The ASCENT Trial: a phase 2 study of induction and consolidation afatinib and chemoradiation with or without surgery in stage III EGFR-mutant NSCLC.

BACKGROUND: The role of tyrosine kinase inhibitors (TKIs) in early-stage and metastatic oncogene-driven non-small cell lung cancer (NSCLC) is established, but it remains unknown how best to integrate TKIs with concurrent chemoradiotherapy (cCRT) in locally advanced disease. The phase 2 ASCENT trial assessed the efficacy and safety of afatinib and cCRT with or without surgery in locally advanced epidermal growth factor receptor (EGFR)-mutant NSCLC. PATIENTS AND METHODS: Adults ≥18 years with histologically confirmed stage III (AJCC 7th edition) NSCLC with activating EGFR mutations were enrolled at Mass General and Dana-Farber/Brigham Cancer Centers, Boston, Massachusetts. Patients received induction afatinib 40 mg daily for 2 months, then cisplatin 75 mg/m2 and pemetrexed 500 mg/m2 IV every 3 weeks during RT (definitive or neoadjuvant dosing). Patients with resectable disease underwent surgery. All patients were offered consolidation afatinib for 2 years. The primary endpoint was the objective response rate (ORR) to induction TKI. Secondary endpoints were safety, conversion to operability, progression-free survival (PFS), and overall survival (OS). Analyses were performed on the intention-to-treat population. RESULTS: Nineteen patients (median age 56 years; 74% female) were enrolled. ORR to induction afatinib was 63%. Seventeen patients received cCRT; 2/9 previously unresectable became resectable. Ten underwent surgery; 6 had a major or complete pathological response. Thirteen received consolidation afatinib. With a median follow-up of 5.0 years, median PFS and OS were 2.6 (95% CI, 1.4-3.1) and 5.8 years (2.9-NR), respectively. Sixteen recurred or died; 6 recurrences were isolated to CNS. The median time to progression after stopping consolidation TKI was 2.9 months (95% CI, 1.1-7.2). Four developed grade 2 pneumonitis. There were no treatment-related deaths. CONCLUSION: We explored the efficacy of combining TKI with cCRT in oncogene-driven NSCLC. Induction TKI did not compromise subsequent receipt of multimodality therapy. PFS was promising, but the prevalence of CNS-only recurrences and rapid progression after TKI discontinuation speak to unmet needs in measuring and eradicating micrometastatic disease.

Impact of the overexpression of the tyrosine kinase receptor RET in the hematopoietic potential of induced pluripotent stem cells (iPSCs).

INTRODUCTION: Previous studies have suggested that the tyrosine kinase receptor RET plays a significant role in the hematopoietic potential in mice and could also be used to expand cord-blood derived hematopoietic stem cells (HSCs). The role of RET in human iPSC-derived hematopoiesis has not been tested so far. METHODS: To test the implication of RET on the hematopoietic potential of iPSCs, we activated its pathway with the lentiviral overexpression of RETWT or RETC634Y mutation in normal iPSCs. An iPSC derived from a patient harboring the RETC634Y mutation (iRETC634Y) and its CRISPR-corrected isogenic control iPSC (iRETCTRL) were also used. The hematopoietic potential was tested using 2D cultures and evaluated regarding the phenotype and the clonogenic potential of generated cells. RESULTS: Hematopoietic differentiation from iPSCs with RET overexpression (WT or C634Y) led to a significant reduction in the number and in the clonogenic potential of primitive hematopoietic cells (CD34+/CD38-/CD49f+) as compared to control iPSCs. Similarly, the hematopoietic potential of iRETC634Y was reduced as compared to iRETCTRL. Transcriptomic analyses revealed a specific activated expression profile for iRETC634Y compared to its control with evidence of overexpression of genes which are part of the MAPK network with negative hematopoietic regulator activities. CONCLUSION: RET activation in iPSCs is associated with an inhibitory activity in iPSC-derived hematopoiesis, potentially related to MAPK activation.

Modeling RET-Rearranged Non-Small Cell Lung Cancer (NSCLC): Generation of Lung Progenitor Cells (LPCs) from Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

REarranged during Transfection (RET) oncogenic rearrangements can occur in 1-2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has yet been described. Patient-derived iPSCs hold great promise for disease modeling and drug screening. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the mutation RETC634Y, commonly associated with medullary thyroid carcinoma. Additionally, we established a RETC634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RETC634Y iPSCs using a 16-day protocol and detected an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RETC634Y mutation, potentially linked to poor NSCLC prognosis. These findings were validated using the RETC634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RETC634Y iPSCs exhibited a positive response to the RET inhibitor pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel patient-derived off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets.

