Analytical tools to determine mycotoxins and modified mycotoxins

El control dels contaminants alimentaris és una prioritat per a la salut humana i animal i és una de les majors preocupacions de les autoritats de tot el món. De tots els compostos no desitjats que es poden trobar de forma quotidiana al menjar i al pinso, les micotoxines són un dels contaminants més estudiats. Les micotoxines són metabòlits secundaris petits produïts per fongs filamentosos que es troben generalment en cereals i derivats, i que presenten propietats tòxiques. El punt central de la recerca de micotoxines es divideix en dos temes principals: la determinació de la seva presència a través del desenvolupament de mètodes de determinació robusts i la recerca de les seves propietats tòxiques. D’aquesta manera la part experimental de la present tesi doctoral es va dividir en aquests dos punts centrals. El principal objectiu de la primera secció va ser el desenvolupament de nous mètodes analítics per determinar la incidència de micotoxines en mostres de cereals i derivats. Aquests, engloben l’optimització de les tècniques d’extracció i separació per cromatografia líquida acoblada a la determinació per espectrometria de masses tàndem. Les micotoxines trobades a les mostres analitzades durant el desenvolupament d’aquesta tesi doctoral demostren la seva prevalença a la cadena alimentària, tan a la d’humans com a la d’animals. A la segona secció es van avaluar les conseqüències relacionades amb el consum d’aliments contaminats per micotoxines. Per això, es va realitzar un estudi metagenòmic de mostres d’intestí de rata després del consum de la micotoxina deoxynivalenol a baixa concentració, per tal de determinar si es produïen canvis bacterians. També es va optimitzar el mètode analític per a mostres de femta. A continuació, es va realitzar un estudi on s’avaluaven mostres d’excreció de rates per estudiar-ne els possibles metabòlits produïts després d’un tractament amb la micotoxina nivalenol. Aquest fet obre la possibilitat d’estudiar nous compostos en un futur.El control de los contaminantes alimentarios es una prioridad para la salud humana y animal, y es una de las mayores preocupaciones de las autoridades de todo el mundo. De todos los compuestos no deseados que se pueden encontrar de un modo cuotidiano en alimentos y pienso, las micotoxinas son uno de los contaminantes más estudiados. Las micotoxinas son metabolitos secundarios pequeños producidos por hongos filamentosos que generalmente se encuentran en cereales y derivados, y que presentan propiedades tóxicas. El punto central de la investigación en micotoxinas se divide en dos ámbitos principales: la determinación de su presencia a través del desarrollo de métodos de determinación robustos y la investigación de sus propiedades tóxicas. De este modo la parte experimental de la presente tesis doctoral se dividió en estos dos puntos centrales. El principal objetivo de la primera sección fue el desarrollo de nuevos métodos analíticos para determinar la incidencia de micotoxinas en muestras de cereales y derivados. Estos, engloban la optimización de las técnicas que extracción y de separación mediante cromatografía líquida acoplada a la determinación por espectrometría de masas tándem. Las micotoxinas halladas en las muestras analizadas durante el desarrollo de esta tesis doctoral demuestran su prevalencia en la cadena alimentaria, de humanos y animales. En la segunda sección se avaluaron las consecuencias relacionadas con el consumo de alimentos contaminados por micotoxinas. Por ello, se realizó un estudio metagenómico de muestras de intestino de rata después del consumo de la micotoxina deoxynivalenol a baja concentración para determinar si se producían cambios bacterianos. También se optimizó el método analítico para muestras de heces. A continuación, se realizó un estudio donde se avaluaron muestras de excreción de ratas para evaluar los posibles metabolitos producidos después de un tratamiento con la micotoxina nivalenol. Este hecho abre la posibilidad de estudiar nuevos compuestos en un futuro.Controlling contaminants in food is a priority for human and animal health and one of the major concerns of authorities across all over the world. In food and feed samples, small and secondary metabolites produced by filamentous fungi, known as mycotoxins, can appear. The central focus of mycotoxin research is divided into two main topics: determining their presence with the development of robust determination methods and studying their toxicological effects. Accordingly, the experimental part of this doctoral thesis is divided into these two central strands. As the major sources of mycotoxin contamination are agricultural products, especially cereals and their derivatives, the main objective of the first section of this thesis is to develop new analytical methods to determine the incidence of targeted mycotoxins, including modified mycotoxins, in cereal and cereal derivative samples. These analytical methods also involved the optimisation of the extraction techniques followed by liquid chromatography coupled to tandem mass spectrometry. The mycotoxins found in the analysed food samples over the course of this doctoral thesis demonstrate their prevalence in the food chain of humans and animals and the importance of control them through robust, selective and simple analytical methods. In the second section, there is an evaluation of the consequences of consuming food contaminated by mycotoxins. To achieve this, for the first time, metagenomic research was performed on rat gut samples after the consumption of a mycotoxin at low concentration levels in order to determine whether mycotoxin consumption can trigger any bacterial changes. The optimisation of the analytical methodology for faecal samples was also explored. Then, a study a large number of possible derivatives biologically generated by rats after nivalenol consumption was also included in the second section. This has led to the identification of new compounds to be explored in further research

