Jean-François Mirjolet, Nicolas Hoffmann, Olivier Duchamp, Marc Hillairet de Boisferon, Cyril Berthet, Francis Bichat, Jan Hoflack Oncodesign ® , Dijon, France Most preclinical investigations in Oncology research are performed using permanent cancer cell lines that have been kept in continuous passages since decades. These plasticcultured cells became very different from their original tumors since accumulated genetic abnormalities and multiple selections of subclones occurred over time. The use of fresh patientderived tumor samples for assays and for establishment of new relevant tumor models allows investigating the antitumor activity of new therapies directly linked with clinical reality. In this respect, Onco ® has developed a large and international network of clinical centers for the collection of a large number of fresh patientderived tumor biopsies from all cancer pathologies and from healthy tissues. The collection of these samples is done under ethically approved master agreements and with the signed consent of each patient. The patient’s clinical history, the serology results (HIV, HBV and HCV) and tissue banking are centralized in our internal approved biological resource center. Examples of assays will be presented based on patientderived acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia and other hemopathologies using chromium release and Annexin V FACS assays. These studies aimed to demonstrate the CDC, ADCC and apoptosis induction of new therapeutics antibodies whereby approved antibodies such as rituximab and alemtuzumab were used as positive controls. Similarly, these fresh patientderived models were used to study the direct doseresponse effect of new chemotherapeutic agents through apoptosis induction (bortezomib used as positive control). The freshly collected tumors were also used to establish new tumor models in different strains of mice and rats. The full characterization of the genetic patterns of the xenograft derived tumors was compared with the original collected human tumors and with the clinical data of the patient. A panel of “standard of care” compounds was tested in these new tumor models for pharmacological characterization. The expansion of such tumors at early passages in mice was used to implement a reproducible 3D assay. The assay was developed to investigate the potential antitumor activity of conventional and targeted therapies, and allows sufficient throughput for use during drug discovery lead optimisation. drug effects were then correlated with results using the same panel of tumorgrafts. The use of fresh patientderived tumors in drug discovery and early preclinical development of new therapies aimed at corroborating results with clinical reality. Altogether, these processes from the clinical tumor collection to the drug efficacy study through assays should help the preclinical drug selection, development and clinical positioning as well as companion biomarker identification. Target identification and patient selection FACS analysis of ALDH positive cells isolated from fresh patientderived lung tumor tissue using Aldefluor ® kit (StemCell Technologies). These ALDH + cells were sorted on a BD FACS Aria ® and derived cell line was established. Control Aldefluor® Tissue microarrays are used to validate clinical relevance of potential biological targets in the development of diagnostics, therapeutics and study new protein markers and genes. Collection opportunities Ex vivo drug evaluation in hematological malignancies ! "# $%& ’( Fresh patientderived CLL cells were treated for 24hr with bortezomib or vehicle and analysed by FACS for apoptosis induction. Results are presented as bivariate distribution of