Adoptive transfer of ex vivo expanded SARS-CoV-2-specific cytotoxic lymphocytes: A viable strategy for COVID-19 immunosuppressed patients?

Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is on focus of research. We evaluate herein the feasibility of expanding virus-specific T cells (VST) against SARS-CoV-2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS-CoV-2 asymptomatic infection/negative serology, (b) SARS-CoV-2 symptomatic infection/positive serology, and (c) no history of SARS-CoV-2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 62.7%, respectively) with CD4 + dominance (60% in both donors). Higher numbers of VST were obtained from the donor 2 as compared to donor 1. T-cell clonality test showed oligoclonal reproducible peaks on a polyclonal background for both donors. In contrast, VST could be neither expanded nor primed in a donor without evidence of prior infection. This proof-of-concept study supports the feasibility of expanding ex vivo SARS-CoV-2-specific VST from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS-CoV-2-specificity for future adoptive transfer to immunosuppressed patients.

Detailed analysis of inversions predicted between two human genomes: errors, real polymorphisms, and their origin and population distribution.

The growing catalogue of structural variants in humans often overlooks inversions as one of the most difficult types of variation to study, even though they affect phenotypic traits in diverse organisms. Here, we have analysed in detail 90 inversions predicted from the comparison of two independently assembled human genomes: the reference genome (NCBI36/HG18) and HuRef. Surprisingly, we found that two thirds of these predictions (62) represent errors either in assembly comparison or in one of the assemblies, including 27 misassembled regions in HG18. Next, we validated 22 of the remaining 28 potential polymorphic inversions using different PCR techniques and characterized their breakpoints and ancestral state. In addition, we determined experimentally the derived allele frequency in Europeans for 17 inversions (DAF = 0.01-0.80), as well as the distribution in 14 worldwide populations for 12 of them based on the 1000 Genomes Project data. Among the validated inversions, nine have inverted repeats (IRs) at their breakpoints, and two show nucleotide variation patterns consistent with a recurrent origin. Conversely, inversions without IRs have a unique origin and almost all of them show deletions or insertions at the breakpoints in the derived allele mediated by microhomology sequences, which highlights the importance of mechanisms like FoSTeS/MMBIR in the generation of complex rearrangements in the human genome. Finally, we found several inversions located within genes and at least one candidate to be positively selected in Africa. Thus, our study emphasizes the importance of careful analysis and validation of large-scale genomic predictions to extract reliable biological conclusions.

[Early diagnosis of HIV in Primary Care in Spain. Results of a pilot study based on targeted screening based on indicator conditions, behavioral criteria and region of origin]. / Diagnóstico precoz del VIH en atención primaria en España. Resultados de una prueba piloto de cribado dirigido basado en condiciones indicadoras, criterios conductuales y de origen.

OBJECTIVES: To estimate the prevalence of HIV infection in patients diagnosed with an indicator condition (IC) for HIV and/or risk behavior for their acquisition and/or coming from high prevalence countries. To determine the acceptability and feasibility of offering HIV testing based on IC and behavioral and origin criteria in Primary Care (PC). DESIGN: Cross-sectional study in a convenience sample. LOCATION: Six PC centers in Spain. PARTICIPANTS: The inclusion criteria were: patients between 16 and 65years old who presented at least one of the proposed ICs and/or at least one of the proposed behavioral and/or origin criteria. A total of 388 patients participated. INTERVENTION: HIV serology was offered to all patients who met the inclusion criteria. MAIN MEASUREMENTS: Description of IC frequency, behavioral and origin criteria. Prevalence of HIV infection. Level of acceptability and feasibility of the HIV screening based on IC and behavioral and origin criteria. RESULTS: A total of 174 patients had an IC (44.84%). The most common behavioral criterion was: having unprotected sex at some time in life with people who did not know their HIV status (298; 76.8%). Four HIV+ patients (1.03%) were diagnosed. All had an IC and were men who had sex with men. The level of acceptability in PC was high. CONCLUSIONS: Offering HIV testing to patients with IC and behavioral criteria is feasible and effective in PC.

Validation and genotyping of multiple human polymorphic inversions mediated by inverted repeats reveals a high degree of recurrence.