Efficacy and safety of native versus pegylated Escherichia coli asparaginase for treatment of adults with high-risk, Philadelphia chromosome-negative acute lymphoblastic leukemia

Native or pegylated (PEG) asparaginase (ASP) are commonly used in treatment of acute lymphoblastic leukemia (ALL), but have been scarcely compared in the same trial in adult patients. Native vs. PEG-ASP administered according to availability in each center were prospectively evaluated in adults with high-risk ALL. Ninety-one patients received native ASP and 35 PEG-ASP in induction. No significant differences were observed in complete remission, minimal residual disease levels after induction and after consolidation, disease-free survival, and overall survival. No significant differences in grades 3–4 toxicity were observed in the induction period, although a trend for higher hepatic toxicity was observed in patients receiving PEG-ASP. In this trial the type of ASP did not influence patient response and outcome.Supported in part with the grants PI10/01417 from Fondo de Investigaciones Sanitarias and RD12/0036/0029 from RTICC, Instituto de Salud Carlos III, 2014 SGR225(GRE), CERCA Program, Generalitat de Catalunya, Spain, and a funding from ‘La Caixa’ Foundation

Genetic and phenotypic characterisation of HIV-associated aggressive B-cell non-Hodgkin lymphomas, which do not occur specifically in this population: diagnostic and prognostic implications

The frequency of aggressive subtypes of B-cell non-Hodgkin lymphoma (B-NHL), such as high-grade B-cell lymphomas (HGBL) with MYC and BCL2 and/or BCL6 rearrangement (HGBL-DH/TH) or Burkitt-like lymphoma (BL) with 11q aberration, is not well known in the HIV setting. We aimed to characterise HIV-associated aggressive B-NHL according to the 2017 WHO criteria, and to identify genotypic and phenotypic features with prognostic impact. Seventy-five HIV-associated aggressive B-NHL were studied by immunohistochemistry (CD10, BCL2, BCL6, MUM1, MYC, and CD30), EBV-encoded RNAs (EBERs), and fluorescence in situ hybridisation (FISH) to evaluate the status of the MYC, BCL2, and BCL6 genes and chromosome 11q. The 2017 WHO classification criteria and the Hans algorithm, for the cell-of-origin classification of diffuse large B-cell lymphomas (DLBCL), were applied. In DLBCL cases, the frequencies of MYC and BCL6 rearrangements (14.9 and 27.7%, respectively) were similar to those described in HIV-negative patients, but BCL2 rearrangements were infrequent (4.3%). MYC expression was identified in 23.4% of DLBCL cases, and coexpression of MYC and BCL2 in 13.0%, which was associated with a worse prognosis. As for BL cases, the expression of MUM1 (30.4%) conferred a worse prognosis. Finally, the prevalence of HGBL-DH/TH and BL-like with 11q aberration are reported in the HIV setting. The phenotypic and genotypic characteristics of HIV-associated aggressive B-NHL are similar to those of the general population, except for the low frequency of BCL2 rearrangements in DLBCL. MYC and BCL2 coexpression in DLBCL, and MUM-1 expression in BL, have a negative prognostic impact on HIV-infected individuals.Peer reviewe

Chemotherapy or allogeneic transplantation in high-risk Philadelphia chromosome–negative adult lymphoblastic leukemia

