FCM marker importance for MRD assessment in T-cell acute lymphoblastic leukemia: An AIEOP-BFM-ALL-FLOW study group report.

T-lineage acute lymphoblastic leukemia (T-ALL) accounts for about 15% of pediatric and about 25% of adult ALL cases. Minimal/measurable residual disease (MRD) assessed by flow cytometry (FCM) is an important prognostic indicator for risk stratification. In order to assess the MRD a limited number of antibodies directed against the most discriminative antigens must be selected. We propose a pipeline for evaluating the influence of different markers for cell population classification in FCM data. We use linear support vector machine, fitted to each sample individually to avoid issues with patient and laboratory variations. The best separating hyperplane direction as well as the influence of omitting specific markers is considered. Ninety-one bone marrow samples of 43 pediatric T-ALL patients from five reference laboratories were analyzed by FCM regarding marker importance for blast cell identification using combinations of eight different markers. For all laboratories, CD48 and CD99 were among the top three markers with strongest contribution to the optimal hyperplane, measured by median separating hyperplane coefficient size for all samples per center and time point (diagnosis, Day 15, Day 33). Based on the available limited set tested (CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99), our findings prove that CD48 and CD99 are useful markers for MRD monitoring in T-ALL. The proposed pipeline can be applied for evaluation of other marker combinations in the future.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles.

Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. Here, we demonstrate that Hecw, a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons.

Activity of regorafenib in advanced pretreated soft tissue sarcoma: Results of a single-center phase II study.

BACKGROUND: Regorafenib, a multitargeted tyrosine kinase inhibitor, proved to be active in patients with soft tissue sarcomas (STS). METHODS: We conducted an open-label, non-randomized, single-center phase II study in advanced pretreated STS patients. Patients received regorafenib 160 mg daily on days 1 enrule 21 of a 28-day cycle. The primary endpoint was the progression-free survival (PFS) at 8 weeks. Toxicity was registered. RESULTS: Between April 2015 and November 2016, 21 patients were enrolled in the trial. A total of 13 out of 21 evaluable patients (61.9%) were progression-free at 8 weeks. Median PFS was 3.8 months (95% CI: 2.1-9.4). Median overall survival was 14.8 months (95% CI: 7.7-27.8). In the intention-to-treat population, we reported a PFS of 66.7% at 3 months (95% CI: 40.4-83.4) and 16.7% at 12 months (95% CI: 4.1-36.5). As per the RECIST criteria, the response rate was 4.7% (1 partial response out of 21 evaluable patients) with a clinical benefit rate of 61.9%; no complete response was observed. Treatment was well tolerated. CONCLUSION: Regorafenib shows signs of clinical activity in patients with advanced STS. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT02307500.

Regorafenib in patients with refractory metastatic pancreatic cancer: a Phase II study (RESOUND).

Aim: Regorafenib may be active in different cancer types. This Phase II trial included patients with various refractory cancer types treated with regorafenib. Here, we report the results of the pancreatic adenocarcinoma cohort. Methods: The primary end point was progression-free survival (PFS) rate at 8 weeks; further investigation of regorafenib would be warranted with a PFS rate ≥50%. Results: A total of 20 patients were enrolled. The best response was stable disease in four patients (20%). The 8-week PFS rate was 25% with a median PFS of 1.7 months (95% CI: 1.5-2.0). A total of 13 patients (65%) experienced grade 3-4 treatment-related adverse events. Conclusion: The study did not meet its primary end point. Further investigation of regorafenib monotherapy in this setting is not recommended. Clinical Trial Registration: NCT02307500.

When ubiquitin meets E-cadherin: Plasticity of the epithelial cellular barrier.