In recent years different types of structural variants (SVs) have been discovered in the human genome and their functional impact has become increasingly clear. Inversions, however, are poorly characterized and more difficult to study, especially those mediated by inverted repeats or segmental duplications. Here, we describe the results of a simple and fast inverse PCR (iPCR) protocol for high-throughput genotyping of a wide variety of inversions using a small amount of DNA. In particular, we analyzed 22 inversions predicted in humans ranging from 5.1 kb to 226 kb and mediated by inverted repeat sequences of 1.6-24 kb. First, we validated 17 of the 22 inversions in a panel of nine HapMap individuals from different populations, and we genotyped them in 68 additional individuals of European origin, with correct genetic transmission in ∼ 12 mother-father-child trios. Global inversion minor allele frequency varied between 1% and 49% and inversion genotypes were consistent with Hardy-Weinberg equilibrium. By analyzing the nucleotide variation and the haplotypes in these regions, we found that only four inversions have linked tag-SNPs and that in many cases there are multiple shared SNPs between standard and inverted chromosomes, suggesting an unexpected high degree of inversion recurrence during human evolution. iPCR was also used to check 16 of these inversions in four chimpanzees and two gorillas, and 10 showed both orientations either within or between species, providing additional support for their multiple origin. Finally, we have identified several inversions that include genes in the inverted or breakpoint regions, and at least one disrupts a potential coding gene. Thus, these results represent a significant advance in our understanding of inversion polymorphism in human populations and challenge the common view of a single origin of inversions, with important implications for inversion analysis in SNP-based studies.

Population genetic analysis of bi-allelic structural variants from low-coverage sequence data with an expectation-maximization algorithm.

BACKGROUND: Population genetics and association studies usually rely on a set of known variable sites that are then genotyped in subsequent samples, because it is easier to genotype than to discover the variation. This is also true for structural variation detected from sequence data. However, the genotypes at known variable sites can only be inferred with uncertainty from low coverage data. Thus, statistical approaches that infer genotype likelihoods, test hypotheses, and estimate population parameters without requiring accurate genotypes are becoming popular. Unfortunately, the current implementations of these methods are intended to analyse only single nucleotide and short indel variation, and they usually assume that the two alleles in a heterozygous individual are sampled with equal probability. This is generally false for structural variants detected with paired ends or split reads. Therefore, the population genetics of structural variants cannot be studied, unless a painstaking and potentially biased genotyping is performed first. RESULTS: We present svgem, an expectation-maximization implementation to estimate allele and genotype frequencies, calculate genotype posterior probabilities, and test for Hardy-Weinberg equilibrium and for population differences, from the numbers of times the alleles are observed in each individual. Although applicable to single nucleotide variation, it aims at bi-allelic structural variation of any type, observed by either split reads or paired ends, with arbitrarily high allele sampling bias. We test svgem with simulated and real data from the 1000 Genomes Project. CONCLUSIONS: svgem makes it possible to use low-coverage sequencing data to study the population distribution of structural variants without having to know their genotypes. Furthermore, this advance allows the combined analysis of structural and nucleotide variation within the same genotype-free statistical framework, thus preventing biases introduced by genotype imputation.

Amino Acid-Metabolizing Enzymes in Advanced High-Grade Serous Ovarian Cancer Patients: Value of Ascites as Biomarker Source and Role for IL4I1 and IDO1.

The molecular mechanisms contributing to immune suppression in ovarian cancer are not well understood, hampering the successful application of immunotherapy. Amino acid-metabolizing enzymes are known to contribute to the immune-hostile environment of various tumors through depletion of amino acids and production of immunosuppressive metabolites. We aimed to collectively evaluate the activity of these enzymes in high-grade serous ovarian cancer patients by performing targeted metabolomics on plasma and ascites samples. Whereas no indication was found for enhanced l-arginine or l-glutamine metabolism by immunosuppressive enzymes in ovarian cancer patients, metabolism of l-tryptophan by indoleamine 2,3-dioxygenase 1 (IDO1) was significantly elevated compared to healthy controls. Moreover, high levels of l-phenylalanine- and l-tyrosine-derived metabolites associated with interleukin 4 induced 1 (IL4I1) activity were found in ovarian cancer ascites samples. While l-tryptophan is a major substrate of both IDO1 and IL4I1, only its enhanced conversion into l-kynurenine by IDO1 could be detected, despite the observed activity of IL4I1 on its other substrates. In ascites of ovarian cancer patients, metabolite levels were higher compared to those in plasma, demonstrating the value of utilizing this fluid for biomarker identification. Finally, elevated metabolism of l-phenylalanine and l-tyrosine by IL4I1 correlated with disease stage, pointing towards a potential role for IL4I1 in ovarian cancer progression.

