Circulating exosomal immuno-oncological checkpoints and cytokines are potential biomarkers to monitor tumor response to anti-PD-1/PD-L1 therapy in non-small cell lung cancer patients

Immune checkpoint inhibitors (ICIs) including anti-PD-1 and anti-PD-L1 antibodies, have significantly changed the treatment outcomes of NSCLC patients with better overall survival. However, 15-40% of the patients still fail to respond to ICIs therapy. Identification of biomarkers associated with responses are mandated in order to increase the efficacy of such therapy. In this study we evaluated 27 serum-derived exosomal immuno-oncological proteins and 44 cytokines/chemokines before and after ICIs therapy in 17 NSCLC patients to identify surrogate biomarkers for treatment/monitoring patient stratification for maximum therapeutic benefit. We first confirmed the identity of the isolated exosomes to have their specific markers (CD63, CD81, HSP70 and CD91). We have demonstrated that baseline concentration of exosomal-PD-L1 (p<0.0001), exosomal-PD-L2 (p=0.0413) and exosomal-PD-1 (p=0.0131) from NSCLC patients were significantly higher than their soluble-free forms. Furthermore, the exosomal-PD-L1 was present in all the patients (100%), while only 71% of patients expressed tissue PD-L1. This indicates that exosomal-PD-L1 is a more reliable diagnostic biomarker. Interestingly, exosomal-PD-L2 expression was significantly higher (p=0.0193) in tissue PD-L1-negative patients compared to tissue PD-L1-positive patients. We have also shown that immuno-oncological proteins isolated from pre-ICIs treated patients were significantly higher in exosomes compared to their soluble-free counterparts (CD152, p=0.0008; CD80, p=0.0182; IDO, p=0.0443; Arginase, p<0.0001; Nectin-2, p<0.0001; NT5E, p<0.0001; Siglec-7, p<0.0001; Siglec-9, p=0.0335; CD28, p=0.0092; GITR, p<0.0001; MICA, p<0.0001). Finally, the changes in the expression levels of exosomal immuno-oncological proteins/cytokines and their correlation with tumor response to ICIs treatment were assessed. There was a significant downregulation of exosomal PD-L1 (p=0.0156), E-Cadherin (p=0.0312), ULBP1 (p=0.0156), ULBP3 (p=0.0391), MICA (p=0.0391), MICB (p=0.0469), Siglec7 (p=0.0078) and significant upregulation of exosomal PD-1 (p=0.0156) and IFN- γ (p=0.0156) in responding patients. Non-responding patients showed a significant increase in exosomal-PD-L1 (p=0.0078). Furthermore, responding-patients without liver-metastasis showed significant-upregulation of PD-1 (p=0.0070), and downregulation of ULBP1 (p=0.0137) and Siglec-7 (p=0.0037). Non-responding patients had significant-downregulation of ULBP3 (p=0.0317) in patient without brain-metastasis and significant-upregulation/downregulation of PD-L1 and ULBP3 (p=0.0262/0.0286) in patients with pulmonary-metastasis. We demonstrated for the first time that exosomal immuno-oncological proteins/cytokines are potential biomarkers to monitor response to ICIs therapy and can predict the clinical outcomes in NSCLC patients

Circulating exosomal immuno-oncological checkpoints and cytokines are potential biomarkers to monitor tumor response to anti-PD-1/PD-L1 therapy in non-small cell lung cancer patients