Annexin VFITC (x axis) 7AAD (y axis) labeling. Apoptosis was induced on CLL cells after bortezomib treatment. Fresh patientderived CLL cells were treated for 24hr with rituximab or isotype control and analysed by FACS for apoptosis induction using 7AAD and FITCconjugated Annexin V. Heterogeneous early apoptosis induction was evidenced on CLL cells after rituximab treatment. Cell cycle analysis (monovariate distribution of propidium iodide staining) after a 24hr treatment with flavopiridol on fresh patientderived CLL cells. Cells from CLL samples exhibit a nonproliferative state with less than 5% of cells in S and G2/M phases of cell cycle. An increase in the percentage of cells in subG1 was observed after treatment with a corresponding decrease in the percentage of cells in G0/G1 phase. various concentrations of each antibody. Thereafter, purified and IL2 activated NK cells (effector cells) were added for 3hr. Dosedependent ADCC of rituximab was specifically demonstrated using CLL cells. CD20-PE-Cy5 (CD19+ sorted) CD20 IgG CD19-PE (whole PBMCs) IgG CD19 FACS analysis of CD19 expression on PBMCs from a fresh patientderived chronic lymphocytic leukemia (CLL). PBMCs were sorted using magnetic beads and then analysed for CD20 expression by FACS. Analysis of antibody dependent cell cytotoxicity (ADCC) of rituximab or irrelevant IgG by 51 Cr release assay on fresh patientderived CLL cells. 51 Cr loaded cells were treated for 30 min with 0 10 20 30 40 50 60 70 80 Untreated IgG1 2 μg/ml IgG1 20 μg/ml Rituximab 2 μg/ml Rituximab 20 μg/ml Mean Early Apoptosis (%) CLL1 CLL2 CLL3 -5 0 5 10 15 20 25 30 0 3 30 300 3000 Concentration (ng/ml) Specific lysis (%) IgG Rituximab 0 20 40 60 80 100 120 Prostate Liver Kidney Lung Bladder Pancreas Stomach Skin Breast Ovary LLC AML Thyroid ORL Colon Sarcoma ALL Number of samples per year Healthy and cancerous tissues ) Onco ® has built up an unique private approved biologic resource center in accordance with European legal and ethical rules, Onco ® ’ BRC has developed an international network to provide (all fresh) biological samples needed for preclinical investigations, The BRC associated to the Chimice ® platform allows the establishment of new advanced and predictive experimental models, The combination of patientderived models with reconstituted human immune system in mice aims to improve efficient translational drug discovery and drug positioning in Oncology and other related pathologies. * Identification number of the patient (patient anonymous) * Patient informed consent * Patient clinical history * Negative HIV, HBV, HCV serology * + • Blood/ Serum and cord blood • Skin, liver … (abdominal and breast skin, healthy tissue) • Solid tumors (kidney, lung, colon, bladder, brain, pancreas, prostate, sarcome) • Hematological malignancies (AML, CLL, ALL, Lymphoma) • Stem cells (HSCUCB) , - Models and/or study results French and International BRC Onco ® BRC Declaration of collections of human tissues for research purposes, storage and preparations (Declaration number: DC20091008) Authorization by the French Research Ministry for human tissue collection, storage and distribution for scientific use (Authorization number: AC20091021) Authorization for import/export activity of human samples for scientific purposes (Authorization number: El2009518) Approval of ethical research projects using human biological ressources by an ethics committee Oncodesign BRC meets legal requirements in ethics, anonymity of samples and associated clinical data, handling safety and tracability of sample. ./ & 0 **0 .0’ 1’23 2 # "4 2 ) , ,’ 0 $ - 0"’)(5 Order upon sponsor Requests Patient selectivities Specific criteria Database 1+2 ’’6731*)7)8 +2)6)" * 2 1 731/2)1 729: 739 0"’) 0"; + Chi-mice ® platform to establish new predictive models Patient: CR-IGR-034P Patient: CR-LRB-010P Patient: CR-LRB-008M P10: CR-LRB-010P P11: CR-LRB-008M P11: CR-IGR-034P Patient Tumor Model Well Differentiated Well to Moderate Differentiated Poor to UnDifferentiated Patient: CR-IGR-034P Patient: CR-LRB-010P Patient: CR-LRB-008M P10: CR-LRB-010P P11: CR-LRB-008M P11: CR-IGR-034P Patient: CR-IGR-034P Patient: CR-LRB-010P Patient: CR-LRB-008M Patient: CR-IGR-034P Patient: CR-IGR-034P Patient: CR-LRB-010P Patient: CR-LRB-010P Patient: CR-LRB-008M Patient: CR-LRB-008M P10: CR-LRB-010P P11: CR-LRB-008M P11: CR-IGR-034P P10: CR-LRB-010P P10: CR-LRB-010P P11: CR-LRB-008M P11: CR-LRB-008M P11: CR-IGR-034P P11: CR-IGR-034P Patient Tumor Model Patient Tumor Model Well Differentiated Well to Moderate Differentiated Poor to UnDifferentiated CRIGR023M GAIN LOSS CRIGR002C GAIN LOSS Histopathological analyses completed for 53 colon tumor models (#) were in concordance to those observed in the corresponding patient’s tumor. CGH analysis showed very similar profile between early (blue) and advance (red) passages Histological and molecular characterization )1< )1< Whole body irradiated NSG newborn mice were inoculated the intracardiac route with CD34+ cells isolated from UCB samples. Peripheral blood chimerism was analysed by FACS on week 12 by detecting both mouse and human leukocytes. Twenty six weeks after humanization, subcutaneous tumours were induced by xenografting human patientderived ovarian tumor fragment (TOVA002) into the right flank of humanized NSG mice (D0). Take and growthrate of TOVA002 patientderived tumor was not modified when xenografted on humanized NSG mice. CRLRB022P in mice 0 500 1000 1500 15 20 25 30 35 Days post implantation Median Tumor volume (mm 3 ) Control Cetuximab(12.5 mg/kg/adm) 5-FU (50.0 mg/kg/ adm) Oxaliplatin (5.0mg/kg/ adm) CetuximabIP treatment 5-FU IV treatment OxaliplatinIV treatment 0 500 1000 1500 15 20 25 30 35 Days post implantation MedianTumorvolume (mm 3 ) Control Cetuximab (12.5 mg/kg/adm) 5-FU (50.0 mg/kg/ adm) Oxaliplatin(5.0mg/kg/ adm) Cetuximab IP treatment 5-FU IV treatment OxaliplatinIV treatment 0 500 1000 1500 15 20 25 30 35 Dayspost implantation MedianTumorvolume (mm 3 ) Control CPT-11 (22mg/kg/ Adm) CPT-11 IV treatment 3 CRLRB022P in rats Control Oxaliplatin (4 mg/kg/adm) CPT-11 (40 mg/kg/ adm) Cetuximab (10 mg/kg/adm) 5-FU (30 mg/kg/ adm) Days post implantation 70 60 50 40 30 20 10 Median tumour volume (mm 3 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Treatment start (D23) Control Oxaliplatin (4 mg/kg/adm) CPT-11 (40 mg/kg/ adm) Cetuximab (10 mg/kg/adm) 5-FU (30 mg/kg/ adm) (4 mg/kg/adm) CPT- adm) (10 mg/kg/ 5- adm) Days post implantation 70 60 50 40 30 20 10 Median tumour volume (mm 3 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Days post implantation 70 60 50 40 30 20 10 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Treatment start (D23) - adm) 5- ) - 5- ) - - ) - - ) - CRLRB022P in mice 0 500 1000 1500 15 20 25 30 35 Days post implantation Median Tumor volume (mm 3 ) Control Cetuximab(12.5 mg/kg/adm) 5-FU (50.0 mg/kg/ adm) Oxaliplatin (5.0mg/kg/ adm) CetuximabIP treatment 5-FU IV treatment OxaliplatinIV treatment 0 500 1000 1500 15 20 25 30 35 Days post implantation MedianTumorvolume (mm 3 ) Control Cetuximab (12.5 mg/kg/adm) 5-FU (50.0 mg/kg/ adm) Oxaliplatin(5.0mg/kg/ adm) Cetuximab IP treatment 5-FU IV treatment OxaliplatinIV treatment CRLRB022P in mice 0 500 1000 1500 15 20 25 30 35 Days post implantation Median Tumor volume (mm 3 ) Control Cetuximab(12.5 mg/kg/adm) 5-FU (50.0 mg/kg/ adm) Oxaliplatin (5.0mg/kg/ adm) CetuximabIP treatment 5-FU IV