The need for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in adults with Philadelphia chromosome–negative (Ph−) acute lymphoblastic leukemia (ALL) with high-risk (HR) features and adequate measurable residual disease (MRD) clearance remains unclear. The aim of the ALL-HR-11 trial was to evaluate the outcomes of HR Ph− adult ALL patients following chemotherapy or allo-HSCT administered based on end-induction and consolidation MRD levels. Patients aged 15 to 60 years with HR-ALL in complete response (CR) and MRD levels (centrally assessed by 8-color flow cytometry) <0.1% after induction and <0.01% after early consolidation were assigned to receive delayed consolidation and maintenance therapy up to 2 years in CR. The remaining patients were allocated to allo-HSCT. CR was attained in 315/348 patients (91%), with MRD <0.1% after induction in 220/289 patients (76%). By intention-to-treat, 218 patients were assigned to chemotherapy and 106 to allo-HSCT. The 5-year (±95% confidence interval) cumulative incidence of relapse (CIR), overall survival (OS), and event-free survival probabilities for the whole series were 43% ± 7%, 49% ± 7%, and 40% ± 6%, respectively, with CIR and OS rates of 45% ± 8% and 59% ± 9% for patients assigned to chemotherapy and of 40% ± 12% and 38% ± 11% for those assigned to allo-HSCT, respectively. Our results show that avoiding allo-HSCT does not hamper the outcomes of HR Ph− adult ALL patients up to 60 years with adequate MRD response after induction and consolidation. Better postremission alternative therapies are especially needed for patients with poor MRD clearance

Single‐agent daratumumab in patients with relapsed and refractory multiple myeloma requiring dialysis: results of a Spanish retrospective, multicentre study

Correspondence

Incidence, clinical and biological characteristics and outcome of secondary acute lymphoblastic leukemia after solid organ or hematologic malignancy

Acute lymphoblastic leukemia (ALL) following solid organ or hematologic malignancy (secondary ALL, s-ALL) is not well characterized. We analyzed the characteristics and outcome of patients with s-ALL and compared them with those of patients with de novo- ALL. Of 448 patients, 24 (5%) had previous neoplasia. Sixteen patients had received previous cytotoxic therapy (therapy-associated ALL, t-ALL), and eight had not (antecedent-malignancy ALL, am-ALL). Except for more advanced age in patients with s-ALL, no statistically significant differences were observed in WBC count, CNS involvement, immunophenotype or cytogenetics between the groups, nor in complete remission (t-ALL: 94%; am-ALL: 75%; de novo-ALL: 85%), 3-year remission duration (58%; 50%; 72%), overall survival (71%; 38%; 60%) or event-free survival (53%, 38%; 53%). Our study did not show poor clinical or cytogenetic features or inferior outcome in ALL patients with antecedent neoplastic disease, irrespective of the type of treatment received for the neoplasia.Peer Reviewe

Chronic lymphocytic leukemia with isochromosome 17q: An aggressive subgroup associated with TP53 mutations and complex karyotypes

Although i(17q) [i(17q)] is frequently detected in hematological malignancies, few studies have assessed its clinical role in chronic lymphocytic leukemia (CLL). We recruited a cohort of 22 CLL patients with i(17q) and described their biological characteristics, mutational status of the genes TP53 and IGHV and genomic complexity. Furthermore, we analyzed the impact of the type of cytogenetic anomaly bearing the TP53 defect on the outcome of CLL patients and compared the progression-free survival (PFS) and overall survival (OS) of i(17q) cases with those of a group of 38 CLL patients harboring other 17p aberrations. We detected IGHV somatic hypermutation in all assessed patients, and TP53 mutations were observed in 71.4% of the cases. Patients with i(17q) were more commonly associated with complex karyotypes (CK) and tended to have a poorer OS than patients with other anomalies affecting 17p13 (median OS, 44 vs 120 months, P = 0.084). Regarding chromosomal alterations, significant differences in the median OS were found among groups (P = 0.044). In conclusion, our findings provide new insights regarding i(17q) in CLL and show a subgroup with adverse prognostic features.Peer Reviewe

Incidence and outcome after first molecular versus overt recurrence in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia included in the ALL Ph08 trial from the Spanish PETHEMA Group

