In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) represents a promising therapeutic option for patients lacking a compatible donor. We previously designed a lentiviral vector (βAS3 LV) expressing a potent anti-sickling βAS3 globin and demonstrated its safety and efficacy in SCD patient cells (Weber et al., 2018). In vitro and in vivo preclinical studies demonstrated the safety and efficacy of a gene therapy (GT) protocol based on the efficient transduction of plerixafor-mobilized SCD HSPCs by βAS3 LV. Based on these results a Phase 1/2 clinical trial (NCT03964792) started in January 2020. Three homozygous SCD patients (P1, P2 and P3) were recruited. Before GT, patients suffered from severe SCD symptoms that were not controlled despite of a strict adherence to the treatment. Plerixafor-mobilized HSPCs were transduced achieving a VCN of 0.9±0.2 in liquid culture, 1.6±0.3 in BFU-E and 0.8±0.2 in CFU-GM. The patients were hypertransfused to reach HbS levels <30%. After busulphan-based myeloablative conditioning, they received a drug product 6 to 8.1x 10 6 per kg. Here, for the 3 patients we report the follow-up of 18 (P1), 10 (P2) and 6 (P3) months. No drug-related severe adverse events were observed. Importantly, no sign of clonal hematopoiesis was detected by NGS sequencing performed on the graft before and after transduction. Despite of the similar VCN in the drug product, gene marking in peripheral blood mononuclear cells was variable and ranged between 0.2 and 0.6, as measured at the last follow-up (0.4 in P1, 0.6 in P2 and 0.2 in P3). These results pinpoint the difficulty to estimate the gene marking, self-renewal and engraftment potential of HSCs in the grafts. P1 and P2 clinically benefit from the GT treatment despite they presented non-severe vaso-occlusive crises (VOCs) not requiring hospitalization (two for P1 at month 6 and 14 and one in P2 at month 8 post-GT). Despite the rapid resolution of these episodes, P1 started hydroxyurea at month 10 and stopped it at month 16. P2 initiated a phlebotomy program to treat liver iron overload and decrease viscosity. Intravascular hemolysis improved in P1 and P2, as shown by a decrease in plasma hemoglobin and heme, and an increase in hemopexin (HPX). Interestingly, in P1 a mild worsening occurred at the time of VOC. In P1 and P2, total bilirubin, a marker of extravascular hemolysis, decreased post-GT. Deformability, measured using osmoscan, improved in P1 and to a lesser extent in P2; however, both still exhibited some degree of dehydration compared to normal RBCs (Fig. 1). Adhesion to thrombospondin decreased after GT with an increase during VOC in P1 or prior the VOC in P2 (Fig. 1). In both patients, markers of inflammation (e.g., C reactive protein) and reticulocyte counts decreased upon GT. An in vitro sickling assays was performed for P1 and showed significant improvement, reaching a frequency of sickling cells similar to SCD heterozygous carriers (from 88% before GT to 45% 6 months post-GT). The GT treatment was not effective in P3 due to the modest intake and rapid decline of gene modified cells. In P1 and P2, who were no longer on transfusions, HbAS3 levels correlated with the VCN (2.1 g/dl for P1 and 3.3 g/dl for P2) and the therapeutic Hb accounted for 21.5%, and 28.7% of the total hemoglobins, respectively. For P3, who remained transfusion-dependent, this frequency was <3% due to the low VCN and the presence of the transfusion-derived Hb. Notably, total Hb concentration was only 8.1 g/dl in P2 before the treatment and reached 11.4 g/dl at the last follow-up, suggesting a better survival of RBCs derived from genetically modified HSPCs. Furthermore, in P1 and P2, HbAS3 levels were significantly higher in mature RBCs compared to reticulocytes, indicating a survival advantage of βAS3 globin-expressing cells. Overall, these clinical data indicate a variable efficacy of the DREPAGLOBE GT treatment, which likely depends on the extent of gene marking achieved in HSCs in vivo and on the engraftment capability of genetically modified HSCs in SCD patients' bone marrow. Single-cell RNA-seq analysis of HSPCs and evaluation of the bone marrow niche in SCD patients will aid to define critical parameters for achieving successful outcomes in GT clinical trials for SCD. Figure 1 Figure 1. Disclosures Joseph: bluebird bio: Consultancy. El Nemer: Hemanext: Consultancy. Bartolucci: INNOVHEM: Other: Co-founder; AGIOS: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; Jazz Pharma: Other: Lecture fees; Fabre Foundation: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Hemanext: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; Emmaus: Consultancy. Cavazzana: Smart Immune: Other: co-founder.
BACKGROUND: Cetuximab, a monoclonal antibody targeting EGFR, improves overall survival in the locally advanced setting in combination with radiotherapy and in first-line recurrent setting in combination with chemotherapy in HNSCC patients. No predictive biomarker of efficacy of cetuximab in these settings. We aimed at identifying biomarkers of sensitivity/resistance to cetuximab. PATIENTS AND METHODS: HNSCC patients treated with cetuximab at Institut Curie, for which complete clinicopathological data and formalin-fixed, paraffin-embedded (FFPE) tumor tissue were available and collected before cetuximab treatment, were included in this retrospective study. Immunohistochemistry analyses of PTEN and EGFR were performed to assess expression levels. PIK3CA and KRAS mutations were analyzed using high resolution melting (HRM) and Sanger sequencing. The predictive value of these alterations on progression-free survival (PFS) was evaluated. RESULTS: PIK3CA and KRAS/HRAS mutations were associated with poor PFS in HNSCC patients treated with cetuximab in first-line recurrence setting but not in patients treated with cetuximab in combination with radiotherapy. PTEN loss of expression had a negative prognostic impact in HNSCC patients treated with cetuximab and radiotherapy, while EGFR high expression was not found to be a sensitivity biomarker for cetuximab in our patient population. CONCLUSIONS: PIK3CA and RAS mutations were resistance biomarkers of cetuximab in first-line recurrent setting, whereas PTEN loss was a resistance biomarker of cetuximab when given in combination with radiotherapy. Citation Format: Maud Kamal, Olivia Leblanc, Sophie Vacher, Charlotte Lecerf, Emmanuelle Jeannot, Jerzy Klijanienko, Frederique Berger, Caroline Hoffmann, Valentin Calugaru, Nathalie Badois, Anne Chilles, Maria Lesnik, Samar Krhili, Thomas Jouffroy, Ivan Bieche, Christophe Le Tourneau. Resistance biomarkers in patients with head and neck squamous cell carcinoma treated with cetuximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2600.
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