Immune checkpoint inhibitors (ICIs) including anti-PD-1 and anti-PD-L1 antibodies, have significantly changed the treatment outcomes of NSCLC patients with better overall survival. However, 15-40% of the patients still fail to respond to ICIs therapy. Identification of biomarkers associated with responses are mandated in order to increase the efficacy of such therapy. In this study we evaluated 27 serum-derived exosomal immuno-oncological proteins and 44 cytokines/chemokines before and after ICIs therapy in 17 NSCLC patients to identify surrogate biomarkers for treatment/monitoring patient stratification for maximum therapeutic benefit. We first confirmed the identity of the isolated exosomes to have their specific markers (CD63, CD81, HSP70 and CD91). We have demonstrated that baseline concentration of exosomal-PD-L1 (p<0.0001), exosomal-PD-L2 (p=0.0413) and exosomal-PD-1 (p=0.0131) from NSCLC patients were significantly higher than their soluble-free forms. Furthermore, the exosomal-PD-L1 was present in all the patients (100%), while only 71% of patients expressed tissue PD-L1. This indicates that exosomal-PD-L1 is a more reliable diagnostic biomarker. Interestingly, exosomal-PD-L2 expression was significantly higher (p=0.0193) in tissue PD-L1-negative patients compared to tissue PD-L1-positive patients. We have also shown that immuno-oncological proteins isolated from pre-ICIs treated patients were significantly higher in exosomes compared to their soluble-free counterparts (CD152, p=0.0008; CD80, p=0.0182; IDO, p=0.0443; Arginase, p<0.0001; Nectin-2, p<0.0001; NT5E, p<0.0001; Siglec-7, p<0.0001; Siglec-9, p=0.0335; CD28, p=0.0092; GITR, p<0.0001; MICA, p<0.0001). Finally, the changes in the expression levels of exosomal immuno-oncological proteins/cytokines and their correlation with tumor response to ICIs treatment were assessed. There was a significant downregulation of exosomal PD-L1 (p=0.0156), E-Cadherin (p=0.0312), ULBP1 (p=0.0156), ULBP3 (p=0.0391), MICA (p=0.0391), MICB (p=0.0469), Siglec7 (p=0.0078) and significant upregulation of exosomal PD-1 (p=0.0156) and IFN- γ (p=0.0156) in responding patients. Non-responding patients showed a significant increase in exosomal-PD-L1 (p=0.0078). Furthermore, responding-patients without liver-metastasis showed significant-upregulation of PD-1 (p=0.0070), and downregulation of ULBP1 (p=0.0137) and Siglec-7 (p=0.0037). Non-responding patients had significant-downregulation of ULBP3 (p=0.0317) in patient without brain-metastasis and significant-upregulation/downregulation of PD-L1 and ULBP3 (p=0.0262/0.0286) in patients with pulmonary-metastasis. We demonstrated for the first time that exosomal immuno-oncological proteins/cytokines are potential biomarkers to monitor response to ICIs therapy and can predict the clinical outcomes in NSCLC patients.This research was funded by Academic Health System, Medical Research Center, Hamad Medical Corporation, Doha, Qatar, grant number MRC-01-20-507 and the Article Processing Charges was funded by Academic Health System, Medical Research Center, Hamad Medical Corporation, Doha, Qatar

Serum immune mediators as novel predictors of response to anti-PD-1/PD-L1 therapy in non-small cell lung cancer patients with high tissue-PD-L1 expression

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related morbidity and mortality worldwide. Immune checkpoint inhibitors (ICIs) including anti-PD-1 and anti-PD-L1 antibodies, have significantly changed the treatment outcomes with better overall survival, but only 15-40% of the patients respond to ICIs therapy. The search for predictive biomarkers of responses is warranted for better clinical outcomes. We aim here to identify pre-treatment soluble immune molecules as surrogate biomarkers for tissue PD-L1 (TPD-L1) status and as predictors of response to anti-PD-1/PD-L1 therapy in NSCLC patients. Sera from 31 metastatic NSCLC patients, eligible for anti-PD-1/PD-L1 or combined chemoimmunotherapy, were collected prior to treatment. Analysis of soluble biomarkers with TPD-L1 status showed significant up/down regulation of the immune inhibitory checkpoint markers (sSiglec7, sSiglec9, sULBP4 and sPD-L2) in patients with higher TPD-L1 (TPD-L1 >50%) expression. Moreover, correlation analysis showed significant positive linear correlation of soluble PD-L1 (sPD-L1) with higher TPD-L1 expression. Interestingly, only responders in the TPD-L1 >50% group showed significant down regulation of the immune inhibitory markers (sPD-L2, sTIMD4, sNectin2 and CEA). When responders vs. non-responders were compared, significant down regulation of other immune inhibitory biomarkers (sCD80, sTIMD4 and CEA) was recorded only in responding patients. In this, the optimal cut-off values of CD80 <91.7 pg/ml and CEA <1614 pg/ml were found to be significantly associated with better progression free survival (PFS). Indeed, multivariate analysis identified the cutoff-value of CEA <1614 pg/ml as an independent predictor of response in our patients. We identified here novel immune inhibitory/stimulatory soluble mediators as potential surrogate/predictive biomarkers for TPD-L1 status, treatment response and PFS in NSCLC patients treated with anti-PD-1/PD-L1 therapy.This research was funded by Academic Health System, Medical Research Center, Hamad Medical Corporation, Doha, Qatar, grant number MRC-01-20-507 and the Article Processing Charges was funded by Academic Health System, Medical Research Center, Hamad Medical Corporation, Doha, Qatar. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. AcknowledgmentsScopu

