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.

The molecular mechanisms of apoptosis accompanied with the epigenetic regulation of the NY-ESO-1 antigen in non-small lung cancer cells treated with decitabine (5-aza-CdR).

Dysregulated epigenetic modifications are common in lung cancer but have been reversed using demethylating agent like 5-Aza-CdR. 5-Aza-CdR induces/upregulates the NY-ESO-1 antigen in lung cancer. Therefore, we investigated the molecular mechanisms accompanied with the epigenetic regulation of NY-ESO-1 in 5-Aza-CdR-treated NCI-H1975 cell line. We showed significant induction of the NY-ESO-1 protein (**p < 0.0097) using Cellular ELISA. Bisulfite-sequencing demonstrated 45.6% demethylation efficiency at the NY-ESO-1 gene promoter region and RT-qPCR analysis confirmed the significant induction of NY-ESO-1 at mRNA level (128-fold increase, *p < 0.050). We then investigated the mechanism by which 5-Aza-CdR inhibits cell proliferation in the NCI-H1975 cell line. Upregulation of the death receptors TRAIL (2.04-fold *p < 0.011) and FAS (2.1-fold *p < 0.011) indicate activation of the extrinsic apoptotic pathway. The upregulation of Voltage-dependent anion-selective channel protein 1 (1.9-fold), Major vault protein (1.8-fold), Bax (1.16-fold), and Cytochrome C (1.39-fold) indicate the activation of the intrinsic pathway. We also observed the differential expression of protein Complement C3 (3.3-fold), Destrin (-5.1-fold), Vimentin (-1.7-fold), Peroxiredoxin 4 (-1.6-fold), Fascin (-1.8-fold), Heme oxygenase-2 (-0.67-fold**p < 0.0055), Hsp27 (-0.57-fold**p < 0.004), and Hsp70 (-0.39-fold **p < 0.001), indicating reduced cell growth, cell migration, and metastasis. The upregulation of 40S ribosomal protein S9 (3-fold), 40S ribosomal protein S15 (4.2-fold), 40S ribosomal protein S18 (2.5-fold), and 60S ribosomal protein L22 (4.4-fold) implied the induction of translation machinery. These results reiterate the decisive role of 5-Aza-CdR in lung cancer treatment since it induces the epigenetic regulation of NY-ESO-1 antigen, inhibits cell proliferation, increases apoptosis, and decreases invasiveness.

Successful rechallenge with cisplatin following cisplatin induced ischemic cerebrovascular accident in a patient with small cell lung cancer.

Cisplatin is a widely used platinum-based chemotherapy agent. Its common adverse effects are neuropathy, nephrotoxicity, electrolyte abnormality, and rarely causing thrombotic vascular toxicity. We present a patient known to have small-cell lung cancer who developed ischemic cerebrovascular accident (CVA) after receiving chemotherapy regimen including cisplatin.

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.

The effect of protein mutations on drug binding suggests ensuing personalised drug selection.

The advent of personalised medicine promises a deeper understanding of mechanisms and therefore therapies. However, the connection between genomic sequences and clinical treatments is often unclear. We studied 50 breast cancer patients belonging to a population-cohort in the state of Qatar. From Sanger sequencing, we identified several new deleterious mutations in the estrogen receptor 1 gene (ESR1). The effect of these mutations on drug treatment in the protein target encoded by ESR1, namely the estrogen receptor, was achieved via rapid and accurate protein-ligand binding affinity interaction studies which were performed for the selected drugs and the natural ligand estrogen. Four nonsynonymous mutations in the ligand-binding domain were subjected to molecular dynamics simulation using absolute and relative binding free energy methods, leading to the ranking of the efficacy of six selected drugs for patients with the mutations. Our study shows that a personalised clinical decision system can be created by integrating an individual patient's genomic data at the molecular level within a computational pipeline which ranks the efficacy of binding of particular drugs to variant proteins.

Thrombocytopenia responding to red blood cell transfusion.

Three patients with severe symptomatic iron deficiency anemia and thrombocytopenia had a significant rise in platelet count a few days following packed red blood cell transfusion. Pre-transfusion platelet count of patient one was 17 x 10(9)/L, 22 x 10(9)/L in patient 2 and 29 x 10(9)/L in patient 3. On the 6th day post transfusion, the platelet count rose to 166 x 10(9)/L in patient one, 830 x 10(9)/L in patient 2 and 136 x 10(9)/L in patient 3. The possible mechanisms behind such an unreported observation are discussed.