Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high ...prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.
Rituximab-containing regimens are becoming a therapeutic standard in chronic lymphocytic leukemia (CLL), so that a validation of flow cytometric minimal residual disease (MRD) quantification (MRD ...flow) in the presence of this antibody is necessary. We therefore compared results obtained by real-time quantitative (RQ)-PCR to MRD flow in 530 samples from 69 patients randomized to receive chemotherapy or chemotherapy plus rituximab. Quantitative MRD levels assessed by both techniques were closely correlated irrespective of therapy (r=0.95). The sensitivity and specificity of MRD flow was not influenced by the presence of rituximab. With 58.9% positive and 26.4% negative samples by both techniques, 85.3% of assessments (452/530) were qualitatively concordant between MRD flow and RQ-PCR. Discordant samples were typically negative by MRD flow and simultaneously positive close to the detection limit of the PCR assays, indicating a higher sensitivity of PCR for very low MRD levels. However, 93.8% of all samples were concordantly classified by both methods using a threshold of 10(-4) to determine MRD positivity. MRD flow and PCR are equally effective for MRD quantification in rituximab-treated CLL patients within a sensitivity range of up to 10(-4), whereas PCR is more sensitive for detecting MRD below that level.
Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain ...reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.
The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples ...of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.
Quantitative reverse transcription polymerase chain reaction (RT-PCR) is being used increasingly as an alternative to Northern blots analysis or RNase protection assays for quantitation of gene ...expression. To quantify different samples, measurements are often normalized using the expression of so-called "housekeeping" genes, such as cytoplasmic β-actin or glyceraldehyde-3-phosphate dehydrogenase. This approach can produce false results because the presence of processed pseudogenes in the genome, which are related to some of the commonly used transcripts of housekeeping genes, leads to coamplification of contaminating genomic DNA. By yielding amplification products of the same or similar size as the reverse-transcribed target, mRNA quantitation of expression is prone to error. In this paper, we report the results of using three sets of β-actin primers for RT-PCR in the presence and absence of genomic DNA. In addition, we propose two new pairs of oligonucleotide primers that specifically amplify the human and rat β-actin reverse-transcribed mRNA but not pseudogene sequences. These primers are especially suitable for quantitation of mRNA in small tissue samples (e.g., biopsies), where DNase digestion is not feasible, and therefore DNA contamination cannot be avoided.
The purpose of this note is to establish a certainty-equivalence feedback design for inverse optimally controlled affine systems. In particular, it is shown that a class of polynomial-type state ...feedbacks in conjunction with a globally asymptotically convergent observer leads to a globally asymptotically stable closed-loop. A key step in the proposed certainty-equivalence feedback design procedure is the identification of a new class of polynomial-type inverse optimal feedbacks which guarantees input-to-state stability (ISS) with respect to measurement errors. As a consequence, the proposed certainty-equivalence feedback design has the important feature that the state feedback is allowed to contain polynomial nonlinearities of arbitrarily high degree in the unmeasured states. This feature is illustrated on an example
Current MRD studies in T-cell acute lymphoblastic leukemia (T-ALL) mainly use T-cell receptor gamma, delta and SIL-TAL1 gene rearrangements as MRD-PCR targets. However, low frequency or limited ...diversity of these markers restricts the number of evaluable patients, particularly because two markers are recommended for MRD monitoring. Hence, we developed a new strategy implementing the TCR beta (TCRB) locus for MRD quantification. The frequency and characteristics of complete and incomplete TCRB rearrangements were investigated in 53 childhood and 100 adult T-ALL patients using the BIOMED-2 multiplex PCR assay. Clonal rearrangements were identified in 92% both childhood and adult T-ALL (Vbeta-Dbeta-Jbeta rearrangements in 80%, Dbeta-Jbeta rearrangements in 53%). Comparative sequence analysis of 203 TCRB recombinations revealed preferential usage of the 'end-stage' segment Jbeta2.7 in childhood T-ALL (27%), whereas Jbeta2.3 was most frequently involved in adult T-ALL (24%). In complete rearrangements, three downstream Vbeta segments (19-1/20-1/21-1) were preferentially used. Subsequently, a TCRB real-time quantitative PCR assay to quantify MRD with 13 germline Jbeta primer/probe combinations and allele-specific oligonucleotides was developed and applied to 60 clonal TCRB rearrangements. The assay allowed the detection of one leukemic cell within at least 10(4) polyclonal cells in 93% of cases and will be of high value for future MRD studies.