Case report: Long-term voluntary Tyrosine Kinase Inhibitor (TKI) discontinuation in chronic myeloid leukemia (CML): Molecular evidence of an immune surveillance.

The classical natural history of chronic myeloid leukemia (CML) has been drastically modified by the introduction of tyrosine kinase inhibitor (TKI) therapies. TKI discontinuation is currently possible in patients in deep molecular responses, using strict recommendations of molecular follow-up due to risk of molecular relapse, especially during the first 6 months. We report here the case of a patient who voluntarily interrupted her TKI therapy. She remained in deep molecular remission (MR4) for 18 months followed by detection of a molecular relapse at +20 months. Despite this relapse, she declined therapy until the occurrence of the hematological relapse (+ 4 years and 10 months). Retrospective sequential transcriptome experiments and a single-cell transcriptome RNA-seq analysis were performed. They revealed a molecular network focusing on several genes involved in both activation and inhibition of NK-T cell activity. Interestingly, the single-cell transcriptome analysis showed the presence of cells expressing NKG7, a gene involved in granule exocytosis and highly involved in anti-tumor immunity. Single cells expressing as granzyme H, cathepsin-W, and granulysin were also identified. The study of this case suggests that CML was controlled for a long period of time, potentially via an immune surveillance phenomenon. The role of NKG7 expression in the occurrence of treatment-free remissions (TFR) should be evaluated in future studies.

Modeling Blast Crisis Using Mutagenized Chronic Myeloid Leukemia-Derived Induced Pluripotent Stem Cells (iPSCs).

PURPOSE: To model CML progression in vitro and generate a blast crisis (BC-CML) model in vitro in order to identify new targets. METHODS: Three different CML-derived iPSC lines were mutagenized with the alkylating agent ENU on a daily basis for 60 days. Cells were analyzed at D12 of hematopoietic differentiation for their phenotype, clonogenicity, and transcriptomic profile. Single-cell RNA-Seq analysis has been performed at three different time points during hematopoietic differentiation in ENU-treated and untreated cells. RESULTS: One of the CML-iPSCs, compared to its non-mutagenized counterpart, generated myeloid blasts after hematopoietic differentiation, exhibiting monoblastic patterns and expression of cMPO, CD45, CD34, CD33, and CD13. Single-cell transcriptomics revealed a delay of differentiation in the mutated condition as compared to the control with increased levels of MSX1 (mesodermal marker) and a decrease in CD45 and CD41. Bulk transcriptomics analyzed along with the GSE4170 GEO dataset reveal a significant overlap between ENU-treated cells and primary BC cells. Among overexpressed genes, CD25 was identified, and its relevance was confirmed in a cohort of CML patients. CONCLUSIONS: iPSCs are a valuable tool to model CML progression and to identify new targets. Here, we show the relevance of CD25 identified in the iPSC model as a marker of CML progression.

Chimeric antigen-receptor (CAR) engineered natural killer cells in a chronic myeloid leukemia (CML) blast crisis model.

During the last two decades, the introduction of tyrosine kinase inhibitors (TKIs) to the therapy has changed the natural history of CML but progression into accelerated and blast phase (AP/BP) occurs in 3-5% of cases, especially in patients resistant to several lines of TKIs. In TKI-refractory patients in advanced phases, the only curative option is hematopoietic stem cell transplantation. We and others have shown the relevance of the expression of the Interleukin-2-Receptor α subunit (IL2RA/CD25) as a biomarker of CML progression, suggesting its potential use as a therapeutic target for CAR-based therapies. Here we show the development of a CAR-NK therapy model able to target efficiently a blast crisis cell line (K562). The design of the CAR was based on the scFv of the clinically approved anti-CD25 monoclonal antibody (Basiliximab). The CAR construct was integrated into NK92 cells resulting in the generation of CD25 CAR-NK92 cells. Target K562 cells were engineered by lentiviral gene transfer of CD25. In vitro functionality experiments and in vivo leukemogenicity experiments in NSG mice transplanted by K562-CD25 cells showed the efficacy and specificity of this strategy. These proof-of-concept studies could represent a first step for further development of this technology in refractory/relapsed (R/R) CML patients in BP as well as in R/R acute myeloblastic leukemias (AML).