Persistent anti-NY-ESO-1-specific T cells and expression of differential biomarkers in a patient with metastatic gastric cancer benefiting from combined radioimmunotherapy treatment: a case report

Combined radioimmunotherapy is currently being investigated to treat patients with cancer. Anti-programmed cell death-1 (PD-1) immunotherapy offers the prospect of long-term disease control in solid tumors. Radiotherapy has the ability to promote immunogenic cell death leading to the release of tumor antigens, increasing infiltration and activation of T cells. New York esophageal squamous cell carcinoma-1 (NY-ESO-1) is a cancer–testis antigen expressed in 20% of advanced gastric cancers and known to induce humoral and cellular immune responses in patients with cancer. We report on the dynamic immune response to the NY-ESO-1 antigen and important immune-related biomarkers in a patient with metastatic gastric cancer treated with radiotherapy combined with anti-PD-1 pembrolizumab antibody.Our patient was an 81-year-old man diagnosed with locally advanced unresectable mismatch repair-deficient gastric cancer having progressed to a metastatic state under a second line of systemic treatment consisting of an anti-PD-1 pembrolizumab antibody. The patient was subsequently treated with local radiotherapy administered concomitantly with anti-PD-1, with a complete response on follow-up radiologic assessment. Disease control was sustained with no further therapy for a period of 12 months before relapse. We have identified an NY-ESO-1-specific interferon-γ (IFN-γ) secretion from the patients’ T cells that was significantly increased at response (****p˂0.0001). A novel promiscuous immunogenic NY-ESO-1 peptide P39 (P153–167) restricted to the four patient’s HLA-DQ and HLA-DP alleles was identified. Interestingly, this peptide contained the known NY-ESO-1-derived HLA-A2-02:01(P157–165) immunogenic epitope. We have also identified a CD107+ cytotoxic T cell subset within a specific CD8+/HLA-A2-NY-ESO-1 T cell population that was low at disease progression, markedly increased at disease resolution and significantly decreased again at disease re-progression. Finally, we identified two groups of cytokines/chemokines. Group 1 contains five cytokines (IFN-γ, tumor necrosis factor-α, interleukin-2 (IL-2), IL-5 and IL-6) that were present at disease progression, significantly downregulated at disease resolution and dramatically upregulated again at disease re-progression. Group 2 contains four biomarkers (perforin, soluble FAS, macrophage inflammatory protein-3α and C-X-C motif chemokine 11/Interferon–inducible T Cell Alpha Chemoattractant that were present at disease progression, significantly upregulated at disease resolution and dramatically downregulated again at disease re-progression. Combined radioimmunotherapy can enhance specific T cell responses to the NY-ESO-1 antigen that correlates with beneficial clinical outcome of the patient

Treatment with decitabine induces the expression of stemness markers, PD-L1 and NY-ESO-1 in colorectal cancer: potential for combined chemoimmunotherapy

