Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia.

Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n = 12 of 21), branching (n = 4 of 21), and clonal evolution (n = 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in ∼80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.

The transcriptomic landscape of elderly acute myeloid leukemia identifies B7H3 and BANP as a favorable signature in high-risk patients.

Acute myeloid leukemia (AML) in the elderly remains a clinical challenge, with a five-year overall survival rate below 10%. The current ELN 2017 genetic risk classification considers cytogenetic and mutational characteristics to stratify fit AML patients into different prognostic groups. However, this classification is not validated for elderly patients treated with a non-intensive approach, and its performance may be suboptimal in this context. Indeed, the transcriptomic landscape of AML in the elderly has been less explored and it might help stratify this group of patients. In the current study, we analyzed the transcriptome of 224 AML patients > 65 years-old at diagnosis treated in the Spanish PETHEMA-FLUGAZA clinical trial in order to identify new prognostic biomarkers in this population. We identified a specific transcriptomic signature for high-risk patients with mutated TP53 or complex karyotype, revealing that low expression of B7H3 gene with high expression of BANP gene identifies a subset of high-risk AML patients surviving more than 12 months. This result was further validated in the BEAT AML cohort. This unique signature highlights the potential of transcriptomics to identify prognostic biomarkers in in elderly AML.

Impact of measurable residual disease by decentralized flow cytometry: a PETHEMA real-world study in 1076 patients with acute myeloid leukemia.

The role of decentralized assessment of measurable residual disease (MRD) for risk stratification in acute myeloid leukemia (AML) remains largely unknown, and so it does which methodological aspects are critical to empower the evaluation of MRD with prognostic significance, particularly if using multiparameter flow cytometry (MFC). We analyzed 1076 AML patients in first remission after induction chemotherapy, in whom MRD was evaluated by MFC in local laboratories of 60 Hospitals participating in the PETHEMA registry. We also conducted a survey on technical aspects of MRD testing to determine the impact of methodological heterogeneity in the prognostic value of MFC. Our results confirmed the recommended cutoff of 0.1% to discriminate patients with significantly different cumulative-incidence of relapse (-CIR- HR:0.71, P < 0.001) and overall survival (HR: 0.73, P = 0.001), but uncovered the limited prognostic value of MFC based MRD in multivariate and recursive partitioning models including other clinical, genetic and treatment related factors. Virtually all aspects related with methodological, interpretation, and reporting of MFC based MRD testing impacted in its ability to discriminate patients with different CIR. Thus, this study demonstrated that "real-world" assessment of MRD using MFC is prognostic in patients at first remission, and urges greater standardization for improved risk-stratification toward clinical decisions in AML.

Measurable residual disease in elderly acute myeloid leukemia: results from the PETHEMA-FLUGAZA phase 3 clinical trial.

The value of measurable residual disease (MRD) in elderly patients with acute myeloid leukemia (AML) is inconsistent between those treated with intensive vs hypomethylating drugs, and unknown after semi-intensive therapy. We investigated the role of MRD in refining complete remission (CR) and treatment duration in the phase 3 FLUGAZA clinical trial, which randomized 283 elderly AML patients to induction and consolidation with fludarabine plus cytarabine (FLUGA) vs 5-azacitidine. After consolidation, patients continued treatment if MRD was ≥0.01% or stopped if MRD was <0.01%, as assessed by multidimensional flow cytometry (MFC). On multivariate analysis including genetic risk and treatment arm, MRD status in patients achieving CR (N = 72) was the only independent prognostic factor for relapse-free survival (RFS) (HR, 3.45; P = .002). Achieving undetectable MRD significantly improved RFS of patients with adverse genetics (HR, 0.32; P = .013). Longer overall survival was observed in patients with undetectable MRD after induction though not after consolidation. Although leukemic cells from most patients displayed phenotypic aberrancies vs their normal counterpart (N = 259 of 265), CD34 progenitors from cases with undetectable MRD by MFC carried extensive genetic abnormalities identified by whole-exome sequencing. Interestingly, the number of genetic alterations significantly increased from diagnosis to MRD stages in patients treated with FLUGA vs 5-azacitidine (2.2-fold vs 1.1-fold; P = .001). This study supports MRD assessment to refine CR after semi-intensive therapy or hypomethylating agents, but unveils that improved sensitivity is warranted to individualize treatment and prolong survival of elderly AML patients achieving undetectable MRD.