Benefits, Facilitators and Barrier Reductions in Physical Activity Programmes for People with Severe Mental Disorder: A Systematic Review.

The current high prevalence of people with Severe Mental Disorder and the high impact the latter has on their quality of life is one of the main problems in terms of health, as it affects both physical and mental health. One of the lines of action to intervene in these factors is the practice of physical activity, as this usually has a low level of participation due to different barriers. However, there are several facilitators that improve adherence to these practices. The main objectives of this work were (1) to conduct a systematic review of the scientific literature on the possible benefits obtained by people with SMD from their participation in physical activity programmes; (2) to identify the characteristics of physical activity programmes and determine the barriers to their implementation that have been considered and (3) the facilitators incorporated. To meet these objectives, the SCOPUS, Web of Science, PubMed, Dialnet and Elsevier online databases were consulted and, following the PRISMA statement, 17 articles were finally selected. Their analysis has revealed various physical, psychological and social benefits, as well as the barriers that appear in the intervention programmes, mostly related to personal factors and the programme itself, and those factors that facilitate their adherence or development, the most common being the carrying out of the activities outdoors, the inclusion of social components and the possibility of adapting the activities. In this way, the results obtained have made it possible to highlight the characteristics that should be taken into account when planning this type of intervention.

Detecting ALK, ROS1, and RET fusions and the METΔex14 splicing variant in liquid biopsies of non-small-cell lung cancer patients using RNA-based techniques.

ALK, ROS1, and RET fusions and MET∆ex14 variant associate with response to targeted therapies in non-small-cell lung cancer (NSCLC). Technologies for fusion testing in tissue must be adapted to liquid biopsies, which are often the only material available. In this study, circulating-free RNA (cfRNA) and extracellular vesicle RNA (EV-RNA) were purified from liquid biopsies. Fusion and MET∆ex14 transcripts were analyzed by nCounter (Nanostring) and digital PCR (dPCR) using the QuantStudio® System (Applied Biosystems). We found that nCounter detected ALK, ROS1, RET, or MET∆ex14 aberrant transcripts in 28/40 cfRNA samples from positive patients and 0/16 of control individuals (70% sensitivity). Regarding dPCR, aberrant transcripts were detected in the cfRNA of 25/40 positive patients. Concordance between the two techniques was 58%. Inferior results were obtained when analyzing EV-RNA, where nCounter often failed due to a low amount of input RNA. Finally, results of dPCR testing in serial liquid biopsies of five patients correlated with response to targeted therapy. We conclude that nCounter can be used for multiplex detection of fusion and MET∆ex14 transcripts in liquid biopsies, showing a performance comparable with next-generation sequencing platforms. dPCR could be employed for disease follow-up in patients with a known alteration. cfRNA should be preferred over EV-RNA for these analyses.

Digital multiplexed analysis of circular RNAs in FFPE and fresh non-small cell lung cancer specimens.

Although many studies highlight the implication of circular RNAs (circRNAs) in carcinogenesis and tumor progression, their potential as cancer biomarkers has not yet been fully explored in the clinic due to the limitations of current quantification methods. Here, we report the use of the nCounter platform as a valid technology for the analysis of circRNA expression patterns in non-small cell lung cancer (NSCLC) specimens. Under this context, our custom-made circRNA panel was able to detect circRNA expression both in NSCLC cells and formalin-fixed paraffin-embedded (FFPE) tissues. CircFUT8 was overexpressed in NSCLC, contrasting with circEPB41L2, circBNC2, and circSOX13 downregulation even at the early stages of the disease. Machine learning (ML) approaches from different paradigms allowed discrimination of NSCLC from nontumor controls (NTCs) with an 8-circRNA signature. An additional 4-circRNA signature was able to classify early-stage NSCLC samples from NTC, reaching a maximum area under the ROC curve (AUC) of 0.981. Our results not only present two circRNA signatures with diagnosis potential but also introduce nCounter processing following ML as a feasible protocol for the study and development of circRNA signatures for NSCLC.

Evaluation of PSA testing by general practitioners: regional study in the autonomic Community of Valencia.

OBJECTIVES: The aim of the study is to compare the use of PSA testing among general practitioners (GPs). METHODS: The number of PSA tests ordered by general practitioners in the years 2008-2009 was examined in a cross-sectional study of nine health districts of Spain. The percentage of PSA ordered to men younger than 50 (PSA<50/PSAtotal) and 40 years (PSA<40/PSAtotal) was calculated. The percentage of men over 50 years who were attended was also calculated and this data was compared with the number of PSA ordered to this population. For two of the departments, these data were also compared between GPs and urologists. RESULTS: PSA testing in 2009 is higher than 2008 in seven health districts. PSA testing in men younger than 50 years was increased along the period of the study and in men younger than 40 years remained steady. The differences between the values of the indicators for urologists and GPs are significant. CONCLUSIONS: The number of PSA tests and the percentage performed to men younger 50 years has been increasing and the variability is high. These data are suggestive for interventions focused on PSA testing and prostate cancer screening in primary care settings.

Comprehensive analysis of the clinicopathological features, targetable profile, and prognosis of mucinous adenocarcinoma of the lung.

PURPOSE: The clinicopathological or genetic features related to the prognosis of mucinous adenocarcinoma are unknown because of its rarity. The clinicopathological or targetable features were investigated for better management of patients with mucinous adenocarcinoma of the lung. METHODS: We comprehensively evaluated the clinicopathological and genetic features of 60 completely resected mucinous lung adenocarcinomas. Targetable genetic variants were explored using nCounter and polymerase chain reaction, PD-L1 and TTF-1 expression were evaluated using immunohistochemistry. We analyzed the prognostic impact using the Kaplan-Meier method and log-rank test. RESULTS: Of the 60 enrolled patients, 13 (21.7%) had adenocarcinoma in situ/minimally invasive adenocarcinoma, and 47 (78.3%) had invasive mucinous adenocarcinoma (IMA). Fifteen patients (25%) showed a pneumonic appearance on computed tomography (CT). CD74-NRG1 fusion, EGFR mutations, and BRAF mutation were detected in three (5%), four (6.7%), and one (1.7%) patient(s), respectively. KRAS mutations were detected in 31 patients (51.7%). Two patients (3.5%) showed immunoreactivity for PD-L1. No in situ or minimally invasive cases recurred. IMA patients with pneumonic appearance had significantly worse recurrence-free survival (RFS) and overall survival (OS) (p < 0.001). Furthermore, IMA patients harboring KRAS mutations had worse RFS (p = 0.211). Multivariate analysis revealed that radiological pneumonic appearance was significantly associated with lower RFS (p < 0.003) and OS (p = 0.012). KRAS mutations served as an unfavorable status for RFS (p = 0.043). CONCLUSION: Mucinous adenocarcinoma had a low frequency of targetable genetic variants and PD-L1 immunoreactivity; however, KRAS mutations were frequent. Pneumonic appearance on CT imaging and KRAS mutations were clinicopathological features associated with a worse prognosis.

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

Functional and structural analysis of five mutations identified in methylmalonic aciduria cblB type.

ATP:cob(I)alamin adenosyltransferase (ATR, E.C.2.5.1.17) converts reduced cob(I)alamin to the adenosylcobalamin cofactor. Mutations in the MMAB gene encoding ATR are responsible for the cblB type methylmalonic aciduria. Here we report the functional analysis of five cblB mutations to determine the underlying molecular basis of the dysfunction. The transcriptional profile along with minigenes analysis revealed that c.584G>A, c.349-1G>C, and c.290G>A affect the splicing process. Wild-type ATR and the p.I96T (c.287T>C) and p.R191W (c.571C>T) mutant proteins were expressed in a prokaryote and a eukaryotic expression systems. The p.I96T protein was enzymatically active with a K(M) for ATP and K(D) for cob(I)alamin similar to wild-type enzyme, but exhibited a 40% reduction in specific activity. Both p.I96T and p.R191W mutant proteins are less stable than the wild-type protein, with increased stability when expressed under permissive folding conditions. Analysis of the oligomeric state of both mutants showed a structural defect for p.I96T and also a significant impact on the amount of recovered mutant protein that was more pronounced for p.R191W that, along with the structural analysis, suggest they might be misfolded. These results could serve as a basis for the implementation of pharmacological therapies aimed at increasing the residual activity of this type of mutations.

RNA-Based Multiplexing Assay for Routine Testing of Fusion and Splicing Variants in Cytological Samples of NSCLC Patients.

The detection of ALK receptor tyrosine kinase (ALK), ROS proto-oncogen1, receptor tyrosine kinase (ROS1), ret proto-oncogen (RET), and MET proto-oncogen exon 14 skipping (METΔex14) allows for the selection of specific kinase inhibitor treatment in patients with non-small cell lung cancer (NSCLC). Multiplex technologies are recommended in this setting. We used nCounter, a multiplexed technology based on RNA hybridization, to detect ALK, ROS1, RET, and METΔex14 in RNA purified from cytological specimens (n = 16) and biopsies (n = 132). Twelve of the 16 cytological samples (75.0%) were evaluable by nCounter compared to 120 out of 132 (90.9%) biopsies. The geometrical mean (geomean) of the housekeeping genes of the nCounter panel, but not the total amount of RNA purified, was significantly higher in biopsies vs. cytological samples. Among cytological samples, we detected ALK (n = 3), METΔex14 (n = 1) and very high MET expression (n = 1) positive cases. The patient with METΔex14 had a partial response to tepotinib, one of the patients with ALK fusions was treated with crizotinib with a complete response. Cell blocks and cytological extensions can be successfully used for the detection of fusions and splicing variants using RNA-based methods such as nCounter.

Community acquired respiratory virus infections in adult patients undergoing umbilical cord blood transplantation.

Characteristics and risk factors (RFs) of community-acquired respiratory virus (CARV) infections after umbilical cord blood transplantation (UCBT) are lacking. We retrospectively analyzed CARV infections in 216 single-unit myeloablative UCBT recipients. One-hundred and fourteen episodes of CARV infections were diagnosed in 62 (29%) patients. Upper respiratory tract disease (URTD) occurred in 61 (54%) whereas lower respiratory tract disease (LRTD) in 53 (46%). The 5-year cumulative incidence of CARV infection was 29%. RFs for developing CARV infections were: prednisone-based graft-versus-host disease (GVHD) prophylaxis and grade II-IV acute GVHD. RFs analysis of CARV progression to LRTD identified 2007-2009 period and absolute lymphocyte count (ALC) < 0.5 × 109/L. ALC < 0.5 × 109/L had a negative impact on day 60 mortality in both overall CARV and those with LRTD, whereas proven LRTD was associated with higher day 60 mortality. CARV infections had a negative effect on non-relapse mortality. Overall survival at day 60 after CARV detection was significantly lower in recipients with LRTD compared with URTD (74% vs. 93%, respectively). In conclusion, CARV infections after UCBT are frequent and may have a negative effect in the outcomes, in particular in the context of lymphocytopenia.

Validation of the Scale of Basic Psychological Needs towards Physical Exercise, with the Inclusion of Novelty.

The purpose of this study was to validate and adapt to the Spanish context of Physical Education, the Spanish version of the Scale of Basic Psychological Needs in the context of physical exercise, with the incorporation of novelty to the scale. The sample that took part in the study was 2372 people from 16 to 48 years old from the province of Almeria. In order to analyze the psychometric properties of the scale, several analyses have been carried out. The results have offered support both for the eight-factor structure and for the higher-order double model where the eight subscales are joined into two constructs called frustration and satisfaction. The structure of both models was invariant with respect to gender and age. Cronbach's alpha values were above 0.70 in the subscales and scales; and adequate levels of temporal stability. In addition, the subfactors pertaining to the satisfaction of basic psychological needs positively predicted the intrinsic motivation for physical activity, while each of the subfactors of the frustration of psychological needs predicted it negatively. The results of this study provide evidence of the reliability and validity of the BPNS in the Spanish context of physical activity.

An Inclusive View of the Disability of Secondary School Students.

Achieving the educational inclusion of students with special educational needs (SEN) is one of the significant challenges of the current Spanish educational system. This is a group of students with a high rate of bullying that leads to academic failure, as well as significant psychological and social consequences. Despite the fact that the behaviours and psychological characteristics of their peers seem to influence the degree of inclusion, there is no detail on this subject. Therefore, the aim of this paper is to determine the relationship between emotional intelligence, psychological flexibility, prosocial behaviour and inclusive behaviour. To carry out this study, a sample of 642 students between the ages of 12 and 19 years old participated and answered four questionnaires, one for each variable under study. The relationships established were extracted from different statistical analyses and a hypothesised predictive model. The results obtained revealed that emotional intelligence is positively related to psychological flexibility and prosocial behaviour and that these, in turn, are positively related to the development of inclusive behaviour. Therefore, the importance of considering the variables under study during the teaching-learning processes carried out in the classroom is highlighted.