On behalf of the PETHEMA Group of the Spanish Society of Hematology. Presented at the 23rd Congress of the European Hematology Association; June 14‐17, 2018; Stockholm, Sweden.[Background] Disease recurrence occurs in 20% to 40% of adults with Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) who are treated with chemotherapy and tyrosine kinase inhibitors (TKIs). In the current study, the authors report the incidence, treatment, and outcome after first disease recurrence in young and older adults treated in the ALL Ph08 trial (ClinicalTrials.gov identifier NCT01491763). [Methods] Patients aged 18 to 55 years with de novo Ph+ ALL were treated with imatinib concurrently with standard‐dose induction and consolidation therapy followed by allogeneic hematopoietic stem cell transplantation (allo‐HSCT) when possible. In patients with first disease recurrence, the authors analyzed the type of recurrence, timing, location, presence of kinase domain mutations, type of treatment, and outcomes. [Results] Of the 125 patients, 28 patients (22%) developed disease recurrence before (4 patients) or after (24 patients) HSCT, with the recurrences being molecular in 11 patients (39%) and overt in 17 patients (61%). T315I was the most common mutation noted at the time of disease recurrence. Change in TKI was the most frequent treatment for patients with molecular disease recurrence whereas rescue chemotherapy and TKI change followed by second allo‐HSCT when possible were performed for the most part in patients with overt disease recurrence. A total of 20 patients (71%) achieved response. The median disease‐free survival (DFS) and overall survival (OS) were 8.5 months and 15.3 months, respectively. A trend for better DFS and OS was observed in patients with molecular recurrence compared with those with overt recurrence (median of 16.9 months vs 6.3 months [P = .05] and 28.7 months vs 11.5 months [P = .05] for DFS and OS, respectively). [Conclusions]Disease recurrence was frequent in young and older adults with Ph+ ALL who were treated with imatinib and chemotherapy with HSCT. Although the majority of patients responded to rescue therapy, their outcomes were poor, especially with regard to overt disease recurrence.Supported in part by grants RD12/0036/0029 (Cancer Cooperative Research Thematic Network/Spanish Federation of Rare Diseases [RTICC/FEDER), PI14/01971 FIS, Carlos III Health Institute, and SGR 288 (GRC) Spain. Pere Barba was supported by grants from the Carlos III Health Institute (FIS16/01433) and PERIS 2018‐2020 from the Generalitat of Catalunya (BDNS357800)

The poor prognosis of low hypodiploidy in adults with B-cell precursor acute lymphoblastic leukaemia is restricted to older adults and elderly patients

The prognostic significance of low‐hypodiploidy has not been extensively evaluated in minimal residual disease (MRD)‐oriented protocols for adult acute lymphoblastic leukaemia (ALL). We analysed the outcome of hypodiploid adult ALL patients treated within Programa Español de Tratamientos en Hematología (PETHEMA) protocols. The 5‐year cumulative incidence of relapse (CIR) of low‐hypodiploid B‐cell precursor (BCP)‐ALL was significantly higher than that of high‐hypodiploids (52% vs. 12%, P = 0.013). Low‐hypodiploid BCP‐ALL patients aged ≤35 years showed superior survival (71% vs. 21%, P = 0.026) and lower 5‐year CIR (17% vs. 66%, P = 0.090) than low‐hypodiploids aged >35 years. Older adults and elderly low‐hypodiploid BCP‐ALL patients show dismal prognosis although achieving an end‐induction good MRD response.This work was supported in part by grants from Asociación Española Contra el Cáncer, AECC (GC16173697BIGA), Instituto de Salud Carlos III (PI14/01971 FI), 2017‐SGR288 (GRC), CERCA Program from Generalitat de Catalunya and “La Caixa” Foundation

Treatment of Frail Older Adults and Elderly Patients With Philadelphia Chromosome-negative Acute Lymphoblastic Leukemia: Results of a Prospective Trial With Minimal Chemotherapy

[Background]: The treatment of acute lymphoblastic leukemia (ALL) in older adults and elderly patients is a challenge, and modern protocols include targeted therapy and immunotherapy in combination with attenuated or minimal chemotherapy. However, frail patients are excluded from these trials, and reports on the outcome of this subgroup of patients are scarce. Our objective was to analyze the outcome of unfit older adults and elderly patients with Philadelphia chromosome-negative ALL included in a prospective trial (ALL-07FRAIL).[Patients and Methods]: Older adults and elderly patients with Charlson Comorbidity Index (CCI) ≥ 4 were included. Induction therapy consisted of vincristine and dexamethasone, and maintenance therapy with mercaptopurine and methotrexate for 2 years.[Results]: Seventy-two patients with a median age of 67 years (range, 57-89 years) and a median CCI of 5 (range, 4-12) were included. The rates of early withdrawal, early death, resistance, and complete response (CR) were 5%, 10%, 31%, and 54%, respectively. Six patients with CR abandoned the study, 5 died in CR, and 23 relapsed (cumulative relapse incidence 75%). The medians of disease-free and overall survival (OS) were 6.9 months (95% confidence interval [CI], 0.3-13.5 months) and 7.6 months (95% CI, 6.3-8.9 months), respectively. The most frequent toxic events were hematologic (neutropenia 77% and thrombocytopenia 54%, of grade III-IV in all cases). Eastern Cooperative Oncology Group score but not the CCI had significant impact on OS.[Conclusion]: Complete remission with very attenuated chemotherapy can be attained in one-half of older or elderly infirm patients with ALL. These results suggest that some of these patients could benefit from the concomitant or subsequent use of immunotherapy and/or targeted therapy.This study was supported in part by the CERCA Program/Generalitat de Catalunya, Spain and the Josep Carreras Leukemia Research Institute, Badalona, Spain