Chemo-Immunotherapy: A New Trend in Cancer Treatment.

Chemotherapy has been the basis of advanced cancer treatment for decades. This therapy has largely been considered immunosuppressive, yet accumulated preclinical and clinical evidence shows that certain chemotherapeutic drugs, under defined conditions, may stimulate antitumor immunity and potentiate immune checkpoint inhibitor (ICI)-based therapy. Its effectiveness has been highlighted by recent regulatory approvals of various combinations of chemotherapy with ICIs in several tumors, particularly in some difficult-to-treat cancers. This review discusses the immune modulatory properties of chemotherapy and how they may be harnessed to develop novel chemo-immunotherapy combinations. It also highlights the key determinants of the success of chemo-immunotherapy and provides an overview of the combined chemo-immunotherapies that have been clinically approved.

BTLA dysregulation correlates with poor outcome and diminished T cell-mediated antitumor responses in chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) progressively develop marked immunosuppression, dampening innate and adaptive-driven antitumor responses. However, the underlying mechanisms promoting immune exhaustion are largely unknown. Herein, we provide new insights into the role of BTLA/HVEM axis promoting defects in T cell-mediated responses against leukemic cells. Increased expression of BTLA, an inhibitory immune checkpoint, was detected on the surface of CD4 + and CD8 + T lymphocytes in patients with CLL. Moreover, high levels of BTLA on CD4 + T cells correlated with diminished time to treatment. Signaling through BTLA activation led to decreased IL-2 and IFN-γ production ex vivo, whereas BTLA/HVEM binding disruption enhanced IFN-γ + CD8 + T lymphocytes. Accordingly, BTLA blockade in combination with bispecific anti-CD3/anti-CD19 antibody promoted CD8 + T cell-mediated anti-leukemic responses. Finally, treatment with an anti-BLTA blocking monoclonal antibody alone or in combination with ibrutinib-induced leukemic cell depletion in vitro. Altogether, our data reveal that BTLA dysregulation has a prognostic role and is limiting T cell-driven antitumor responses, thus providing new insights about immune exhaustion in patients with CLL.

Impact of pre- and/or post-autologous stem cell transplantation exposure to brentuximab vedotin on survival outcomes in patients with high-risk Hodgkin lymphoma.

The AETHERA trial demonstrated that brentuximab vedotin (BV) consolidation after autologous stem cell transplantation (ASCT) in patients with Hodgkin lymphoma (HL) at high risk of relapse/progression increases progression-free survival (PFS). Patients previously exposed to BV were excluded from that trial. However, BV alone or in combination with chemotherapy is frequently used as front-line treatment and/or pre-ASCT salvage therapy. We analyzed data from 156 patients with high-risk HL who underwent ASCT with (BV-CON, n = 62) or without (non-BV, n = 94) BV consolidation. Fifty-seven patients received BV-based salvage regimens before ASCT. The 3-year overall survival and PFS for all patients were 91.6% and 70.0%, respectively. Multivariate analysis showed that BV-CON was associated with better PFS (HR 0.39, p = 0.01), whereas positive PET at transplant leaded to worse PFS (HR 2.71, p = 0.001). BV-CON improved PFS in PET-positive patients (72.2% vs. 43.0%, p = 0.05), with a beneficial trend observed in PET negative (88.8% vs. 75.2%, p = 0.09). BV-CON patients with or without BV exposure pre-ASCT had a significantly better PFS than non-BV with or without BV pretransplant treatment (HR 0.36, p = 0.004). The efficacy of real-life BV consolidation therapy was similar to that in the AETHERA trial. This therapeutic strategy improves survival independently of BV exposure prior to ASCT.

BTLA/HVEM Axis Induces NK Cell Immunosuppression and Poor Outcome in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by progressive immunosuppression and diminished cancer immunosurveillance. Immune checkpoint blockade (ICB)-based therapies, a major breakthrough against cancer, have emerged as a powerful tool to reinvigorate antitumor responses. Herein, we analyzed the role of the novel inhibitory checkpoint BTLA and its ligand, HVEM, in the regulation of leukemic and natural killer (NK) cells in CLL. Flow cytometry analyses showed that BTLA expression is upregulated on leukemic cells and NK cells from patients with CLL, whereas HVEM is downregulated only in leukemic cells, especially in patients with advanced Rai-Binet stage. In silico analysis revealed that increased HVEM, but not BTLA, mRNA expression in leukemic cells correlated with diminished overall survival. Further, soluble BTLA (sBTLA) was found to be increased in the sera of patients with CLL and highly correlated with poor prognostic markers and shorter time to treatment. BTLA blockade with an anti-BTLA monoclonal antibody depleted leukemic cells and boosted NK cell-mediated responses ex vivo by increasing their IFN-γ production, cytotoxic capability, and antibody-dependent cytotoxicity (ADCC). In agreement with an inhibitory role of BTLA in NK cells, surface BTLA expression on NK cells was associated with poor outcome in patients with CLL. Overall, this study is the first to bring to light a role of BTLA/HVEM in the suppression of NK cell-mediated immune responses in CLL and its impact on patient's prognosis, suggesting that BTLA/HVEM axis may be a potential therapeutic target in this disease.

