Receptor Genotyping Research Articles

O-034 Specific KIR gene profiles and recurrent implantation failure

Abstract Study question Is there a link between killer cell immunoglobulin-like receptors (KIRs) genes, haplotypes, or genotypes and recurrent implantation failure (RIF)? Summary answer The KIRAA1 genotype significantly increases the risk of RIF. The presence of the full-length receptor KIR2DS4norm in KIRAA haplotype patients is indicative of RIF development. What is known already KIRs are expressed by a subset of uterine natural killer cells (uNK cells). The KIRs family consists of 16 genes with high polymorphism that are divided into two main haplotypes: KIRAA and KIRBx. The KIRAA haplotype consists mostly of inhibitory KIRs and only one activating receptor, KIR2DS4, whose function is uncertain, whereas the Bx haplotype has a diverse gene composition, with a focus on activating receptor genes. The primary difference is in the balance of KIRs, which can activate or inhibit uNKcells’ ability to produce components required for embryo-implantation, and hence they are thought to being implicated in RIF. Study design, size, duration A three-year observational cohort research was conducted at a private infertility centre. A total of 82 patients were genotyped for activating or inhibiting KIR genes. The study included 60 ovarian normo-responders under the age of 40, with 28 women with repeated implantation failure serving as the study group and 32 as controls. Participants/materials, setting, methods DNA was extracted from blood samples of all participants, including RIF patients and controls. The 16 KIR genes were investigated utilizing PCR with Sequence-Specific Primer (SSP) technique. Individual KIR gene profiles, KIRAA and KIRBx haplotypes, and their unique genotypes were detected by software analyses of the collected data based on the reaction pattern when compared to patterns associated with described KIR gene sequences. All data were statistically analysed with the Fisher Exact Test. Main results and the role of chance The frequencies of the two main haplotypes, KIR AA and KIR Bx, were 28% and 72%, respectively. Patients with more inhibitory KIR genes (haplotype KIR AA) had a higher rate of RIF (60% vs 40% in controls; P = 0.2) than those with KIR Bx phenotypes (37.5% RIF vs 62.5% controls; p = 0.026). Further analysis of KIR AA revealed two genotypes: KIR AA 1 (31.82%) and KIR AA195 (68.18%). The KIR2DS4 locus in the full-length receptor (KIR2DS4-norm) was only expressed in KIR AA1. The distribution of RIF occurrences in the two different genotypes demonstrates a significant increase in the number of RIF in the possessors of KIR AA1 (75% vs 15%, p = 0.01), with no significant difference between the two groups in the KIR AA195. Limitations, reasons for caution The study encompasses small samples but the association strength was noticeably high, which confers the findings more confidence. Wider implications of the findings These findings imply that women with a high proportion of inhibited KIR genes (haplotype KIR AA) and KIR2DS4norm are more likely to develop RIF. Trial registration number NA

A prospective phase 2 study of expeditious EGFR genotyping and immediate therapeutic initiation through extracellular vesicles (EV)-based bronchoalveolar lavage fluid (BALF) liquid biopsy in advanced NSCLC patients.

In our previous study, epidermal growth factor receptor (EGFR) genotyping using extracellular vesicles (EV)-derived DNA isolated from bronchoalveolar lavage fluid (BALF) was proven to be highly concordant with conventional tissue-based genotyping and its turn-around-time (TAT) was only 1-2 days. On this background, we prospectively validated the performance of EV-based BALF liquid biopsy for EGFR genotyping in the real practice of advanced non-small cell lung cancer (NSCLC) patients. After screening 120 newly diagnosed stage III-IV NSCLC patients, 51 cases were detected as EGFR-mutated by EV-based BALF EGFR genotyping and 40 patients were enrolled for gefitinib treatment. BALF EV were isolated by ultracentrifuge method and EGFR genotyping was performed with PCR-based PNA-clamping assisted fluorescence melting curve analysis. The objective response rate, progression-free survival (PFS), TAT, time to treatment initiation (TTI), and concordance rate were analyzed with clinical parameters. There was only one false positive case among the 120 screened patients and the overall concordance rate between tissue biopsy and EV-based BALF liquid biopsy was 99.2% including the subtype of EGFR mutations. TAT for EV-based BALF EGFR genotyping was 1.9±1.1 days, while tissue-based TAT was 12.1±7.2 days (P<0.001). EGFR genotyping was determined even before obtaining histopathologic report in most cases. TTI in BALF EGFR genotyping was faster than tissue genotyping (7.8±6.5 vs. 13.8±12.9 days). Therapeutic outcomes of response rate and PFS were almost similar to tissue-based results. We demonstrated, for the first time, that EV-based BALF liquid biopsy should be an excellent platform for expeditious EGFR genotyping and rapid therapeutic intervention even before obtaining the result of histopathology in advanced NSCLC patients.

