Generation of Functional CLL-Specific Cord Blood CTL Using CD40-Ligated CLL APC

PMCID: PMC3526610This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Patient specific real-time PCR in precision medicine – Validation of IG/TR based MRD assessment in lymphoid leukemia

Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients’ disease characteristics. The calibration range is linear between 10-1 and 10-5 for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation

A model for predicting effect of treatment on progression-free survival using MRD as a surrogate end point in CLL

Our objective was to evaluate minimal residual disease (MRD) at the end of induction treatment with chemoimmunotherapy as a surrogate end point for progression-free survival (PFS) in chronic lymphocytic leukemia (CLL) based on 3 randomized, phase 3 clinical trials (ClinicalTrials.gov identifiers NCT00281918, NCT00769522, and NCT02053610). MRD was measured in peripheral blood (PB) from treatment-naïve patients in the CLL8, CLL10, and CLL11 clinical trials, and quantified by 4-color flow cytometry or allele-specific oligonucleotide real-time quantitative polymerase chain reaction. A meta-regression model was developed to predict treatment effect on PFS using treatment effect on PB-MRD. PB-MRD levels were measured in 393, 337, and 474 patients from CLL8, CLL10, and CLL11, respectively. The model demonstrated a statistically significant relationship between treatment effect on PB-MRD and treatment effect on PFS. As the difference between treatment arms in PB-MRD response rates increased, a reduction in the risk of progression or death was observed; for each unit increase in the (log) ratio of MRD2 rates between arms, the log of the PFS hazard ratio decreased by 20.188 (95% confidence interval, 20.321 to 20.055; P 5 .008). External model validation on the REACH trial and sensitivity analyses confirm the robustness and applicability of the surrogacy model. Our surrogacy model supports use of PB-MRD as a primary end point in randomized clinical trials of chemoimmunotherapy in CLL. Additional CLL trial data are required to establish a more precise quantitative relationship between MRD and PFS, and to support general applicability of MRD surrogacy for PFS across diverse patient characteristics, treatment regimens, and different treatment mechanisms of action

Schwann-cell cylinders grown inside hyaluronic-acid tubular scaffolds with gradient porosity

[EN] Cell transplantation therapies in the nervous system are frequently hampered by glial scarring and cell drain from the damaged site, among others. To improve this situation, new biomaterials may be of help. Here, novel single-channel tubular conduits based on hyaluronic acid (HA) with and without poly-l-lactide acid fibers in their lumen were fabricated. Rat Schwann cells were seeded within the conduits and cultured for 10days. The conduits possessed a three-layered porous structure that impeded the leakage of the cells seeded in their interior and made them impervious to cell invasion from the exterior, while allowing free transport of nutrients and other molecules needed for cell survival. The channel's surface acted as a template for the formation of a cylindrical sheath-like tapestry of Schwann cells continuously spanning the whole length of the lumen. Schwann-cell tubes having a diameter of around 0.5mm and variable lengths can thus be generated. This structure is not found in nature and represents a truly engineered tissue, the outcome of the specific cell-material interactions. The conduits might be useful to sustain and protect cells for transplantation, and the biohybrids here described, together with neuronal precursors, might be of help in building bridges across significant distances in the central and peripheral nervous system.The authors acknowledge financing through projects MAT2011-28791-C03-02 and 03, and ERA-NET NEURON project PRI-PIMNEU-2011-1372. We thank the Cytomics Core Facility at Principe Felipe Research Center (CIPF, Valencia, Spain) for their support and advice in flow cytometry experiments, and the Electron Microscopy Service at the UPV, where the SEM images were obtained. The authors thankfully acknowledge the reviewers' comments, which have helped to improve the clarity of the paper's presentation.Vilariño Feltrer, G.; Martínez Ramos, C.; Monleon De La Fuente, A.; Vallés Lluch, A.; Moratal Pérez, D.; Barcia Albacar, JA.; Monleón Pradas, M. (2016). Schwann-cell cylinders grown inside hyaluronic-acid tubular scaffolds with gradient porosity. Acta Biomaterialia. 30:199-211. https://doi.org/10.1016/j.actbio.2015.10.040S1992113

Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study

Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing 9 disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis, we subsequently used canonical correlation analysis of 176 training cases to project the multidimensional space of all 26 immunophenotypic parameters into 36 2-dimensional plots for each possible pairwise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%), and mantle cell lymphoma (95.4%). Burkitt and CD101 diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD102 diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms

Coincidence of congenital left-sided diaphragmatic hernia and ductus venosus agenesis: Relation between altered hemodynamic flow and lung-to-head-ratio?

Left-sided diaphragmatic hernia (CDH) as well as ductus venosus agenesis (ADV) are rare complex congenital malformations. We present a case of coincidence of these malformations and an abnormally high lung-head-ratio (LHR). The left-sided liver-up CDH and the ADV were diagnosed in prenatal ultrasound examination. In CDH cases lung volume is decreased due to the herniation of abdominal organs into the thorax. With 1.4 the LHR of our patient exceeded the normal ratio in liver-up CDH cases considerably. One explanation for this unusually high LHR might be an altered blood flow due to the coinciding ADV. In ADV cases less blood bypasses the lung through the foramen ovale. Consecutively pulmonary circulation is improved which may constitute as an advantage in CDH cases. Diagnosis, prognostic factors, physiology, and therapy strategy are discussed