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.

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BackgroundAnemia is the most common hematologic abnormality that a pediatrician encounters in clinical practice. Alpha-Thalassemia (a-thal) is widely reported in the Arabian Peninsula as one of the main causes ofasymptomatic microcytic hypochromic red blood cells with or without anemia in the pediatric population. The most common cause of anemia is either iron deficiency or thalassemia trait. Majority of the carriers for beta-thalassemia and some forms of alpha-thalassemia are clinically asymptomatic and although their hematologic phenotypes (i.e. microcytic hypochromic red blood cells) with or without anemia are predictive markers, they are not specific and overlap with conditions such as iron deficiency. Precise diagnosis of alpha-thalassemia is particularly important in the Gulf region where certain forms of alpha-thalassemia (especially non-deletional) are found, resulting in more severe phenotypes. Geographical prevalence's in the region are also variable as seen in a recent large survey of cord blood from Oman at the Sultan Qaboos University Hospital revealing a minimal frequency of approximately 50% of alpha-thalassemia of which hematologically relevant form is expected to represent a significant proportion. Unlike beta-thalassemia for which differential diagnosis can be made by measuring HbA2 levels, it is difficult to distinguish between alpha-thalassemia and iron deficiency, and DNA based diagnosis is essential. Furthermore, DNA diagnostics can provide insight into the commonly observed discrepancies between phenotypes and genotypes, especially in cases of sickle cell disease and beta-thalassemia, where alphathalassemia is known to act as a genetic modifier. To date neither diagnosis of alpha-thalassemia (trait) nor its molecular spectrum had been accurately performed and described in Qatar. This is despite the fact that in Qatar, both beta and alpha-thalassemias (Hb Barts at birth) are not uncommon and nutritional anemia can rarely be expected as cause of such microcytic hypochromic red cells because of therelatively high socioeconomic status of the country. The only published work on alpha-thalassemia in Qatar is a hospital based study in which 1,702 Qatari nationals (905 females, 797 males) were investigated for hemoglobinopathies and of which 8.05% of the patients were suspected to be alpha-thalassemia trait. Preliminary hematology results from the first year of the national premarital program in Qatar further support these results (unpublished data). Our study is the first of its kind in attempting to characterize the molecular spectrum of alpha-thalassemia in Qatar. For this purpose the study population were selected from Qatari school children. According to Qatar's national preschool assessmentprogram, all students are required to undergo routine physical checkup and blood analysis for anemia, prior to the start of their school. Results are then forwarded to the school where the school nurse will review and refer students with low Hb leveland/or low normal Hb along with low MCV values, to the Consultant Pediatrician at Qatar's Pediatric Clinic at the School Objectives:● Determine the mutation spectrum of alpha-thalassemia in Qatari school children population.● Making a preliminary assessment of the prevalence of inherited alpha-thalassemia in Qatari school children with hypochromic microcytic red cell with or without anemia.● Establish molecular diagnostic protocols for diagnosis of alpha-thalassemia in Qatar, more specifically in HMC.● Improving the healthcare management (treatment options) of individuals with hypochromic microcytic red cell with or without anemia.● Depending upon the spectrum obtained, making recommendations regarding expansion of the current premarital and genetic counseling services in Hamad Medical Corporation.MethodologyThis was a prospective study that began in May 2012 for duration of two years and extended for an extra year searching for further mutations and analysis. Random samples were selected through the pre-school health assessment program at Qatari public schools. It is mandatory for all students to submit a full blood work-up on admission, to be held on record at their respective schools. The designated health professionals at these schools evaluate the complete blood count (CBC), and identify students with hypochromic mycrocytic red cells with or without anemia. Those with definite clinical and laboratory determination of iron-deficiency anemia are treated first for nutritional anemia. For this study, children that continued to have microcytosis (low MCV) were randomly selected. Hemoglobin (Hb) electrophoresis was performed to rule out other causes of microcytic anemia, including beta-thal. The group was ultimately narrowed down to a pool of 127 individuals with findings highly suggestive of alpha-thal, although not yet molecularly diagnosed. The samples were screened for the presence of the –alpha3.7 deletion and the alpha–5 nt, alphapoly A1 (alphaT-Saudi) and alphapolyA2 nondeletional mutations. A second group of 110 children who attended the pediatric clinic for reasons other than anemia were randomly selected. and included in this study in order to determine the alleles and genotype frequencies of the –alpha3.7 deletion in the Qatari population.ResultsAmong the samples of 127 individuals, a total of 50 samples exhibited the 3.7 kb deletion; 38 (30.0%) subjects were heterozygous for the mutation, while 12 (9.4%) subjects were homozygous for the mutation. Only one subject (0.8%) was found to be heterozygous for the alpha–5 nt mutation. Three samples were positive for the alphapolyA1 (alphaT-Saudi) mutation, two (1.6%) of the three were heterozygotes and one was a compound heterozygote with the –alpha3.7 deletion. None of the subjects carried the apolyA2 mutation. Fifteen (11.8%) subjectswith mild indices who tested negative for the presence of the–alpha3.7 deletion were considered normal for alpha-thal, while 58 (45.6%) subjects were negative for all four alpha-thal mutations. These subjects were characterized by severe hypochromic microcytic anemia necessitating the extension of the screening for other alpha-thal mutations by sequencing both alpha-globingenes for nondeletional mutations or by multiplex ligand dependent probe amplification (MLPA) (P140 HBA kit; MRC-Holland, Amsterdam, The Netherlands) (http://www.mlpa.com) analysis of the a locus for large deletions. The 110 individuals who constituted the random sample were subsequently screened for the presence of the –alpha3.7deletion that was the most common alpha-thal mutation in the 127 individuals with hypochromic microcytic anemia. Twenty nine (26.4%) out of 110 samples were found to be heterozygous, while five (4.5%) samples were homozygous for the–alpha3.7 deletion. The –alpha3.7 allele frequency in the Qatari population was calculated from these data as 17.7%. Using this frequency, the expected carrier frequencies in the Qatari population were calculated as 3.13% for homozygotes and 29.13% for heterozygotes.Conclusionwere found positive for one of the three mutations, the ? α3.7 deletion, αpolyA1 and the α?5 nt mutations. In this pool of individuals, the ? α3.7 deletion was the most common with 9.4% homozygotes, 30.0% heterozygotes and an allele frequency of 19.7%. 57.2% of children failed to exhibit any mutation despite their clinical presentations highly suggest alpha-thalassemia. This suggests the possibility of other existing mutations in Qatari populations that are yet to be elicited. The mutations found in this study are ground breaking for the Qatari medical community, and have provided a solid explanation for patients with findings suggestive of alpha-thal. This study has paved the way for diagnostic testing for alpha-thal in the Qatari pediatric population. This data will be shared in order to establish an algorithm for prevention, treatment and counseling related to alpha-thal in the Qatari pediatric population. This study has provided important clinical diagnostic insight for physicians treating patients suspected of alpha-thalassemia in Qatari pediatric population and also molecular diagnostic tools for laboratories to test such patients.