Risk assessment of trihalomethanes exposure by consumption of IV gamma products: Evidences from a Portuguese regional survey.

Due to the current demand for healthier food, the IV gamma products industry has grown over the years. Additionally, it is well known that chlorine is a disinfectant largely used in this industry. However, the risk assessment associated with the formation of carcinogenic by-products from this halogenated disinfection has not been widely studied. The aim of this study was to assess the trihalomethanes (THMs) exposure through the consumption of IV gamma products, more specifically, salad mixes, and potential health risk in Portuguese population using a deterministic method. The quantification of THMs in salad mixes from the most representative Portuguese brands was performed by gas chromatography coupled to mass spectrometry (GC-MS). The THMs exposures were estimated by combining the THMs concentration data with a ready-to-eat salad mix consumption survey (case study applied in the Lisboa e Vale do Tejo area; n = 271). The concentration of THMs in all samples was below the limit of quantification of the equipment (1 µg/L), and this limit was used for the evaluation of the exposure. It was found that it is very small for the consumption pattern of these products in the studied population categories, suggesting that there is no risk for consumers due to the halogenated disinfection process. The highest estimated daily exposure was 7,65X10-5 mg/ kg bw/ week, which corresponds to a person who consumes such products daily.

Comparação dos níveis de ansiedade e depressão entre idosos ativos e sedentários / Comparison of levels of anxiety and depression among active and sedentary elderly

CONTEXTO: A ansiedade e a depressão constituem um problema de saúde pública nos idosos. Atualmente, tem-se demonstrado uma associação positiva entre a prática de atividade física e a boa saúde mental.OBJETIVO: Comparar a relação entre os níveis de ansiedade e depressão entre idosos ativos e sedentários.MÉTODOS: A amostra foi constituída por 72 idosos, divididos num grupo de 38 idosos sedentários (GS) e outro grupo (GA) de 34 fisicamente ativos. Estes responderam a um questionário com características sociodemográficas e a Escala de Ansiedade e Depressão para Hospital Geral.RESULTADOS: As variáveis que influenciaram a presença de ansiedade ou depressão foram o nível de atividade física (p < 0,001) e de escolaridade (p < 0,01). O GS apresentou 38 vezes mais de probabilidade de vir a desenvolver sintomas de ansiedade e depressão e os indivíduos sem habilitações apresentam 11 vezes mais de probabilidade de vir a desenvolver esses sintomas. No GS, 35 (92,1%) idosos apresentaram níveis de ansiedade ou depressão e 3 (7,9%) não apresentaram. No GA apenas 8 (23,5%) apresentaram níveis de ansiedade ou depressão e 26 (76,5%) não revelaram esses sintomas (p < 0,05).CONCLUSÃO: A prática da atividade física pode representar um fator importante para reduzir os níveis de ansiedade e depressão em idosos.

BACKGROUND: The anxiety and depression are a public health problem in the elderly. Currently some studies have been shown a positive association between physical activity and good mental health.OBJECTIVE: To compare the possible relationship between levels of anxiety and depression among active and sedentary elderly.METHODS: The sample comprised a total of 72 patients divided into one group of 38 elderly subjects (GS) and another group of 34 physically assets (GA). These answered a questionnaire with socio-demographic characteristics and the Hospital Anxiety and Depression Scale.RESULTS: The variables that influenced the presence of anxiety and depression were the level of physical activity (p < 0,001) and the level of instruction (p < 0,01). The GS showed 38 more probability to develop symptoms of anxiety and depression and subjects without qualifications had 11 more probability to develop these symptoms. For the GS 35 (92,1%) patients had levels of anxiety or depression and 3 (7,9%) did not. In GA, the presence of anxiety or depression were observed only in 8 (23,5%) and the remaining 26 (76,5%) revalidates not any kind of these symptoms (p < 0,05).DISCUSSION: Practicing physical activity may represent a very important factor to reduce levels of anxiety and depression in elderly.