Cellular plasticity is, by definition, the ability of cells to adapt to a dynamic micro-environment by changing their phenotype. E-cadherin is the key organizer of the epithelial cell barrier, and it is required at the cell surface to preserve epithelial tissue integrity and homeostasis, since it not only organizes the adherens junctions, but also transfers intracellular signals that provide cues to regulate cell survival, morphology and polarity. As such, de-regulation of E-cadherin has deleterious effects on cells and whole tissues. The availability of cadherin at the cellular junctions is determined by the rates of new protein synthesis and degradation, as well as of internalization and recycling. Indeed, E-cadherin is subjected to a constant and a signal-mediated turnover due to trafficking and recycling between the cell surface and the cytoplasm. Importantly, the turnover of E-cadherin is required for both cell adhesion and cell plasticity within a tissue. Understanding the pathways and molecular mechanisms that E-cadherin undertakes to move in and out of adherens junctions, through which epithelial cells communicate with each other, has, thus, been a major research focus over the past decade, but several issues remain unresolved. Here, we review major advances and remaining open questions in the understanding of E-cadherin trafficking, with a particular focus on its ubiquitination.

Time point-dependent concordance of flow cytometry and real-time quantitative polymerase chain reaction for minimal residual disease detection in childhood acute lymphoblastic leukemia.

BACKGROUND: Flow cytometric analysis of leukemia-associated immunophenotypes and polymerase chain reaction-based amplification of antigen-receptor genes rearrangements are reliable methods for monitoring minimal residual disease. The aim of this study was to compare the performances of these two methodologies in the detection of minimal residual disease in childhood acute lymphoblastic leukemia. DESIGN AND METHODS: Polymerase chain reaction and flow cytometry were simultaneously applied for prospective minimal residual disease measurements at days 15, 33 and 78 of induction therapy on 3565 samples from 1547 children with acute lymphoblastic leukemia enrolled into the AIEOP-BFM ALL 2000 trial. RESULTS: The overall concordance was 80%, but different results were observed according to the time point. Most discordances were found at day 33 (concordance rate 70%) in samples that had significantly lower minimal residual disease. However, the discordance was not due to different starting materials (total versus mononucleated cells), but rather to cell input number. At day 33, cases with minimal residual disease below or above the 0.01% cut-off by both methods showed a very good outcome (5-year event-free survival, 91.6%) or a poor one (5-year event-free survival, 50.9%), respectively, whereas discordant cases showed similar event-free survival rates (around 80%). CONCLUSIONS: Within the current BFM-based protocols, flow cytometry and polymerase chain reaction cannot simply substitute each other at single time points, and the concordance rates between their results depend largely on the time at which they are used. Our findings suggest a potential complementary role of the two technologies in optimizing risk stratification in future clinical trials.

Late brain metastases from colorectal cancer a case report and review of the literature.

Metastatic brain tumors from colorectal cancer are relatively rare. In previous reports the incidence ranged from 1.9 to 3.5 percent of all metastatic brain tumors. In another recent article the cumulative incidence of brain metastasis was estimated at 1.2%. The prognosis for patients with even a single resectable brain metastasis is poor. This paper presents the case report of a 72-year-old woman with a solitary brain metastasis 16 years after a diagnosis of colorectal cancer. She remained asymptomatic for 16 years. The first sign of a secondary tumor was diplopia. Brain scan demonstrated a space-occupying lesion in the occipital area. A solitary tumor was removed by craniotomy. Histological examination showed that it was a metastasis from the intestinal primary tumor. The patient subsequently underwent whole brain radiotherapy for a total dose of 30 Gy in 10 fractions.

Human neural stem cells express extra-neural markers.

Neural stem cells can be derived from the adult/embryonic nervous system as well as from more primitive embryonic stem cells but, because of the lack of specific markers, only their differentiated progeny can be characterized. We here report the presence of several endothelial and hematopoietic receptors (at protein and mRNA level) on the surface of embryonic human neural stem cells, which are partially maintained during differentiation. This suggests that neural stem cells have a greater potential than previously thought, which involves the ability to respond to different and so far unconsidered environmental signals and may be responsible for the recently discovered process of stem cell-fate conversion.