solid tumors (26), yet there is not enough evidence to use these markers in clinical follow-up of leukemias.

The type of cells expressing immune checkpoint markers and their ligands are given in Figure 1. As will be observed, only drugs blocking PD-1/PD-L1, and CTLA-4/CD80-CD86 have been currently approved for clinical use. Other ICIs are on the line, and we need to explore the roles of these markers for clinical laboratories as well.

This review is focused on immunology- and hematology-related tests for ICIs since the biochemistry tests which are an important part of the follow-up are handled by other laboratory specialists, and they require extensive knowledge in these tests (Table 1).

A comprehensive study published after analysis of >10,000 cancer samples proposed that there are six different immune subtypes across different cancer types, and wound healing, IFN-γ dominant, inflammatory, lymphocyte depleted, immunologically quiet and TGF-beta dominant subtypes differ by somatic aberrations, microenvironment and survival (78). In another study of two hundred (200) acute myeloid leukemia samples, the results for genetic and epigenetic properties of acute myeloid leukemias were published by The Cancer Genome Atlas Research Network in 2013 (79). Studies designed for understanding the immune landscape of hematological malignancies could contribute to use of ICIs considering these immune signatures. It is noteworthy to emphasize that acute myeloid leukemias have less somatic mutations than other cancer types (18 mutations vs. >50 mutations in other cancer types) (79).

Another challenge for clinical laboratories is bridging the genomic information with other clinical features of leukemias. Germline variants are not always detectable for all leukemias, and there are ongoing studies aiming to gain more precise information on the subject (80, 81). Clinical evaluation and use of these tests are challenging because of the lack of familiarity with this type of testing, the requirement for DNA samples that reasonably approximate the germline state and absence of standardization across the different brands of diagnostic platforms for genes that are sequenced as well as their capabilities for detection of full range of variants (82).

There is an unmet need for development of predictive markers for serious side effects such as myocarditis, encephalitis or acute hypophysitis, which could lead to death. Since the action mechanism of ICI treatments relies on the inhibition of immune response, treatment may end up with inflammation of tissues and organs (83). To be able to make predictions currently, available tests and novel tests for prediction of the inflammation should be included in routine testing. A guideline was developed for response criteria for use in clinical trials testing immunotherapies for solid tumors: iRECIST by Response Evaluation Criteria in Solid Tumors Working Group (RECIST) (84). Up to date, there is limited guideline information for use of ICIs in leukemias and for follow-up therapy in laboratories (85).

An experimental study has reported that the Teffs/Tregs ratio can be used as a predictive marker, and with a higher ratio, a better response is expected (86). Other studies on solid tumors also provide support to this finding (87). Exhausted T cells (CD8⁺PD-1⁺CTLA⁺ cells) are known to influence immune response to immunotherapy, and due to possibility of performing functional measurements such as cytokine measurements, it is possible to identify these exhausted T cells in leukemias (87–89). Only, there is a need for ready-to-use individual testing kits with reasonable prices that can be adapted to clinical laboratories from clinical trials. As mentioned by other authors, lack of “Conformite Europeenne” (CE) or “In Vitro Diagnostics” (IVD) labels for the reagents to be used in detection ICs and their counterparts requires more standardization and acceptance of the methods used for the standardization by clinical laboratories that are normally required to use the CE or IVD labeled products only (90). Extensive non-CE and non-IVD panels suggested by the same authors should be evaluated by more laboratories, as the clones chosen for this type of analysis may be different in laboratories, standardization suggestions are cumbersome and challenging with many variations in the measurement systems, reagents, preparation, and analysis methods.

In some solid tissue cancers, after ICI treatment a pseudo progression (an initial increase in tumor volume) may be observed, and pseudo progression should be distinguished from progressive disease for an accurate clinical decision (91). Detailed immunophenotyping of leukemias in a well-structured and standardized framework along with markers of ICP inhibitors will help to make decision on progression and pseudo progression. Employing a full panel of ICIs is suggested, since one marker disappears or is expressed low after treatment while other markers increase as a compensation mechanism.

A study performed for assessment of baseline serological autoantibody profile in prediction of responders to ICIs included lymphoma cases with other solid tumors and found that PD-1 and PD-L1 IgG2 autoantibodies were highly produced in approximately 20% of lymphoma responders. The authors suggest that using high-throughput protein microarrays for selected autoantibody markers can predict anti-PD-1 therapeutic response and guide the therapy (92). To implement this approach, there is a need for more evidence-based studies to confirm the prediction of response to ICIs in leukemias and other hematological malignancies. In a recently published study, measurement of CD8⁺LAG3⁺ cells in peripheral blood of solid tissue tumors were significantly correlated with response, survival and progression-free survival (73). Authors suggested that pretreatment measurement of these cells could be used for identifying actionable immune targets. Clinical trials using this panel and evaluating outcome of patients could provide more insight into use of LAG3+ cells as a marker for clinical decisions (88, 93).

Immunophenotyping of leukemias in pediatric and adult patients differ from several aspects. ACCELERATE and European Medicines Agency Pediatric Strategy Forum for medicinal product development of ICIs has published a statement paper for use of ICIs in pediatric patients and declared that; “The major challenge for developing ICIs for pediatric cancers is the lack of neoantigens (based on mutations) and corresponding antigen-specific T cells. Progress critically depends on understanding the immune macroenvironment and microenvironment and the ability of the adaptive immune system to recognize pediatric cancers in the absence of high neoantigen burden. Future clinical studies regarding administration of ICIs in children need to build upon strong biological hypotheses that take into account the distinctive immunobiology of childhood cancers in comparison to that of ICI responsive adult cancers” (94). Extensive profiling of patients in well-structured clinical trials will help understand immunobiology better and allow reaching to a consensus for follow-up.

In this article, we have focused on the possibilities and challenges ahead in clinical immunology and hematology laboratories. There are much more to be explored in imaging and other laboratories (clinical chemistry, clinical microbiology) along with the genetic laboratories in order to make the most accurate assessments for immunotherapies with ICIs. What we need to do in clinical laboratories is to develop standardized methods for all of the above-mentioned testing, work to offer cheaper and more accessible services to patients and develop new methods to be delivered in routine laboratories. We have come a long way since Rituximab was commercialized more than twenty years ago, and we have learned much about immunity during the COVID-19 pandemic period, but we still need to synchronize the tasks of clinical laboratories with the ever-expanding range of immunotherapies.

Drafting manuscript: BA, MS, CP, EG, MG-H, and GYD. Preparing the figures: CP and EG. Editing and revising the manuscript: GYD. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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