Chimeric antigen-receptor (CAR)-engineered natural killer (NK) cells targeting chronic myeloid Leukemia (CML) blast crisis

Background The use of tyrosine kinase inhibitors (TKIs) has dramatically modified the therapy of chronic myeloid leukemia (CML), generating durable remissions and prolonging survival in TKI-responders. However, progression to blast crisis (BC) still occurs especially in TKI-resistant patients and represents a clinical challenge. We and others have identified IL2RA/CD25 as a typical cell surface marker of BC-CML and reported that its overexpression is correlated with the progression of CML from CP-CML to BC-CML (Imeri et al, Cells 2023). Here we show the experimental development of a third-generation CAR-NK therapy strategy against the CD25 based on the scFV of the clinically approved monoclonal humanized antibody, Basiliximab. Methods: As NK cell model, we have used the NK92 cell line which has a well-established and clinically demonstrated NK cell activity. We have lentivirally transduced NK92 cells with the CAR construct containing a selectable gene (GFP). After FACS-sorting of GFP-positive cells, phenotypical characterization was performed by FACS. The expression of the CAR-CD25 at the surface of the cells was demonstrated an anti-Fab antibody and double-positive (Fab/GFP) cells were further purified. The functionality of the cells was evaluated using CD107a degranulation assay after 3h co-culture and ELISA for IFN-gamma release. We have in parallel engineered K562 cells expressing CD25 by lentiviral transduction (K562-CD25) as well as also a second target cell line (RAJI) using the same strategy. Annexin V staining of target K562 cells was used for in vitro cytotoxicity assessments. For in vivo assays, NSG mice were intraperitoneally (IP) injected with K562-CD25 cells expressing Luciferase at Day-3 (3.10 6 cells/mouse, n = 13). At Days 0, 3, and 7, mice were treated by IP injection of either irradiated CD25 CAR-NK92 cells (10 .10 6/mouse n=6) or irradiated Wild-type (WT)-NK92 cells (n=5). The clinical evolution of mice transplanted mice was followed weekly by luminescence (IVIS 200). Results: After cell sorting, we obtained more than 90% of double-positive NK92 CAR+/GFP+ cells. Lentiviral transduction did not affect the activatory or inhibitory signals of NK92 cells. No statistical differences were observed between CD25 CAR-NK92 and WT- NK92 cells for the expression of NKp30, NKp46, KirDl2-3, TIGIT, and DNAM. However, we observed a strong increase in the Granzyme B and Perforin in CD25 CAR-NK92 cells after co-culture with K562-CD25 as compared to WT NK92 cells (p<0.001). Importantly, we have found increased levels of degranulation after co-culture of target K562-CD25 with CD25-CAR-NK92 cells (40%) as compared to cells co-cultured with WT NK92 alone (20% ) suggesting strongly the occurrence of an additional specific effect due to CAR-CD25. IFN-gamma levels after co-culture of CAR CD25 NK92 cells were also found to be significantly increased ( 400 pg/ml) in as compared to co-cultures of target cells with WT-NK92 (200 pg/ml)(p<0.0001). Similarly,in vitro cytotoxicity assays showed induction of higher levels of apoptosis in target cells (K562-CD25 and Raji-CD25) when co-cultured with CD25 CAR-NK92 as compared to NK92 WT (p<0.0001). In in vivo experiments, we have analyzed K562-CD25 leukemia-bearing mice treated with CAR-NK92 cells (n=6) or WT-NK92 cells (n=5). These experiments analyzed at D+30 post-transplant showed stronger anti-leukemia effect of CAR-NK therapy by IVIS imaging with a survival rate of 84% for mice treated with CD25 CAR-NK92 versus 40% for those treated with WT-NK92. All control mice transplanted with K562-CD25 cells and left untreated died by D+20. Conclusion: We show here for the first time the potential use of an NK cell-mediated CAR therapy strategy targeting CD25 which has been shown to be upregulated in CML blast crisis. The experimental data show a significantly increased and selective in vitro and in vivo cytotoxicity of CD25 CAR-NK92 cells against CD25-expressing leukemia cells as compared to WT-NK92 cells. These results suggest that targeting CD25 by a CD25 CAR based on Basilixiamb's scFV might be an interesting tool in BC-CML and in all acute leukemias overexpressing CD25. In order to translate these findings to NK cells derived from induced pluripotent stem cells (iPSCs), we have produced iPSCs expressing CAR-CD25 constructs and experiments are underway to evaluate the therapeutic potential of iPSC-derived CD25 CAR-NK cells in CML blast crisis or AML models.

Treatment of a genetic brain disease by CNS-wide microglia replacement.