treatment OxaliplatinIV treatment 0 500 1000 1500 15 20 25 30 35 Days post implantation MedianTumorvolume (mm 3 ) Control Cetuximab (12.5 mg/kg/adm) 5-FU (50.0 mg/kg/ adm) Oxaliplatin(5.0mg/kg/ adm) Cetuximab IP treatment 5-FU IV treatment OxaliplatinIV treatment 0 500 1000 1500 15 20 25 30 35 Dayspost implantation MedianTumorvolume (mm 3 ) Control CPT-11 (22mg/kg/ Adm) CPT-11 IV treatment 3 0 500 1000 1500 15 20 25 30 35 Dayspost implantation MedianTumorvolume (mm 3 ) Control CPT-11 (22mg/kg/ Adm) CPT-11 IV treatment 3 CRLRB022P in rats Control Oxaliplatin (4 mg/kg/adm) CPT-11 (40 mg/kg/ adm) Cetuximab (10 mg/kg/adm) 5-FU (30 mg/kg/ adm) Days post implantation 70 60 50 40 30 20 10 Median tumour volume (mm 3 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Treatment start (D23) Control Oxaliplatin (4 mg/kg/adm) CPT-11 (40 mg/kg/ adm) Cetuximab (10 mg/kg/adm) 5-FU (30 mg/kg/ adm) (4 mg/kg/adm) CPT- adm) (10 mg/kg/ 5- adm) Days post implantation 70 60 50 40 30 20 10 Median tumour volume (mm 3 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Days post implantation 70 60 50 40 30 20 10 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Treatment start (D23) - adm) 5- ) - 5- ) - - ) - - ) - CRLRB022P in rats Control Oxaliplatin (4 mg/kg/adm) CPT-11 (40 mg/kg/ adm) Cetuximab (10 mg/kg/adm) 5-FU (30 mg/kg/ adm) Days post implantation 70 60 50 40 30 20 10 Median tumour volume (mm 3 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Days post implantation 70 60 50 40 30 20 10 Median tumour volume (mm 3 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Treatment start (D23) Control Oxaliplatin (4 mg/kg/adm) CPT-11 (40 mg/kg/ adm) Cetuximab (10 mg/kg/adm) 5-FU (30 mg/kg/ adm) (4 mg/kg/adm) CPT- adm) (10 mg/kg/ 5- adm) Days post implantation 70 60 50 40 30 20 10 Median tumour volume (mm 3 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Days post implantation 70 60 50 40 30 20 10 )10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Treatment start (D23) - adm) 5- ) - 5- ) - - ) - - ) - In vivo drug evaluation Similar tumor response ranges have been observed in mice and rats in 53 colorectal patientderived models (#) with some advantages for rat models as seen in this example (CRLRB022 model is sensitive to CPT11 both in mice and rats and sensitivity to cetuximab was only seen in rats). In this example, the TREI002 renal patientderived xenograft tumor was sensitive to RAD001, sorafenib and sunitinib and was not sensitive to lapatinib treatments. Tumors, collected in patient or in tumorbearing mice, were cut in small fragments of 12 mm and cultured for 72hr in 96well polyHEMA coated plates. Tumor fragments were treated for 24hr with either vehicle (DMSO) or tested drugs (5 concentrations between 0100 LM). MTS/collagenase was used before OD analysis. 1 2 3 4 5 Tumor sample 3D culture Drugs Tumorgraft Tumor cell viability MTS assay OD analysis Patient Surgery Ex vivo drug evaluation in solid tumors Collection opportunities using CLL cells. (#) Oncodesign®is a member of the consortium CReMEC providing the colorectal cancer models and associated data used in this publication. The authors are thankful for the support of Medicen-Paris Region and French Ministery of Industry. ).;."&== ).;."&== CRIGR023M CRIGR002C 0 20 40 60 80 100 120 T72 Time (h) % Lapatinib 0 20 40 60 80 100 120 T72 Time (h) % Sunitinib 0 20 40 60 80 100 120 T72 Time (h) % RAD001 100 μM 50 μM 25 μM 12.5 μM 6.25 μM 0 20 40 60 80 100 120 T72 Time (h) % Drug concentrations (μM) Cell viability (%) Sorafenib 100 μM 50 μM 25 μM 12.5 μM 6.25 μM 100 μM 50 μM 25 μM 12.5 μM 6.25 μM 25 μM 12.5 μM 6.25 μM 3.12 μM 1.56 μM 0 500 1000 1500 2000 2500 0 5 10 15 20 25 30 35 40 45 Time (Days) TV (mm 3 )