Background: The mechanism of tumor immune escape and progression in colorectal cancer (CRC) is widely investigated in-vitro to help understand and identify agents that might play a crucial role in response to treatment and improve the overall survival of CRC patients. Several mechanisms of immune escape and tumor progression, including expression of stemness markers, inactivation of immunoregulatory genes by methylation, and epigenetic silencing, have been reported in CRC, indicating the potential of demethylating agents as anti-cancer drugs. Of these, a chemotherapeutic demethylating agent, Decitabine (DAC), has been reported to induce a dual effect on both DNA demethylation and histone changes leading to an increased expression of target biomarkers, thus making it an attractive anti-tumorigenic drug. Methods: We compared the effect of DAC in primary 1076 Col and metastatic 1872 Col cell lines isolated and generated from patients' tumor tissues. Both cell lines were treated with DAC, and the expression of the NY-ESO-1 cancer-testis antigen, the PD-L1 immunoinhibitory marker, and the CD44, Nanog, KLF-4, CD133, MSI-1 stemness markers were analyzed using different molecular and immunological assays. Results: DAC treatment significantly upregulated stemness markers in both primary 1076 Col and meta-static 1872 Col cell lines, although a lower effect occurred on the latter: CD44 (7.85 fold; ***p = 0.0001 vs. (4.19 fold; *p = 0.0120), Nanog (4.1 fold; ***p < 0.0001 vs.1.69 fold; ***p = 0.0008), KLF-4 (4.33 fold; ***p < 0.0001 vs.2.48 fold; ***p = 0.0005), CD133 (16.77 fold; ***p = 0.0003 vs.6.36 fold; *p = 0.0166), and MSI-1 (2.33 fold; ***p = 0.0003 vs.2.3 fold; ***p = 0.0004), respectively. Interestingly, in the metastatic 1872 Col cells treated with DAC, the expression of both PD-L1 and NY-ESO-1 was increased tenfold (*p = 0.0128) and fivefold (***p < 0.0001), respectively. Conclusions: We conclude that the upregulation of both stemness and immune checkpoint markers by DAC treatment on CRC cells might represent a mechanism of immune evasion. In addition, induction of NY-ESO-1 may represent an immuno-therapeutic option in metastatic CRC patients. Finally, the combination of DAC and anti-PD-1/anti-PD-L1 antibodies treatment should represent a potential therapeutic intervention for this group of patients. 2023, The Author(s).Open Access funding provided by the Qatar National Library. The study was supported by the Medical Research Center, Academic Health System, at Hamad Medical Corporation as part of the approved funded IRGC project # IRGC-04-SI-17-142. The Open Access funding is provided by Qatar National Library, Doha, Qatar.Scopu

Persistence of spike-specific immune responses in BNT162b2-vaccinated donors and generation of rapid ex-vivo T cells expansion protocol for adoptive immunotherapy: A pilot study

Introduction: The BNT162b2 mRNA-based vaccine has shown high efficacy in preventing COVID-19 infection but there are limited data on the types and persistence of the humoral and T cell responses to such a vaccine. Methods: Here, we dissect the vaccine-induced humoral and cellular responses in a cohort of six healthy recipients of two doses of this vaccine. Results and discussion: Overall, there was heterogeneity in the spike-specific humoral and cellular responses among vaccinated individuals. Interestingly, we demonstrated that anti-spike antibody levels detected by a novel simple automated assay (Jess) were strongly correlated (r=0.863, P<0.0001) with neutralizing activity; thus, providing a potential surrogate for neutralizing cell-based assays. The spike-specific T cell response was measured with a newly modified T-spot assay in which the high-homology peptide-sequences cross-reactive with other coronaviruses were removed. This response was induced in 4/6 participants after the first dose, and all six participants after the second dose, and remained detectable in 4/6 participants five months post-vaccination. We have also shown for the first time, that BNT162b2 vaccine enhanced T cell responses also against known human common viruses. In addition, we demonstrated the efficacy of a rapid ex-vivo T cell expansion protocol for spike-specific T cell expansion to be potentially used for adoptive-cell therapy in severe COVID-19, immunocompromised individuals, and other high-risk groups. There was a 9 to 13.7-fold increase in the number of expanded T cells with a significant increase of anti-spike specific response showing higher frequencies of both activation and cytotoxic markers. Interestingly, effector memory T cells were dominant in all four participants’ CD8+ expanded memory T cells; CD4+ T cells were dominated by effector memory in 2/4 participants and by central memory in the remaining two participants. Moreover, we found that high frequencies of CD4+ terminally differentiated memory T cells were associated with a greater reduction of spike-specific activated CD4+ T cells. Finally, we showed that participants who had a CD4+ central memory T cell dominance expressed a high CD69 activation marker in the CD4+ activated T cells.This research was funded by Academic Health System, Medical Research Center, Hamad Medical Corporation, Doha, Qatar, grant number MRC-01-21-113, and the Article Processing Charges was funded by Academic Health System, Medical Research Center, Hamad Medical Corporation, Doha, Qatar. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication