Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, ...and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting.
We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design.
PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp).
We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.
Exploring subjective elements of the food environment remains key to understand why and how residents purchase food. Our aim was to explore and describe the social norms relating to the local food ...environment and food purchasing behaviors, as perceived by residents and food traders in Madrid, Spain. This qualitative study took place in a middle socioeconomic status neighborhood of Madrid between January 2015 and May 2016. We conducted 35 semi-structured interviews. We used stratified purposive sampling to recruit residents, neighborhood workers (
= 20) and food traders (
= 15) representing different levels of involvement with food purchasing behaviors. We analyzed these data using an interpretative phenomenological analysis approach. Participants highlighted social aspects of the food environment in relation to food purchasing behaviors. First, interpersonal and relational food environment elements were emphasized, including trust and tradition. Participants also identified generational demographic trends in relation to changes in the way residents purchased food: the new pace of life and the lack of time to buy fresh food and to cook at home. All these elements were influenced by the economic crisis. Food environment interventions aiming to improve food purchasing behaviors and residents' diets should consider intermediate social aspects of the food environment like trust and tradition and the fast pace of life of younger generations.
The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of ...these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early‐onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.‐287C>T and PAX5 binding to BRCA2:c.‐296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.
We have identified rare single nucleotide variants and small indels mapping to the 5’ non‐coding regions of BRCA1 and BRCA2 through targeted sequencing of over 6000 early onset/familial breast cancer patients. We describe four variants in minimal promoter regions that alter gene activity and identify specific transcription factor disruptions for two of these variants. In parallel, we have assessed whether these variants exhibit features expected for high‐risk pathogenic BRCA1 or BRCA2 variants, using available clinical and population data.
To describe severe infection, foci of infection, microorganisms, associated factors, and impact on mortality in patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD).
The ...study was based on a multicenter prospective cohort of patients with RA-ILD followed up from 2015 to 2023. The main outcome measures were incident severe infection and fatal infection. We evaluated infectious foci, etiologic agents, vaccination status, variables associated with lung function, and clinical-therapeutic variables in RA. The incidence rate (IR) for infection and mortality was calculated per 100 person-years, and 3 multivariate models were constructed to explore factors associated with infection.
We followed up 148 patients with RA-ILD for a median 56.7 months (699.3 person-years). During this period, 142 patients (96%) had at least 1 infection. A total of 368 infectious episodes were recorded, with an IR of 52.6 per 100 person-years. Of the 48 patients who died, 65% did so from infection. Respiratory infections were the most common first infection (74%), infection overall (74%), and fatal infection (80%) and were caused mostly by SARS CoV-2
, and influenza A virus. The factors associated with an increased risk of infection and death in patients with RA-ILD were age, inflammatory activity, and therapy with corticosteroids and immunosuppressants.
Patients with RA-ILD have a high risk of serious infection, especially respiratory infection. Infection develops early, is recurrent, and is frequently fatal. The presence of associated factors such as advanced age, joint inflammation, and treatment highlight the importance of integrated and preventive medical care.
Purpose
To obtain reference norms of EORTC QLQ-C30, EORTC QLQ-BR23, and EQ-5D-5L, based on a population of Spanish non-metastatic breast cancer patients at diagnosis and 2 years after, according to ...relevant demographic and clinical characteristics.
Methods
Multicentric prospective cohort study including consecutive women aged ≥ 18 years with a diagnosis of incident non-metastatic breast cancer from April 2013 to May 2015. Health-related quality of life (HRQoL) questionnaires were administered between diagnosis and beginning the therapy, and 2 years after. HRQoL differences according to age, comorbidity and stage were tested with ANOVA or Chi Square test and multivariate linear regression models.
Results
1276 patients were included, with a mean age of 58 years. Multivariate models of EORTC QLQ-C30 summary score and EQ-5D-5L index at diagnosis and at 2-year follow-up show the independent association of comorbidity and tumor stage with HRQoL. The standardized multivariate regression coefficient of EORTC QLQ-C30 summary score was lower (poorer HRQoL) for women with stage II and III than for those with stage 0 at diagnosis (− 0.11 and − 0.07,
p
< 0.05) and follow-up (− 0.15 and − 0.10,
p
< 0.01). The EQ-5D-5L index indicated poorer HRQoL for women with Charlson comorbidity index ≥ 2 than comorbidity 0 both at diagnosis (− 0.13,
p
< 0.001) and follow-up (− 0.18,
p
< 0.001). Therefore, we provided the reference norms at diagnosis and at the 2-year follow-up, stratified by age, comorbidity index, and tumor stage.
Conclusion
These HRQoL reference norms can be useful to interpret the scores of women with non-metastatic breast cancer, comparing them with country-specific reference values for this population.
Abstract
Background
The incidence of non-AIDS defining cancer (NADC) is higher in people living with HIV (PLWH) than in the general population, and it is already one of the leading causes of death in ...the HIV-infected population. It is estimated that the situation will be aggravated by the progressive aging of PLWH. Early diagnosis through intensive cancer screening may improve the ability for therapeutic interventions and could be critical in reducing mortality, but it might also increase expenditure and harms associated with adverse events. The aim of this study is to evaluate an enhanced screening program for early diagnosis of cancer in PLWH compared to standard practice. The specific objectives are (1) to compare the frequency of cancer diagnosed at an early stage, (2) to analyze safety of the enhanced program: adverse events and unnecessary interventions, (3) to analyze the cost-utility of the program, and (4) to estimate the overall and site-specific incidence of NADC in PLWH.
Methods
We will conduct a multicenter, non-blinded, randomized, controlled trial, comparing two parallel arms: conventional vs enhanced screening. Data will be recorded in an electronic data collection notebook. Conventional intervention group will follow the standard of care screening in the participating centers, according to the European AIDS Clinical Society recommendations, and the enhanced intervention group will follow an expanded screening aimed to early detection of lung, liver, anal, cervical, breast, prostate, colorectal, and skin cancer. The trial will be conducted within the framework of the Spanish AIDS Research Network Cohort (CoRIS).
Discussion
The trial will evaluate the efficacy, safety, and efficiency of an enhanced screening program for the early diagnosis of cancer in HIV patients compared to standard of care practice. The information provided will be relevant since there are currently no studies on expanded cancer screening strategies in patients with HIV, and available data estimating cost effectiveness or cost-utility of such as programs are scarce. An enhanced program for NADC screening in patients with HIV could lead to early diagnosis and improve the prognosis of these patients, with an acceptable rate of unnecessary interventions, but it is critical to demonstrate that the benefits clearly outweigh the harms, before the strategy could be implemented.
Trial registration
ClinicalTrials.gov
NCT04735445. Registered on 25 June 2019
Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission ...in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.
Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural ...protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (
= 36/111, 32.4%) or prolonged (5 to 20 days) (
= 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the
gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the
E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication.
This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the
pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.
Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic ...modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8
+
T cells. These results support the development of IgTT-1E for the treatment of EGFR
+
cancers.
Mutations in
BRCA1
and
BRCA2
predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical ...significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of
BRCA1
and
BRCA2
variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25
BRCA1
and
BRCA2
variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (
BRCA1
c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C,
BRCA2
c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (
BRCA2
c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.
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