LAG-3 Blockade with Relatlimab (BMS-986016) Restores Anti-Leukemic Responses in Chronic Lymphocytic Leukemia.

The inclusion of monoclonal antibodies targeting immune checkpoints such PD-1/PD-L1 or CTLA-4 has revolutionized the landscape of anti-cancer therapy. However, PD-1 and CTLA-4 blockade failed to achieve clinical benefit in CLL, thus attention has been focused on emerging checkpoints in this malignancy. LAG-3 is an immune checkpoint receptor that negatively regulates T cell-mediated responses by inducing an hyporesponsive state, thus promoting tumor escape. Patients with chronic lymphocytic leukemia (CLL) develop a profound immune suppression that leads to lessened immunosurveillance and increased risk of developing a secondary neoplasia. In the study herein, we report the profound dysregulation of LAG-3 on leukemic cells in CLL. Likewise, natural killer (NK) and T cells showed increased LAG-3 expression, hence suggesting a role for this checkpoint in CLL-associated immunosuppression. High LAG-3 expression, as well as high levels of soluble LAG-3 (sLAG-3), correlated with adverse cytogenetics and poor outcome in patients with CLL, highlighting the clinical relevance of this immune checkpoint. Treatment of peripheral blood mononuclear cells (PBMCs) from patients with CLL with relatlimab, a new anti-LAG-3 blocking antibody currently evaluated in numerous clinical trials, depleted leukemic cells and restored NK cell- and T cell-mediated responses. Moreover, combination of LAG-3 with the immunomodulatory drug (IMiD) lenalidomide significantly increased IL-2 production by T cells and antibody-dependent cytotoxicity (ADCC) mediated by NK cells. Altogether, these data provide new insights into the potential anti-leukemic effects of relatlimab, currently in clinical trials in CLL, and provides the rationale to further investigate its combination with IMiDs for the management of hematological malignancies.

Driver Mutations and Single Copy Number Abnormalities Identify Binet Stage A Patients with Chronic Lymphocytic Leukemia with Aggressive Progression.

The correlation between progression and the genetic characteristics of Binet stage A patients with chronic lymphocytic leukemia (CLL) detected by whole exome sequencing (WES) was analyzed in 55 patients. The median follow-up for the patients was 102 months. During the follow-up, 24 patients (43%) progressed. Univariate Cox analysis showed that the presence of driver mutations, the accumulation of two or more mutations, the presence of adverse mutations, immunoglobulin heavy chain genes (IGHV) mutation status and unfavorable single copy number abnormalities (SCNAs) were associated with a higher risk of progression. Particularly, the occurrence of an adverse mutation and unfavorable SCNAs increased the risk of progression nine-fold and five-fold, respectively. Nevertheless, only the occurrence of adverse mutations retained statistical significance in the multivariate analysis. All patients carrying an unfavorable mutation progressed with a median progression-free survival (PFS) of 29 months. The accumulation of two or more mutations also increased the risk of progression with a median PFS of 29 months. The median PFS of patients with unfavorable SCNAs was 38 months. Combining mutations and SCNAs, patients may be stratified into three groups with different prognostic outcomes: adverse (17% probability of five-year PFS), protective (86% probability of five-year PFS) and neither (62% probability of five-year PFS, p < 0.001). Overall, the analysis of the mutational status of patients with CLL at an early stage of the disease may allow the identification of patients with a high risk of progression. The feasibility of an early therapeutic intervention in these particular patients requires further investigation.

The Mithralog EC-7072 Induces Chronic Lymphocytic Leukemia Cell Death by Targeting Tonic B-Cell Receptor Signaling.

B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic lymphocytic leukemia (CLL) treated with novel agents targeting components of this pathway. Herein, we demonstrate that the mithralog EC-7072 displays high ex vivo cytotoxic activity against leukemia cells from CLL patients independently from high-risk prognostic markers and IGHV mutational status. EC-7072 was significantly less toxic against T cells and NK cells and did not alter the production of the immune effector molecules IFN-γ and perforin. EC-7072 directly triggered caspase-3-dependent CLL cell apoptosis, which was not abrogated by microenvironment-derived factors that sustain leukemia cell survival. RNA-sequencing analyses revealed a dramatic EC-7072-driven reprograming of the transcriptome of CLL cells, including a wide downregulation of multiple components and targets of the BCR signaling pathway. Accordingly, we found decreased levels of phosphorylated signaling nodes downstream of the BCR. Crosslinking-mediated BCR activation antagonized CLL cell death triggered by EC-7072, increased the phosphorylation levels of the abovementioned signaling nodes and upregulated BCL2 expression, suggesting that the mithralog disrupts CLL cell viability by targeting the BCR signaling axis at multiple levels. EC-7072 exerted similar or higher antileukemic activity than that of several available CLL therapies and displayed additive or synergistic interaction with these drugs in killing CLL cells. Overall, our findings provide rationale for future investigation to test whether EC-7072 may be a potential therapeutic option for patients with CLL and other B-cell malignancies.

NK Cells in the Treatment of Hematological Malignancies.

Natural killer (NK) cells have the innate ability to kill cancer cells, however, tumor cells may acquire the capability of evading the immune response, thereby leading to malignancies. Restoring or potentiation of this natural antitumor activity of NK cells has become a relevant therapeutic approach in cancer and, particularly, in hematological cancers. The use of tumor-specific antibodies that promote antibody-dependent cell-mediated cytotoxicity (ADCC) through the ligation of CD16 receptor on NK cells has become standard for many hematologic malignancies. Hematopoietic stem cell transplantation is another key therapeutic strategy that harnesses the alloreactivity of NK cells against cancer cells. This strategy may be refined by adoptive transfer of NK cells that may be previously expanded, activated, or redirected (chimeric antigen receptor (CAR)-NK cells) against cancer cells. The antitumor activity of NK cells can also be boosted by cytokines or immunostimulatory drugs such as lenalidomide or pomalidomide. Finally, targeting immunosubversive mechanisms developed by hematological cancers and, in particular, using antibodies that block NK cell inhibitory receptors and checkpoint proteins are novel promising therapeutic approaches in these malignant diseases.

Bendamustine as part of conditioning of autologous stem cell transplantation in patients with aggressive lymphoma: a phase 2 study from the GELTAMO group.

We conducted a phase 2 trial to evaluate the safety and efficacy of bendamustine instead of BCNU (carmustine) in the BEAM (BCNU, etoposide, cytarabine and melphalan) regimen (BendaEAM) as conditioning for autologous stem-cell transplantation (ASCT) in patients with aggressive lymphomas. The primary endpoint was 3-year progression-free survival (PFS). Sixty patients (median age 55 [28-71] years) were included. All patients (except one who died early) engrafted after a median of 11 (9-72) and 14 (4-53) days to achieve neutrophil and platelet counts of >0.5 × 109 /l and >20 × 109 /l, respectively. Non-relapse mortality at 100 days and 1 year were 3.3% and 6.7%, respectively. With a median follow-up of 67 (40-77) months, the estimated 3-year PFS and overall survival (OS) were 58% and 75%, respectively. Patients in partial response at study entry had significantly worse PFS and OS than patients who underwent ASCT in complete metabolic remission, and this was the only prognostic factor associated with both PFS (Relative risk [RR], 0.27 [95% confidence interval {CI} [0.12-0.56]) and OS (RR, 0.40 [95% CI 0.17-0.97]) in the multivariate analysis. BendaEAM conditioning is therefore a feasible and effective regimen in patients with aggressive lymphomas. However, patients not in complete metabolic remission at the time of transplant had poorer survival and so should be considered for alternative treatment strategies.

Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia.