Adaptive immune receptor genotyping using the corecount program.

We present a new Rep-Seq analysis tool called corecount, for analyzing genotypic variation in immunoglobulin (IG) and T cell receptor (TCR) genes. corecount is highly efficient at identifying V alleles, including those that are infrequently used in expressed repertoires and those that contain 3' end variation that are otherwise refractory to reliable identification during germline inference from expressed libraries. Furthermore, corecount facilitates accurate D and J gene genotyping. The output is highly reproducible and facilitates the comparison of genotypes from multiple individuals, such as those from clinical cohorts. Here, we applied corecount to the genotypic analysis of IgM libraries from 16 individuals. To demonstrate the accuracy of corecount, we Sanger sequenced all the heavy chain IG alleles (65 IGHV, 27 IGHD and 7 IGHJ) from one individual from whom we also produced two independent IgM Rep-seq datasets. Genomic analysis revealed that 5 known IGHV and 2 IGHJ sequences are truncated in current reference databases. This dataset of genomically validated alleles and IgM libraries from the same individual provides a useful resource for benchmarking other bioinformatic programs that involve V, D and J assignments and germline inference, and may facilitate the development of AIRR-Seq analysis tools that can take benefit from the availability of more comprehensive reference databases.

Clinical impact of KIR haplotypes in 10/10 HLA-matched unrelated donor-recipient pairs undergoing allogeneic hematopoietic stem cell transplantation

To evaluate the impact of killer immunoglobulin-like receptor (KIR) genotyping in allogeneic hematopoietic stem cell transplantation for myeloid disorders at our institution, retrospective KIR genotyping was performed on 77 patients and their 10/10 matched unrelated donors. In a multivariate model including donor age, HLA-DPB1 permissiveness, and presence of donor KIR B/x, an association with overall survival was observed (p = .047). Within the model, increasing donor age increased risk (RR 1.03 [1.00–1.06]/year, p = .046), while donor KIR and HLA-DPB1 permissiveness were not associated with risk (RR 0.51 [0.26–1.03] and RR 0.68 [0.34–1.36]). Grouping recipients by conditioning regimen or limiting the analysis to recipients of peripheral blood stem cells, no association between donor KIR and survival or relapse was identified. No significant associations were observed between overall survival, relapse, grade III-IV acute, or chronic graft versus host disease and presence of KIR B (B/x), quantity of donor KIR B haplotype motifs, or centromeric KIR type (all p > .05).

Predicting EGFR and PD-L1 Status in NSCLC Patients Using Multitask AI System Based on CT Images.

BackgroundEpidermal growth factor receptor (EGFR) genotyping and programmed death ligand-1 (PD-L1) expressions are of paramount importance for treatment guidelines such as the use of tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) in lung cancer. Conventional identification of EGFR or PD-L1 status requires surgical or biopsied tumor specimens, which are obtained through invasive procedures associated with risk of morbidities and may be unavailable to access tissue samples. Here, we developed an artificial intelligence (AI) system that can predict EGFR and PD-L1 status in using non-invasive computed tomography (CT) images.MethodsA multitask AI system including deep learning (DL) module, radiomics (RA) module, and joint (JO) module combining the DL, RA, and clinical features was developed, trained, and optimized with CT images to predict the EGFR and PD-L1 status. We used feature selectors and feature fusion methods to find the best model among combinations of module types. The models were evaluated using the areas under the receiver operating characteristic curves (AUCs).ResultsOur multitask AI system yielded promising performance for gene expression status, subtype classification, and joint prediction. The AUCs of DL module achieved 0.842 (95% CI, 0.825–0.855) in the EGFR mutated status and 0.805 (95% CI, 0.779–0.829) in the mutated-EGFR subtypes discrimination (19Del, L858R, other mutations). DL module also demonstrated the AUCs of 0.799 (95% CI, 0.762–0.854) in the PD-L1 expression status and 0.837 (95% CI, 0.775–0.911) in the positive-PD-L1 subtypes (PD-L1 tumor proportion score, 1%–49% and ≥50%). Furthermore, the JO module of our AI system performed well in the EGFR and PD-L1 joint cohort, with an AUC of 0.928 (95% CI, 0.909–0.946) for distinguishing EGFR mutated status and 0.905 (95% CI, 0.886–0.930) for discriminating PD-L1 expression status.ConclusionOur AI system has demonstrated the encouraging results for identifying gene status and further assessing the genotypes. Both clinical indicators and radiomics features showed a complementary role in prediction and provided accurate estimates to predict EGFR and PD-L1 status. Furthermore, this non-invasive, high-throughput, and interpretable AI system can be used as an assistive tool in conjunction with or in lieu of ancillary tests and extensive diagnostic workups to facilitate early intervention.

A Phase I Clinical Trial Testing the Safety of IL-21-Expanded, Universally Alloreactive Donor-Derived Natural Killer Cells for Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Background: Natural Killer (NK) cells are cytotoxic lymphocytes that are able to exert an anti-tumor effect in an MHC-I independent manner, but are dysfunctional and reduced in number in patients with leukemia. Several studies have shown therapeutic potential for related donor NK cells expanded and/or activated ex vivo when administered to cancer patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, personalized, donor-derived cell therapies require time for donor identification, manufacturing and product release, resulting in patient attrition. As NK alloreactivity and NKG2C expression play critical roles in mediating anti-tumor effects, we identified ‘ideal’ NK cell donors (in partnership with “Be the Match Biotherapies”) for collection. Human leukocyte antigen (HLA) and Killer immunoglobulin receptor (KIR) genotyping were done to screen and select donors. Following this, a sample was collected from donors to expand NK cells under small-scale conditions. If donor NK cells showed robust expansion, they proceeded with apheresis. To ensure these therapeutic cells would be readily-available, we established a third-party NK cell bank through scalable, affordable mass-production with membrane-bound IL-21 feeder cells (FC21), and cryopreserved large numbers of these NK cells for immediate ‘off-the-shelf’ administration to recipients. Here, we report our initial experience in a Phase I trial of these off-the-shelf (OTS) alloreactive NK cells as immunotherapy for patients with relapsed/refractory AML and MDS.Methods: Patients were treated at Dose level 1, to allow infusion at a dose of 1 x 10e7 NK cells/ kg. Each dose level had two cohorts, stratified by age. Patients were enrolled into Cohort 1 (patients <60 yrs. received Fludarabine (Flu) 30 mg/m2/day & Cytarabine 2 g/m2/day on days -6 to -2) and Cohort 2 (patients ≥ 60 yrs. received Flu 30 mg/m2/day (days -5 to -2) & Decitabine 20 mg/m2/day (days -6 to -2). Patients were evaluated to see if they had any donor-directed antibodies. After chemotherapy, NK cells were infused thrice weekly for 6 doses, to be given over a period of 2 weeks (Clinicaltrials.gov: NCT04220684). Persistence of donor NK cells was determined by flow cytometry using haplotype-discriminating anti-HLA antibodies (Figure 1, 2). NK cells were completely HLA-mismatched between donor and recipients.Results: 6 patients (3/cohort) were treated at the first dose level of 1x10 7 NK cells/kg/dose. All 6 patients had relapsed/refractory AML and had received at least two prior lines of treatment. One patient (005) withdrew from the study prior to infusion of NK cells. 6 patients received the first NK cell dose as an inpatient and were discharged to receive the remaining 5 doses as an outpatient. 5 patients tolerated infusion of all 6 doses of NK cells administered over 2 weeks. One patient (006) developed a Cytokine response (CRS) - like syndrome in the context of streptococcus bacteremia and received only one dose of NK cells. Symptoms suggestive of CRS responded fully with steroids and patient was able to successfully wean off steroids and discharged to the outpatient setting. One patient proceeded to allogeneic hematopoietic cell transplant. No infusion-related reactions, neurotoxicity or graft versus host disease was observed. In vivo persistence/expansion NK cells were superior with FLU/CY lymphodepletion (cohort 1).Conclusions: Cryopreserved, third-party donor-derived NK cells are safe and feasible in relapsed/refractory AML patients, and even at this first dose level, completely HLA-mismatched NK cells can persist and expand to high levels in vivo. [Display omitted] DisclosuresVasu: Kiadis, Inc.: Research Funding; Seattle Genetics: Other: travel support; Boehringer Ingelheim: Other: Travel support; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Walker: Newave: Other: clinical trial support; Geron: Other: clinical trial support; Novartis: Other: clinical trial support. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. de Lima: Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding. Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

Contribution of the IdyllaTM System to Improving the Therapeutic Care of Patients with NSCLC through Early Screening of EGFR Mutations.

Epidermal growth factor receptor (EGFR) genotyping, a critical examen for the treatment decisions of patients with non-small cell lung cancer (NSCLC), is commonly assayed by next-generation sequencing (NGS), but this global approach takes time. To determine whether rapid EGFR genotyping tests by the IdyllaTM system guides earlier therapy decisions, EGFR mutations were assayed by both the IdyllaTM system and NGS in 223 patients with NSCLC in a bicentric prospective study. IdyllaTM demonstrated agreement with the NGS method in 187/194 cases (96.4%) and recovered 20 of the 26 (77%) EGFR mutations detected using NGS. Regarding the seven missed EGFR mutations, five were not detected by the IdyllaTM system, one was assayed in a sample with insufficient tumoral cells, and the last was in a sample not validated by the IdyllaTM system (a bone metastasis). IdyllaTM did not detect any false positives. The average time between EGFR genotyping results from IdyllaTM and the NGS method was 9.2 ± 2.2 working days (wd) (12.6 ± 4.0 calendar days (cd)). Subsequently, based on the IdyllaTM method, the timeframe from tumor sampling to the initiation of EGFR-TKI was 7.7 ± 1.2 wd (11.4 ± 3.1 cd), while it was 20.3 ± 6.7 wd (27.2 ± 8.3 cd) with the NGS method (p < 0.001). We thus demonstrated here that the IdyllaTM system contributes to improving the therapeutic care of patients with NSCLC by the early screening of EGFR mutations.

Immune cell profiling of the cerebrospinal fluid enables the characterization of the brain metastasis microenvironment

Brain metastases are the most common tumor of the brain with a dismal prognosis. A fraction of patients with brain metastasis benefit from treatment with immune checkpoint inhibitors (ICI) and the degree and phenotype of the immune cell infiltration has been used to predict response to ICI. However, the anatomical location of brain lesions limits access to tumor material to characterize the immune phenotype. Here, we characterize immune cells present in brain lesions and matched cerebrospinal fluid (CSF) using single-cell RNA sequencing combined with T cell receptor genotyping. Tumor immune infiltration and specifically CD8+ T cell infiltration can be discerned through the analysis of the CSF. Consistently, identical T cell receptor clonotypes are detected in brain lesions and CSF, confirming cell exchange between these compartments. The analysis of immune cells of the CSF can provide a non-invasive alternative to predict the response to ICI, as well as identify the T cell receptor clonotypes present in brain metastasis.

Genomic profiling of extracellular vesicle-derived DNA from bronchoalveolar lavage fluid of patients with lung adenocarcinoma.

BackgroundExtracellular vesicles (EVs) are membrane-bound and nanometer-sized particles released from most types of cells, containing double-stranded DNA reflecting mutational status of the parental tumor cells. Furthermore, epidermal growth factor receptor (EGFR) genotyping using EV-derived DNA (EV DNA) in bronchoalveolar lavage fluid (BALF) showed almost 100% sensitivity in patients with advanced non-small cell lung cancer (NSCLC).MethodsWe assessed the technical performance of DNA derived from BALF-EV (BALF EV DNA) in targeted next-generation sequencing (NGS) for detection and quantification of mutations compared with the matching tissue DNA in 20 lung adenocarcinomas.ResultsDNA yields, tumor purity, and depth of coverage were higher using the tissue DNA than using the BALF EV DNA. However, estimated library size was not significantly different between the two samples, and BALF EV DNA yielded longer fragments than tissue DNA. Overall mutation concordance between the two samples were 56% for nonsynonymous somatic mutations and increased to 81% for clinically significant mutations. By-variant sensitivity for clinically significant somatic mutations increased from 62% to 83% in the NGS of BALF EV DNA. Allele frequencies of EGFR and TP53 were higher in tissue DNA (10–25%) than in BALF EV DNA (<5%). Tumor mutation burden of BALF EV DNA correlated with that of tissue DNA.ConclusionsOur findings demonstrate, for the first time, that BALF EV DNA in patients with NSCLC can be a reliable DNA source for targeted NGS for the identification of actionable genetic alterations and that this approach has high clinical feasibility and utility.

Selection of allogeneic hematopoietic cell transplant donors to optimize natural killer cell alloreactivity

Natural killer (NK) cells are potent mediators of the graft versus leukemia phenomenon critical to the success of allogeneic hematopoietic cell transplantation. Central to calibrating NK effector function via their interaction with class I human leukocyte antigens are the numerous inhibitory killer Ig-like receptors (KIR). The KIR receptors are encoded by a family of polymorphic genes, whose expression is largely stochastic and uninfluenced by human leukocyte antigens genotype. These features provide the opportunity to select hematopoietic cell donors with favorable KIR genotypes that confer enhanced protection from relapse via NK-mediated graft versus leukemia. Over the last 2 decades, a large body of work has emerged examining the use of KIR genotyping to stratify potential donors based on anticipated NK alloreactivity. Overall, these results support KIR-based donor selection for patients undergoing allogeneic hematopoietic cell transplantation for a diagnosis of acute myelogenous leukemia. Despite this, the underlying factors that control NK cell responsiveness are not completely understood, and opportunities remain to refine donor selection using NK cell receptor genotyping. In this review, we will summarize the relevant findings with respect to KIR genotyping as a selection parameter for allogeneic hematopoietic cell donors and address practical considerations with respect to KIR-based selection of donors for patients with myeloid neoplasia.

Alterations in NKG2A and NKG2C Subsets of Natural Killer Cells Following Epstein–Barr Virus Reactivation in CTLA4Ig-based Haploidentical Transplantation Is Associated With Increased Chronic Graft-Versus-Host Disease

The impact of newer approaches to haploidentical transplantation on Epstein-Barr virus (EBV) is largely unknown. We prospectively evaluated the incidence of EBV reactivation and its impact on transplantation outcomes in 71 patients undergoing haploidentical transplantation with posttransplantation cyclophosphamide in combination with CTLA4Ig-based T-costimulation blockade. Eight patients developed EBV reactivation at a median of 96 days with no incidence of lymphoproliferative disorder. There was no impact of EBV reactivation on acute graft-versus-host disease (GVHD), nonrelapse mortality, progression-free, or overall survival. Despite an overall incidence of 19%, there was a significant increase in chronic GVHD following EBV reactivation (62.5% versus 8%; P = 0.01). NKG2A subset of CD56 natural killer cells increased substantially and persisted following EBV reactivation and chronic GVHD, with a reciprocal decrease in NKG2C subset, whereas the reverse was witnessed in those without chronic GVHD (P < 0.01). Increase in NKG2C subset and a decrease in the NKG2A subset were witnessed within 3 months of subsidence of chronic GVHD. Thus, CTLA4Ig-based haploidentical transplantation was associated with a low incidence of EBV reactivation without EBV-lymphoproliferative disorder. However, EBV reactivation was associated with a sustained alteration in NKG2A and NKG2C subsets of CD56 natural killer cells which might have a pathogenic role in chronic GVHD.

Extracellular vesicle-based EGFR genotyping in bronchoalveolar lavage fluid from treatment-naive non-small cell lung cancer patients.

Extracellular vesicles (EV) have been proven to contain double-stranded DNA reflecting the mutational status of the parental tumor cells in non-small cell lung cancer (NSCLC), which can be translated into clinically useful EV-based liquid biopsy for Epidermal growth factor receptor (EGFR) genotyping using bronchoalveolar lavage fluid (BALF) obtained from tumor site. Patients subjected for an initial lung cancer work-up underwent bronchoscopy and BALF was obtained from tumor site. After isolating EVs from BALF by ultracentrifugation, EV-derived DNA (EV DNA) was extracted for subsequent EGFR genotyping performed through peptide nucleic acid (PNA)-mediated Real-Time PCR. The sensitivity, specificity, and concordance rate of BALF EV-based EGFR genotyping were calculated in comparison to tissue genotyping. The average sensitivity and specificity of BALF EV-based EGFR genotyping were 76% and 87%, respectively, while the sensitivity significantly increased as the stage progressed. Especially, in stage IV, BALF EV-based EGFR typing identified all tissue-proven EGFR mutant cases (n=31) and detected 6 additional mutant cases. The concordance rate was 79% in stage I, 100% in stage II, 74% in stage III, and 92% in stage IV. As TNM stage advanced, especially in the presence of metastasis, concordance rate significantly increased (P<0.05). The use of BALF for the collection of EV DNA in lung cancer patients resulted in a highly accurate diagnosis. The establishment of a fast and reliable method to identify target genes using EV DNA illustrated that it can overcome the problems of low sensitivity and instability in using cell-free DNA (cfDNA).

Estrogen receptors genotypes and canine mammary neoplasia

BackgroundEstrogens are essential for the development and proper function of several hormone-dependent organs. There are, however, several lines of evidence associating estrogens with mammary carcinogenesis. A marked individual genetic variability concerning estrogens biosynthesis, metabolism and mechanism of action was recognized and associated with human breast cancer susceptibility, clinical features and progression. Although some genetic variations in canine ESR1 gene were reported, their influence in clinicopathological features and progression of canine mammary tumors has not been fully evaluated. This study aims to assess the influence of SNPs in ESR1 gene (rs397512133, rs397510462, rs851327560, rs397510612, rs852887655, rs852684753 and rs852398698) in canine mammary tumors characteristics and progression. A group of 155 non-neutered bitches with mammary tumors was included in the study. Follow-up information was assessed 24 months after surgery.ResultsGenetic profiles associated with a later onset of mammary tumors and less aggressive clinicopathological features, namely smaller tumor size (≤ 3 cm) with extensive tubular differentiation and low canine-adapted prognostic index (vet-NPI), were identified in this study.ConclusionsOur data suggest that the ESR1 genetic profile may help on the decision regarding the selection of individual tailored preventive measures against canine mammary tumors development, such as early neutering.

HLA and killer cell immunoglobulin-like receptor (KIRs) genotyping in patients with acute ischemic stroke

IntroductionIn humans, a major component of natural killer (NK) and T cell target recognition depends on the surveillance of human leukocyte antigen (HLA) class I molecules by killer immunoglobulin-like receptors (KIRs).AimsTo implement the knowledge about the immunological genetic background of acute ischemic stroke susceptibility in relation to the frequency of the KIR genes and HLA alleles.MethodsSubjects with acute ischemic stroke and subjects without stroke were genotyped for the presence of KIR genes and of the three major KIR ligand groups, HLA-C1, HLA-C2, and HLA-Bw4, both HLA-B and HLA-A loci.ResultsBetween November 2013 and February 2016, consecutive patients with acute ischemic stroke were recruited. As healthy controls, we enrolled subjects without acute ischemic stroke. Subjects with acute ischemic stroke in comparison with controls showed a higher frequency of 2DL3, 2DL5B, 2DS2, and 2DS4 KIR genes and a lower frequency of HLA-B-Bw4I alleles. Subjects without acute ischemic stroke showed a higher frequency of interaction between KIR 2DS2 and HLAC2. We also observed a higher frequency of 2DL3 and 2 DL4 KIR genes in subjects with atherosclerotic (LAAS) subtype. Multiple logistic regression analysis showed a protective effect towards stroke of HLA-B-Bw4I and interaction between KIR 2DL2 and HLAC1 and 2DS2-HLAC2 and a detrimental effect of 2DL2-HLA-C1_A interactions.ConclusionOur findings of a higher frequency of activating KIR genes seem to be consistent with findings previously reported patients with coronary syndrome. This higher frequency of “proinflammatory” genes in subjects with ischemic stroke could also explain the immunoinflammatory activation of the acute phase of stroke.

Circulating innate immune markers and outcomes in treatment-naïve advanced non–small cell lung cancer patients

IntroductionInnate immunity represents the first step of activation of the immune system and dictates the quality of adaptive immune responses. Studies have reported links between systemic inflammatory or innate immune markers and prognosis in patients with lung cancer. To our knowledge, the prospective and concomitant study of these systemic markers has never been performed. MethodsAdvanced treatment-naive non–small cell lung cancer (NSCLC) patients eligible for first-line platinum-based chemotherapy were prospectively included from December 2012 to July 2015 (N = 148). Blood samples of patients were collected before the first cycle for fresh NK cell phenotyping. Peripheral blood mononuclear cells were cryopreserved for natural cytotoxicity receptor (NCR) genotyping as well as sera for NCR's ligand quantification. Data on leukocytes, neutrophils and monocyte counts and lactate dehydrogenase (LDH) levels were extracted from electronic medical records. ResultsAmong all studied markers, monocytosis, neutrophilia, leucocytosis, high LDH and sBAG6 levels and reduced levels of NCR3 transcripts were associated with poor overall survival (OS) in univariate analysis. The levels of NCR3 transcripts was linked to age, number of metastatic sites, monocyte counts, LDH and sBAG6 levels. Neutrophilia was associated to high sBAG6 levels. NCR3 was the unique innate immune parameter that remained as an independent factor associated with both OS (P = 0.003) and progression-free survival (P = 0.009) in the multivariate analysis. ConclusionThis study brought evidence that these biomarkers are entangled; parameters associated with an inflammatory process were related to reduced levels of NCR3 transcripts. Finally, the level of NCR3 transcripts was independently associated with outcomes in treatment-naive patients with advanced NSCLC.

Predicting EGFR mutation status in lung adenocarcinoma on computed tomography image using deep learning.

Epidermal growth factor receptor (EGFR) genotyping is critical for treatment guidelines such as the use of tyrosine kinase inhibitors in lung adenocarcinoma. Conventional identification of EGFR genotype requires biopsy and sequence testing which is invasive and may suffer from the difficulty of accessing tissue samples. Here, we propose a deep learning model to predict EGFR mutation status in lung adenocarcinoma using non-invasive computed tomography (CT).We retrospectively collected data from 844 lung adenocarcinoma patients with pre-operative CT images, EGFR mutation and clinical information from two hospitals. An end-to-end deep learning model was proposed to predict the EGFR mutation status by CT scanning.By training in 14 926 CT images, the deep learning model achieved encouraging predictive performance in both the primary cohort (n=603; AUC 0.85, 95% CI 0.83–0.88) and the independent validation cohort (n=241; AUC 0.81, 95% CI 0.79–0.83), which showed significant improvement over previous studies using hand-crafted CT features or clinical characteristics (p<0.001). The deep learning score demonstrated significant differences in EGFR-mutant and EGFR-wild type tumours (p<0.001).Since CT is routinely used in lung cancer diagnosis, the deep learning model provides a non-invasive and easy-to-use method for EGFR mutation status prediction.

HLA and killer cell immunoglobulin-like receptor (KIRs) genotyping in patients with acute viral encephalitis.

IntroductionThe HLA genes, as well as the innate immune KIR genes, are considered relevant determinants of viral outcomes but no study, to our knowledge, has evaluated their role in the clinical setting of acute viral encephalitis.ResultsSubjects with acute viral encephalitis in comparison to subjects without acute viral encephalitis showed a significantly higher frequency of 2DL1 KIR gene and AA KIR haplotypes and of HLA-C2 and HLA-A-Bw4 alleles. Subjects without acute viral encephalitis showed a higher frequency of interaction between KIR2DL2 and HLAC1. Multiple logistic regression analysis showed the detrimental effect of HLA-A haplotype and HLA-C1, HLA-A-BW4 HLA-B-BW4T alleles, whereas multiple logistic regression showed a protective effect of AB+BB KIR haplotype and a detrimental effect of interaction between KIR3DL1 and HLA-A-Bw4.DiscussionOur findings of a lower frequency of activating receptors in patients with acute encephalitis compared to controls could result in a less efficient response of NK cells. This finding could represent a possible pathogenetic explanation of susceptibility to acute symptomatic encephalitis in patients with viral infection from potentially responsible viruses such as Herpes virus.Materials and Methods30 Consecutive patients with symptomatic acute viral encephalitis and as controls, 36 consecutive subjects without acute encephalitis were analyzed. The following KIR genes were analyzed, KIR2DL1, 2DL2, 2DL3, 2DL5, 3DL1, 3DL2, 3DL3, 2DL4, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DS1, 2 pseudogenes (2DP1 and 3DP1) and the common variants of KIR2DL5 (KIR2DL5A, KIR2DL5B).

Endogenous antibody responses to mucin 1 in a large multiethnic cohort of patients with breast cancer and healthy controls: Role of immunoglobulin and Fcγ receptor genes

High levels of naturally occurring IgG antibodies to mucin 1 (MUC1), a membrane-bound glycoprotein that is overexpressed in patients with breast cancer, are associated with good prognosis. This suggests that endogenous anti-MUC1 antibodies have a protective effect and, through antibody-mediated host immunosurveillance mechanisms, might contribute to a cancer-free state. To test this possibility, we characterized a large number of multiethnic patients with breast cancer and matched controls for IgG antibodies to MUC1. We also aimed to determine whether the magnitude of anti-MUC1 antibody responsiveness was associated with particular immunoglobulin GM (γ marker), KM (κ marker), and Fcγ receptors (FcγR) genotypes. After adjusting for the confounding variables in a multivariate analysis, we found no significant difference in the levels of anti-MUC1 IgG antibodies between patients and cancer-free controls. However, in patients and controls, particular GM, KM, and FcγR genotypes—individually or epistatically—were significantly associated with the levels of anti-MUC1 IgG antibodies in a racially restricted manner. These findings, if confirmed in an independent investigation, could help identify individuals most likely to benefit from a MUC1-based therapeutic or prophylactic vaccine for MUC1-overexpressing malignancies.

Report From the First and Second Spanish Killer Immunoglobulin-Like Receptor Genotyping Workshops: External Quality Control for Natural Killer Alloreactive Donor Selection in Haploidentical Stem Cell Transplantation

An important factor affecting the success in the setting of related haploidentical hematopoietic stem cell transplantation (HSCT) is the graft-versus-leukemia effect mediated by natural killer (NK) cells when the donor displays NK alloreactivity versus the recipient. NK cell function is regulated by killer immunoglobulin-like receptors (KIR) and it has been described that donor KIR genotype influences transplantation outcome. This has led to a requirement of laboratories to have a quality assurance program for validation and control of their KIR genotyping methods. The goal of the 1st and 2nd Spanish KIR Genotyping Workshops was to provide an external proficiency testing program in KIR genotyping for Spanish immunology and transplant laboratories. These workshops were conducted during the years 2014–2016 and consisted of 17 participating laboratories typing a set of 20 samples. The presence/absence of 16 mandatory KIR loci (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 2DP1, 3DL1, 3DL2, 3DL3, 3DS1, and 3DP1) was evaluated per sample. Methods for KIR genotyping included polymerase chain reaction with the use of sequence-specific primers and sequence-specific oligoprobes. Consensus typing was reached in all samples, and the performance of laboratories in external proficiency testing was satisfactory in all cases. The polymorphism detected in the small sample studied in both workshops is indicative of an ample variety of KIR gene profiles in the Spanish population.

Epidermal growth factor receptor (EGFR) genotyping of matched urine, plasma and tumor tissue from non-small cell lung cancer (NSCLC) patients (pts) treated with rociletinib.

9001 Background: Rociletinib is an oral inhibitor of mutant EGFR, including T790M. We compared EGFR mutation detection in circulating tumor DNA from blood and urine to that in matched tissue in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in pts with mutant EGFRpositive advanced NSCLC. Methods: EGFR status was assessed by the therascreen EGFR test (Qiagen) in tissue, BEAMing (Sysmex) in plasma, and a quantitative short footprint assay method that uses next-generation sequencing (Trovagene) in urine. Results: Of 417 pts in the 500 and 625 mg BID dosing groups (data cut-off date: 18 Sept 2015; pts enrolled as of 1 July 2015), 331 of 417 were T790M+ by central tissue genotyping; 189 of 242 were T790M+ by plasma genotyping; and 136 of 169 were T790M+ by urine genotyping. Confirmed investigator-assessed objective response rate (ORR) and median duration of response (mDOR) were similar regardless of sample type (Table). Four of 14 pts who were T790M+ in plasma but T790M− in tissue responded; 3 of 7 pts who were T790M+ in urine but T790M− in tissue responded. Shrinkage of target lesions correlated with higher T790M:activating mutation ratio in plasma (P=0.006). With tissue as reference, positive percent agreement for T790M status between matched plasma and tissue was 81.5% (n=195) and 83.8% (n=136) between matched urine and tissue. In both dosing groups, the most common treatment-related adverse events were hyperglycemia, diarrhea, nausea, and fatigue. Conclusions: In T790M+ pts, response was similar whether T790M status was identified by tissue, plasma or urine. Plasma and urine testing identified T790M mutations missed by biopsy due to tumor heterogeneity or inadequate sample quality. These data suggest plasma and urine EGFRanalyses complement tissue biopsies in EGFR TKI resistant NSCLC. Clinical trial information: NCT01526928. 500 mg BID 625 mg BID Tissue Plasma Urine Tissue Plasma Urine n (T790M+) 156 61 50 175 128 86 ORR, % 25.0 27.9 32.0 39.4 33.6 40.7 95% CI 18.4-32.6 17.1-40.8 19.5-46.7 32.1-47.1 25.5-42.5 30.2-51.8 mDOR, mo 9.0 9.0 9.0 7.9 6.7 8.0 95% CI 4.6-11.0 4.4-11.0 2.6-11.0 5.6-9.2 4.8-9.0 6.8-11.2 CI, confidence interval; mo, months; NR, not reached.