Alicia Robles

Drug Discovery Testing Compounds in Patient Samples by Automated Flow Cytometry

Functional ex vivo assays that predict a patient’s clinical response to anticancer drugs for guiding cancer treatment have long been a goal, but few have yet proved to be reliable. To address this, we have developed an automated flow cytometry platform for drug screening that evaluates multiple endpoints with a robust data analysis system that can capture the complex mechanisms of action across different compounds. This system, called PharmaFlow, is used to test peripheral blood or bone marrow samples from patients diagnosed with hematological malignancies. Functional assays that use the whole sample, retaining all the microenvironmental components contained in the sample, offer an approach to ex vivo testing that may give results that are clinically relevant. This new approach can help to predict the patients’ response to existing treatments or to drugs under development, for hematological malignancies or other tumors. In addition, relevant biomarkers can be identified that determine the patient’s sensitivity, resistance, or toxicity to a given treatment. We propose that this approach, which better recapitulates the human microenvironment, constitutes a more predictive assay for personalized medicine and preclinical drug discovery.

SAT0706 End-stage organ failure in sarcoidosis: characterization and predictive factors in 1082 patients

Objectives To characterize the main features at presentation of sarcoidosis associated with the development of end-stage organ failure in a large multicenter cohort of patients from Southern Europe. Methods In January 2017, the Spanish National Registry of Sarcoidosis (SARCOGEAS-SEMI) included 1082 consecutive patients diagnosed with sarcoidosis according to the ATS/ERS/WASOG 1999 statement and extrathoracic involvement to the 2014 WASOG instrument. The development of end-stage organ failure was assessed at the last visit. Results The cohort consisted of 618 (57%) women and 464 (43%) men, with a mean age at diagnosis of 47yrs. After a mean follow-up of 82 months, 90 (8%) patients developed end-stage organ failure, including respiratory failure (n=56), chronic renal failure (n=13), cardiac failure/permanent cardiac device (n=8) and liver cirrhosis (n=3). The following baseline features were associated with end-stage organ failure in the univariate analysis: patients born in Spain (p=0.008), a higher mean age at diagnosis (p<0.001) and a radiological stage III/IV (p<0.001) With respect to extrathoracic involvement, spleen (p=0.015), renal (p=0.001), cardiac (p=0.028) and bone marrow (p=0.003) involvements, hypercalcemia (p=0.018) and use of corticosteroids (p<0.001) were associated with end-stage organ failure, while patients with cutaneous sarcoidosis had a lower risk (p=0.029). Multivariate analysis identified age at diagnosis (OR 1.05), radiological stages III/IV (OR 3.12) and use of corticosteroids (OR 4.55) as independent variables associated with the development of end-stage organ failure. Conclusions Nearly 10% of patients with sarcoidosis developed end-stage organ failure. Respiratory failure represented two thirds of cases of sarcoidosis-related organ failure, followed by renal (15% of cases) and cardiac (9%). Older patients, as well as those presenting with advanced radiological stages, had an enhanced risk of developing end-stage organ failure. Disclosure of Interest None declared

A novel ex vivo high-throughput assay reveals antiproliferative effects of idelalisib and ibrutinib in chronic lymphocytic leukemia

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect.

THU0539 Sarcoidosis in spain: clinical and epidemiological characteristics at diagnosis in 1082 patients