One of the cardinal features of chronic lymphocytic leukemia (CLL) is its association with a profound immunosuppression. NK cell function is markedly impaired in CLL patients, who show a significant dysregulation of the expression of activating and inhibitory receptors. Here, we analyzed the role of the novel inhibitory receptor Ig-like transcript 2 (ILT2, also termed LIR-1, LILRB1) in the regulation of NK cells in CLL. Our results show that ILT2 expression was significantly decreased on leukemic cells and increased on NK cells of CLL patients, particularly in those with advanced disease and with bad prognostic features, such as those carrying chromosome del(11q). The immunomodulatory drug lenalidomide may regulate the expression of ILT2 and its ligands in CLL since it significantly increased the expression of ILT2 and partially reestablished the expression of its ligands on leukemic cells. Furthermore, lenalidomide significantly increased the activation and proliferation of NK cells, which was strongly enhanced by ILT2 blockade. Combining ILT2 blockade and lenalidomide activated NK cell cytotoxicity resulting in increased elimination of leukemic cells from CLL patients. Overall, we describe herein the role of an inhibitory receptor involved in the suppression of NK cell activity in CLL, which is restored by ILT2 blockade in combination with lenalidomide, suggesting that it may be an interesting therapeutic strategy to be explored in this disease.

NK Cell-Based Immunotherapy in Cancer Metastasis.

Metastasis represents the leading cause of cancer-related death mainly owing to the limited efficacy of current anticancer therapies on advanced malignancies. Although immunotherapy is rendering promising results in the treatment of cancer, many adverse events and factors hampering therapeutic efficacy, especially in solid tumors and metastases, still need to be solved. Moreover, immunotherapeutic strategies have mainly focused on modulating the activity of T cells, while Natural Killer (NK) cells have only recently been taken into consideration. NK cells represent an attractive target for cancer immunotherapy owing to their innate capacity to eliminate malignant tumors in a non-Major Histocompatibility Complex (MHC) and non-tumor antigen-restricted manner. In this review, we analyze the mechanisms and efficacy of NK cells in the control of metastasis and we detail the immunosubversive strategies developed by metastatic cells to evade NK cell-mediated immunosurveillance. We also share current and cutting-edge clinical approaches aimed at unleashing the full anti-metastatic potential of NK cells, including the adoptive transfer of NK cells, boosting of NK cell activity, redirecting NK cell activity against metastatic cells and the release of evasion mechanisms dampening NK cell immunosurveillance.

Ig-like transcript 2 (ILT2) suppresses T cell function in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is associated with a profound dysregulation of the immune system. Loss of T cell function is frequently caused in cancer by sustained signaling of inhibitory receptors. Here, we analyzed the role of the novel inhibitory receptor Ig-like transcript 2 (ILT2) in the pathogenesis of CLL. We observed that ILT2 expression was markedly reduced on leukemic cells, whereas it was increased on CD8 and CD4 T cells from CLL patients, particularly in those patients harboring chromosome 11q deletion, which includes the ATM gene. A deep dysregulation of ILT2 ligands expression in leukemia cells was also observed. ILT2 impaired the activation and proliferation of CD4 and CD8 T cells in CLL patients, but it had no effect in leukemic cells. ILT2 downregulated the production of IL-2 by CD4 T cells of CLL patients and induced the expression of cytokines that promote the survival of leukemic cells, such as IFN-γ, by T cells. Importantly, ILT2 blockade restored the activation, proliferation and cytokine production of T cells. In conclusion, we describe a novel immune inhibitory pathway that is upregulated in CLL and delineate a new potential target to be explored in this disease.

Analysis of clinical prognostic variables for Chronic Lymphocytic Leukemia decision-making problems.

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is a disease with highly heterogeneous clinical course. A key goal is the prediction of patients with high risk of disease progression, which could benefit from an earlier or more intense treatment. In this work we introduce a simple methodology based on machine learning methods to help physicians in their decision making in different problems related to CLL. MATERIAL AND METHODS: Clinical data belongs to a retrospective study of a cohort of 265 Caucasians who were diagnosed with CLL between 1997 and 2007 in Hospital Cabueñes (Asturias, Spain). Different machine learning methods were applied to find the shortest list of most discriminatory prognostic variables to predict the need of Chemotherapy Treatment and the development of an Autoimmune Disease. RESULTS: Autoimmune disease occurrence was predicted with very high accuracy (>90%). Autoimmune disease development is currently an unpredictable severe complication of CLL. Chemotherapy Treatment has been predicted with a lower accuracy (80%). Risk analysis showed that the number of false positives and false negatives are well balanced. CONCLUSIONS: Our study highlights the importance of prognostic variables associated with the characteristics of platelets, reticulocytes and natural killers, which are the main targets of the autoimmune haemolytic anemia and immune thrombocytopenia for autoimmune disease development, and also, the relevance of some clinical variables related with the immune characteristics of CLL patients that are not taking into account by current prognostic markers for predicting the need of chemotherapy. Because of its simplicity, this methodology could be implemented in spreadsheets.