Hematopoietic cell transplantation after myeloablative conditioning has been used to treat various genetic metabolic syndromes but is largely ineffective in diseases affecting the brain presumably due to poor and variable myeloid cell incorporation into the central nervous system. Here, we developed and characterized a near-complete and homogeneous replacement of microglia with bone marrow cells in mice without the need for genetic manipulation of donor or host. The high chimerism resulted from a competitive advantage of scarce donor cells during microglia repopulation rather than enhanced recruitment from the periphery. Hematopoietic stem cells, but not immediate myeloid or monocyte progenitor cells, contained full microglia replacement potency equivalent to whole bone marrow. To explore its therapeutic potential, we applied microglia replacement to a mouse model for Prosaposin deficiency, which is characterized by a progressive neurodegeneration phenotype. We found a reduction of cerebellar neurodegeneration and gliosis in treated brains, improvement of motor and balance impairment, and life span extension even with treatment started in young adulthood. This proof-of-concept study suggests that efficient microglia replacement may have therapeutic efficacy for a variety of neurological diseases.

Molecular investigation of adequate sources of mesenchymal stem cells for cell therapy of COVID-19-associated organ failure.

The use of mesenchymal stem cells (MSC) derived from several sources has been suggested as a major anti-inflammation strategy during the recent outbreak of coronavirus-19 (COVID-19). As the virus enters the target cells through the receptor ACE2, it is important to determine if the MSC population transfused to patients could also be a target for the virus entry. We report here that ACE2 is highly expressed in adult bone marrow, adipose tissue, or umbilical cord-derived MSC. On the other hand, placenta-derived MSC express low levels of ACE2 but only in early passages of cultures. MSC derived from human embryonic stem cell or human induced pluripotent stem cells express also very low levels of ACE2. The transcriptome analysis of the MSCs with lowest expression of ACE2 in fetal-like MSCs is found to be associated in particularly with an anti-inflammatory signature. These results are of major interest for designing future clinical MSC-based stem cell therapies for severe COVID-19 infections.

A phase I trial of high dose gefitinib for patients with leptomeningeal metastases from non-small cell lung cancer.

INTRODUCTION: There are few effective treatment options for leptomeningeal metastasis (LM) in non-small-cell lung cancer (NSCLC). This study assessed the feasibility of high-dose gefitinib in patients with LM from NSCLC harboring EGFR mutations or prior systemic response to EGFR-TKI. METHODS: This phase I open-label trial of a novel gefitinib dosing schedule employed a 3+3 design. Eligible NSCLC patients with LM had known EGFR mutations and/or prior response to EGFR-TKI. Patients alternated 2 weeks of high-dose daily gefitinib (dose levels: 750 mg, 1000 mg, 1250 mg) with 2 weeks of maintenance therapy (500 mg daily). Primary endpoints were safety and toxicity. Secondary endpoints included overall survival (OS), neurological progression-free survival, radiological response, and cytological response in cerebrospinal fluid (CSF). RESULTS: Seven patients were treated: 3 at 750 mg dose level, 4 at 1000 mg dose level. There were no DLTs at the 750 mg dose level, and one DLT (toxic epidermal necrolysis) at the 1000 mg dose level. The study was closed due to slow accrual. Median neurological PFS was 2.3 months (range 1.6-4.0 months); median OS was 3.5 months (range 1.6-5.1 months). Though there were no radiologically documented remissions of LM disease, four patients had improvement in neurological symptoms. One patient cleared their CSF of NSCLC cells, while 2 others had decrease in malignant cells in CSF. CONCLUSION: Although the MTD was not defined due to slow accrual, this study provides important information about the tolerability and CSF penetration of high-dose gefitinib as a therapeutic option for modest palliation for NSCLC patients with LM and a known EGFR mutation.

Recurrent thrombotic thrombocytopenic purpura (TTP) as a complication of acute relapsing pancreatitis.

Pancreatitis is a known complication of thrombotic thrombocytopenic purpura (TTP) found in approximately 2% of cases. The development of TTP as a clinical sequelae of acute pancreatitis has also been reported, including one patient with chronic pancreatitis who developed TTP on two occasions following acute exacerbations of pancreatitis. We describe a case in which multiple distinct episodes of TTP have followed the clinical and laboratory demonstration of acute pancreatitis in the same patient. Supportive care of the patient's pancreatitis and plasmapheresis in each case resulted in clinical improvement and resolution of TTP. While the pathophysiologic mechanism explaining this association remains unclear, the recurrence of TTP associated with the "rechallenge" of relapsing episodes of pancreatitis in our patient suggests that a cause-and-effect relationship does exist.