Objectives To characterize the main features at presentation of sarcoidosis in a large multicenter cohort from Southern Europe. Methods In January 2016, the Autoimmune Diseases Study Group (GEAS-SEMI) created a national registry (SARCOGEAS) of patients with sarcoidosis. Sarcoidosis was diagnosed in agreement with the criteria proposed by the ATS/ERS/WASOG 1999 statement, and extrathoracic disease was classified with the 2014 WASOG instrument. Results The cohort consisted of 1082 patients (82% biopsy-proven), including 618 (57%) women and 464 (43%) men, with a mean age at diagnosis of 47yrs; 140 (13%) patients were born outside Spain, 965 (89%) were White, 69 (6%) Hispanic, 30 (3%) Black/African American and 18 (2%) Asian. Thoracic involvement was present at diagnosis in 979 (90%) patients, including 437 (40%) patients with stage I, 374 (35%) with stage II, 123 (11%) with stage III and 26 (2%) with stage IV. The most frequently reported extrathoracic involvements at diagnosis were cutaneous in 385 (36%) patients, extrathoracic lymph nodes in 218 (20%), liver involvement in 151 (14%) and ocular involvement in 118 (11%). Potentially life-threatening WASOG involvements were reported in frequencies less than 10%, including neurological involvement in 77 (7%) patients, kidney involvement in 59 (5%) or cardiac involvement in 21 (2%). Therapeutic approaches at diagnosis included the use of oral glucocorticosteroids in 637 (59%) patients, immunosuppressive agents in 84 (8%, mainly methotrexate in 63 patients) and biological agents in 15 (1%, mainly infliximab in 10 cases). Conclusions In this large series of sarcoidosis from Southern Europe, clinical presentation is dominated by adenopathies (both thoracic and extrathoracic) and cutaneous involvement (erythema nodosum), with lower frequencies in the main extrathoracic involvements than that reported in US and Japanese series. Disclosure of Interest None declared

An ex vivo native environment precision medicine test shows high clinical correlation with responses to first line acute myeloid leukemia treatment

P Preventive and Personalized Medicine (PPPM) as the healthcare model of the near future and its tool, i.e., Translational Medicine (TraMed), represent an innovative model of healthcare services to consolidate advanced healthcare and robust platform for relevant branches of predictive diagnostics, personalized therapeutics and preventive drugs. To achieve the implementation of PPPM concept into practice, it is necessary to create a new strategy based upon the sub-clinical recognition of biomarkers long before the disease clinically manifests itself. This strategy would secure preventive measures whose personalization could have a significant influence on demographics! Meanwhile, penetration of new technologies into the market would demand the reforms not only in the area of healthcare but in medical education as well. Therefore, the problem of the preparation of specialists of the newest generation to secure priority in growing up medical doctors as creative artists is becoming particularly urgent and would require significant revision of training programs and curricula of the higher education as applicable to the medical schools. Modernization and integration of widely accepted medical and teaching standards require consolidation of both the life sciences and medicine that may become the conceptual basis for the medical curricula. The main goal of this training is not to achieve advanced training and expansion of technological skills but to provide development of novel multifaceted approaches to build academic schools of the newest generations and to outline curricula and courses to suit markets of the newest medical platforms. PPPM consists of a wide variety of tests and tools including so much complicated areas as networking, mathematic modeling, nanotools and nanotechnologies, cloudy and mobile technologies to suit the requests and standards of the new healthcare model. Coordinated measures to optimize the progress should be well-focused on solving the accumulating problems in healthcare and the concomitant economic burden that societies across the globe are facing more and more. Taking into consideration the current trends and personal experience, we have made first steps towards direct involvement in the modernization of the healthcare model. Guided by the above-mentioned facts, a non-canonical approach has become setting up under the aegis of EPMA (Brussels, EU), PMC (Washington, DC, USA) and ISPM (Tokyo, Japan) a unique team of medical students, young researchers, entrepreneurs in drug designing, clinicians and administrators of the future to come. Used as an educational-methodical kernel is a three-level basic education system (undergraduate, graduate, and postgraduate) to suit the continuing education. Group and individual vectors as part of the basic inventory are represented by translational medicine, bioinformatics, drug design, translational tools, regulatory courses, etc. The model for accelerated development of continuous vocational education (CVE) in PPPM and TraMed is based on the combinatorial approaches (competence, moduletype approach, personal activity, program-design and problem-oriented) to the elucidation of innovative processes of modernization of the existing educational model. The application of the model for development of CVE has required a new type of the infrastructure of the curricula. PPPM whilst secured by the upgraded educational system would offer great and real promise for the future. And the next generations will speak about the XXI century as a time, when healthcare services became predictive and preventive and its outcomes secured and guaranteed!RESULTS Background: We have overcome the limitations of 40 years of ex vivo testing. The aim of this study is to determine the ability of Vivias novel test (based on studying the ex-vivo sensitivity to drugs) to predict the complete remission (CR) rates after induction chemotherapy with cytarabine (Ara-C) and idarubicin (Ida) in 1st line AML.. Material and Methods: This has been an observational clinical trial where bone marrow samples from adult patients diagnosed with de novo AML in Spanish centers from the PETHEMA group were included. Whole marrow samples maintaining their Native Environment were incubated for 48h in well plates containing Ara-C, Ida, or their combination. Pharmacological responses are calculated using population models. Induction response was assessed according to the Cheson criteria (2003). Patients attaining a CR/CRi were classified as responders and the remaining as resistant. Results: 390 patient samples were used to calculate the dose response (DR) curves for Ara-C alone, Ida alone, and their synergism. For clinical correlation we used 142 patients with median 56 years. The strongest clinical predictors were the Area Under the Curve (AUC) of the DR of Ara-C (P=1.34E-05), and the AUC of IDA (P=3.9E-05). The GAM models revealed a significant relationship (RSquare=0.452 and deviance explained=45%) between these predictors and higher probabilities of post-induction resistance. Fig 1A shows a table illustrating the correlation between clinical outcome (columns) and the test predictions (lines). Using the cut off determined by the GAM models. The test obtain a high Specificity and Positive Protective Value (95% and 80,77%) and a lower sensitivity (50%) with a general prediction of a 81,69%. Interestingly, the 5 cases that the test identify as resistant but were clinically sensitive have high level of minimal residual disease. On the other hand, the test does not properly identify 21/142 that are clinically resistant and the test predicts as sensitive (bottom left quadrant right panel). This mismatched subgroup mimics the problems from molecular markers where a resistant clone present in a minority of leukemic cells cannot be detected yet drives the patient response. Consistent with this analysis, adding the cytogenetic risk factor to the ex vivo results, identifying the high risk population by molecular markers that might be present in a minority of the cells, significantly improves the correlation; Fig. 1B shows the 90% overall correlation achieved in 117 patient samples adding the cytogenetic risk factor, with a major improvement in the sensitivity from 50% to 72%. Both approaches lead to substantial improvements in estimated overall survival. ABSTRACT© 2019 The Egyptian Journal of Internal Medicine | Publ Background/purpose of the study Chronic hepatitis C virus (HCV) infection is considered one of the major healthproblems.About170millionpatientswere infectedwithHCVworldwide.Till few years, Egypt was considered the highest HCV prevalent country worldwide, with predominant genotype number 4. The host immunity plays a major role in HCV infection with evolving data confirming the role of T-helper 17 cells in the formation of chronic HCV infection. The aim of our work was to determine the role of interleukins 17A (IL17A), 17F (IL17F) in the formation of chronic hepatitis C infection. Patients and methods We classify the patients into two groups: the first group included 51 chronic HCV patients who did not take antiviral therapy (the study group) and the second group included 51 healthy blood donors (as a control group). The levels of IL17A and IL17F in the serum of both groups were measured using the sandwich enzymelinked immunosorbent assay method. Results The serum values of IL17A were higher in patients with chronic HCV than the other group with the mean values being 52.9±32.6 pg/ml in the patient group and 17.1 ±10.4 pg/ml in the control group. IL17F was slightly higher in the HCV patient group than the control group, but it was statistically insignificant. Moreover, there were significant positive correlations between IL17A and alanine aminotransferase, viral load, and degree of liver fibrosis. Conclusion Patients with chronic HCV infections had a higher serum level of IL17A than the normal persons and it is positively correlated with alanine aminotransferase, viral load, and degree of liver fibrosis. This suggests its pivotal role in the formation of chronic HCV infection; so, it can be used as a new marker for disease progression due to its positive correlation with the severity of liver injury.