Using the multiplex PCR tubes of the BIOMED-2 Concerted Action, TCRB gene rearrangements were detected in 35% of childhood (n=161) and adult (n=172) precursor-B-ALL patients (Vbeta-(Dbeta)-Jbeta in ...25%; Dbeta-Jbeta in 15%). The presence of TCRB rearrangements showed a significant relation with age (highest frequency of 46% between 5 and 10 years of age) and the presence of TEL-AML1 transcripts, and was associated with relatively high frequencies of IGK-Kde, TCRG, and Vdelta2-Jalpha rearrangements. In 62 out of 65 patients with Southern blot-detected Vbeta-(Dbeta)-Jbeta and/or Dbeta-Jbeta rearrangements, at least one TCRB gene rearrangement was detected by PCR. Based on combined Southern blot and PCR analysis, oligoclonal TCRB gene rearrangements were observed in only 12% of patients. Analysis of paired diagnosis and relapse samples (n=26) showed that 20 out of 24 (83%) Vbeta-(Dbeta)-Jbeta rearrangements and eight out of 14 (57%) Dbeta-Jbeta rearrangements remained stable. Using real-time quantitative PCR, a quantitative range < or =10(-4) was obtained in 64% of TCRB gene rearrangements and in 86% of cases a sensitivity < or =10(-4) was obtained. In conclusion, TCRB gene rearrangements occur in 35% of precursor-B-ALL patients and are relatively stable and sensitive PCR targets for detection of minimal residual disease, particularly if this concerns complete Vbeta-(Dbeta)-Jbeta rearrangements.
We sought to evaluate safety of electrical cardioversion (ECV) for patients with acute atrial fibrillation (AF) or atrial flutter (AFL) in the emergency department (ED).
This was an analysis of data ...from 4 multicentre AF/AFL studies conducted from 2008 to 2019 at 23 large EDs. We included adult patients who received attempts at ECV and who had presented acutely after symptom onset. Staff manually reviewed study and clinical records to abstract data.
We evaluated 1736 ECV cases with a mean age of 60.1 years and 67.1% male. The overall success of ECV was 90.2% (95% confidence interval 88.7%-91.6%), with 4.9% of patients admitted. ED physicians performed the ECV in 95.2% and provided sedation in 96.5%; 13.9% (12.3%-15.7%) of cases experienced important adverse events that required treatment, and 0.4% were classified as life threatening. Another 5.6% had adverse events that did not require treatment. Logistic regression found that the RAFF-3 study cohort (odds ratio OR 2.0), age ≥ 85 years (OR 2.1), coronary artery disease (OR 1.5), midazolam (OR 1.9), and fentanyl (OR 1.5) were associated with important adverse events.
This large evaluation of the safety of ECV for acute AF/AFL in the ED found that while serious adverse events were rare, there were a concerning number of events following sedation that required intervention. Physicians should be aware that older age, coronary artery disease, and fentanyl are associated with higher risks of important adverse events. This study provides more information for shared decision making discussions with patients when choosing between drug-shock and shock-only cardioversion strategies.
Nous avons cherché à évaluer la sûreté de la cardioversion électrique (CVE) pour les patients présentant des épisodes de fibrillation auriculaire (FA) ou de flutter auriculaire au sein de services d'urgences (SU).
Il s'agit d'une analyse des données de quatre études multicentriques portant sur la FA/le flutter menées de 2008 à 2019 dans 23 grands SU. Nous avons inclus des patients adultes qui ont bénéficié d'essais de CVE et qui s'étaient présentés en urgence après l'apparition de symptômes. Le personnel a parcouru manuellement les fiches d'étude et les dossiers cliniques pour en extraire les données.
Nous avons évalué 1 736 cas de CVE avec un âge moyen de 60,1 ans et 67,1 % d'hommes. La réussite globale de la CVE était de 90,2 % (intervalle de confiance à 95 % : 88,7 %-91,6 %), avec 4,9 % des patients admis. Les urgentistes ont pratiqué la CVE dans 95,2 % des cas et ont assuré la sédation dans 96,5 % des cas; 13,9 % (12,3 %-15,7 %) des cas ont éprouvé des effets indésirables importants qui ont nécessité un traitement, et 0,4 % ont été classés comme étant une menace vitale. Par ailleurs, 5,6 % des patients ont expérimenté des effets indésirables qui n'ont pas nécessité de traitement. Une régression logistique a révélé que la cohorte de l'étude RAFF-3 (rapport de cotes RC 2,0), l'âge ≥ 85 ans (RC 2,1), la coronaropathie (RC 1,5), le midazolam (RC 1,9) et le fentanyl (RC 1,5) étaient associés à des effets indésirables importants.
Cette vaste évaluation de la sécurité de la CVE pour des épisodes de FA/flutter aux urgences a révélé que, si les effets indésirables graves étaient rares, un nombre inquiétant d'événements survenus après sédation ont nécessité une intervention. Les médecins doivent savoir que l'âge avancé, la maladie coronarienne et le fentanyl sont associés à des risques plus élevés d'effets indésirables importants. Cette étude fournit davantage d'informations pour les discussions en vue d'une prise de décision partagée avec les patients lors du choix entre les stratégies de choc électrique externe associé à un traitement médicamenteux ou par choc seul.
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