Prospective analysis of liquid biopsies of advanced non-small cell lung cancer patients after progression to targeted therapies using GeneReader NGS platform.

BACKGROUND: In a significant percentage of advanced non-small cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses at time to progression. We prospectively analyzed the appearance of genetic alterations associated with resistance in liquid biopsies of advanced NSCLC patients progressing to targeted therapies using the NGS platform. METHODS: A total of 24 NSCLC patients were included in the study, 22 progressing to tyrosine kinase inhibitors and two to other treatments. Liquid biopsies samples were obtained and analyzed using the GeneReadTM QIAact Lung DNA UMI Panel, designed to enrich specific target regions and containing 550 variant positions in 19 selected genes frequently altered in lung cancer tumors. Previously, a retrospective validation of the panel was performed in clinical samples. RESULTS: Of the 21 patients progressing to tyrosine kinase inhibitors with valid results in liquid biopsy, NGS analysis identified a potential mechanism of resistance in 12 (57%). The most common were acquired mutations in ALK and EGFR, which appeared in 8/21 patients (38%), followed by amplifications in 5/21 patients (24%), and KRAS mutations in one patient (5%). Loss of the p.T790M was also identified in two patients progressing to osimertinib. Three of the 21 (14%) patients presented two or more concomitant alterations associated with resistance. Finally, an EGFR amplification was found in the only patient progressing to immunotherapy included in the study. CONCLUSIONS: NGS analysis in liquid biopsies of patients progressing to targeted therapies using the GeneReader platform is feasible and can help the oncologist to make treatment decisions.

Response to crizotinib in a non-small-cell lung cancer patient harboring an EML4-ALK fusion with an atypical LTBP1 insertion.

Fusion of the anaplastic lymphoma receptor tyrosine kinase gene (ALK) with the echinoderm microtubule-associated protein 4 gene (EML4) is the second most common actionable alteration in non-small-cell lung cancer, with a frequency of 5%. Here, we present a case of an EML4-ALK-positive patient with an atypical in-frame insertion from the LTBP1 gene in the canonical junction of variant 1. The patient was a 39-year-old never-smoker female diagnosed with Stage IV lung adenocarcinoma. A core biopsy was negative for EGFR and KRAS mutations but positive for ALK immunohistochemistry and fluorescence in situ hybridization. When submitted to nCounter, the sample showed a 3'/5' imbalance indicative of an ALK rearrangement, but failed to give a positive signal for any of the variants tested. Finally, a band with a molecular weight higher than expected appeared after reverse transcriptase-polymerase chain reaction analysis. When Sanger sequencing was performed, the band revealed an atypical EML4-ALK fusion gene with an in-frame 129 bp insertion. A 115 bp segment of the insertion corresponded to an intronic region of LTBP1, a gene located in the short arm of chromosome 2, between ALK and EML4. The patient received crizotinib and showed a good therapeutic response that is still ongoing after 12 months. Our result suggests that short in-frame insertions of other genes in the EML4-ALK junction do not affect the sensitivity of the EML4-ALK fusion protein to crizotinib.

BH4 responsiveness associated to a PKU mutation with decreased binding affinity for the cofactor.

BACKGROUND: Tetrahydrobiopterin (BH4), cofactor of phenylalanine hydroxylase, can be used to treat a subset of phenylketonuria (PKU) patients as it results in a reduction in blood phenylalanine levels. The molecular basis of the response appears to be multifactorial. METHOD: A standard BH4 loading test (20 mg/kg) was performed. Genotyping was performed by DGGE and sequencing analysis. Expression analysis of the D129G mutation was performed in E. coli (expression as fusion protein MBP-PAH) and in a human hepatoma cell line with an N-terminal FLAG epitope. RESULTS: We report the positive response and long-term treatment of a patient functionally hemizygous for the D129G mutation in the phenylalanine hydroxylase gene. Expression in the prokaryotic system revealed partial activity and a decreased binding affinity for BH4 of the mutant protein. In the eukaryotic system the mutant protein shows reduced stability. CONCLUSION: The D129G mutation which confers a BH4-responsive phenotype, has a decreased binding affinity for BH4.