Serum ferritin as prognostic marker in classical Hodgkin lymphoma treated with ABVD-based therapy.

Ferritin levels might correlate with disease activity in classical Hodgkin lymphoma (cHL). We analyzed the prognostic significance of the ferritin value at diagnosis in 173 cHL patients treated with ABVD between 2003 and 2013. The 5-year overall survival (OS) and progression-free survival (PFS) probabilities were 80% and 64%, respectively. Patients with ferritin ≥ 350 µg/l [high ferritin group (HF), n = 62] were more likely to have advanced stage disease, B-symptoms and higher International Prognostic Score (IPS) compared with patients with ferritin < 350 µg/l [low ferritin group (LF), n = 111]. The complete remission (CR) rate and 5-year PFS and OS probabilities were lower in HF vs. LF patients (69% vs. 89%, p = 0.025; 40% vs. 78%, p < 0.001; 61% vs. 90%, p = 0.001; respectively). Multivariate analysis revealed that advanced stage (p = 0.001) and ferritin levels ≥ 350 µg/l (p = 0.002) were independent predictors for PFS. In conclusion, the ferritin level at diagnosis is a useful prognostic marker for cHL.

Expression of ERp5 and GRP78 on the membrane of chronic lymphocytic leukemia cells: association with soluble MICA shedding.

MICA is a ligand of the activating receptor NKG2D, expressed by NK and T cells. MICA expression is induced in cancer cells favoring their elimination by the immune system; however, many advanced tumors shed soluble MICA (sMICA), which impairs NKG2D-mediated cytotoxicity. ERp5 and GRP78 are endoplasmic reticulum-resident proteins that are translocated to the surface of epithelial tumor cells where they interact with MICA and are involved in sMICA shedding. In this study, we analyze the role of ERp5 and GRP78 in sMICA shedding in chronic lymphocytic leukemia (CLL). Immunofluorescence and flow cytometry analyses showed that ERp5 and GRP78 were significantly expressed on the surface of B cells and leukemia cells, but they were not expressed on T cells. The expression of ERp5 and GRP78 was significantly higher in leukemia cells than in B cells from controls. ERp5 and GRP78 co-localized with MICA on the surface of leukemia cells and the levels of expression of ERp5 and GRP78 correlated with the level of expression of membrane-bound MICA in CLL patients. Associated with higher expression of membrane-bound ERp5 and GRP78, serum sMICA levels were approximately threefold higher in patients than in controls. Elevated sMICA levels in CLL patients were associated with the down-modulation of NKG2D surface expression on CD8 T cells. Finally, pharmacological inhibition of B cell lines and stimulated leukemia cells showed that ERp5 activity is involved in sMICA shedding in CLL. In conclusion, these results uncover a molecular mechanism which regulates MICA protein shedding and immune evasion in CLL.

Prognostic significance of CD8 and CD4 T cells in chronic lymphocytic leukemia.

The prognostic value of the number of T cells and NK cells at diagnosis in CLL was analyzed in a cohort of 256 patients with CLL diagnosed between 1997 and 2007. Patients with leukemia showed elevated NK cells and T cell populations and CD4/CD8 ratio was inverted in 39.7% cases. Prognostic significance of lymphocytes was analyzed as a ratio of relative number of T cells to the size of the malignant monoclonal B-cell pool (T/NK cells:Malignant monoclonal B-cells ratio). Patients showed higher relative number of CD4 (p = 0.03), CD8 (p = 0.02), and NK cells (p = 0.01) in early Rai stage of disease. The multivariate Cox analysis identified the relative number of CD8 (hazard ratio (HR) = 1.464; p = 0.006) and CD4 T cells (HR = 0.091; p < 0.01) as independent predictors for survival. Additionally, patients with relative CD8 count > 0.074 or CD4 count > 0.1 had higher 10-year overall survival than patients with CD8 count ≤0.074 or CD4 count ≤0.1 (p = 0.002). Higher CD8 count was associated with significantly higher median time of survival of patients (149.33 vs. 82.06 months). Finally, association of the good prognostic factor of leukemia cells (CD38⁻ with high relative CD8 count identified a group of patients with an indolent clinical course with an overall survival probability at 10 years of 95%.