Significant Prolongation Of Survival Research Articles

De Novo Malignancies After Liver Transplantation: Experience of a High-Volume Center.

Patient care, newer immunosuppressive medications, and advances in surgical technique, have resulted in significant prolongation of survival after liver transplantation in recent years. However, as life expectancy increased and the early mortality rates have decreased, different problems have evolved due to chronic immunosuppressive therapy. The aim of the present study is to evaluate patients who were transplanted and then developed de novo malignancies, in terms of the type of malignancies and the follow-up period. The study was conducted on 2814 patients who received liver transplantation between 2008 and 2020 in Inonu University Liver Transplant Institute. In total, the data of 23 patients were evaluated retrospectively. Non-melanoma skin cancer was the most common de novo malignancy (21.7%), followed by gynecological cancers (17.3%). The interval between the time of transplantation until the development of de novo malignancy was 36 (6-75) months. The median follow-up period after the diagnoses of the de novo malignancies was 4.11years. One, 3-, 5-year survival rates of patients after the diagnoses of de novo malignancies were 69.6%, 56.5%, and 41.9%; respectively. Non-melanotic skin cancers were the most common de novo cancers in liver transplant recipients. A strict surveillance program is very important in the follow-up of liver transplant recipients.

DDRE-25. WSD0628: A BRAIN PENETRABLE ATM INHIBITOR AS A RADIOSENSITIZER FOR THE TREATMENT OF GBM AND METASTATIC CNS TUMOR

Abstract ATM (ataxia telangiectasia mutated) kinase, activated by DNA double-strand breaks, promotes DNA repair as well as activates DNA damage checkpoint and plays a key role for resistance to radiotherapy and chemotherapy. ATM function loss confers hypersensitivity to ionizing radiation evidenced by ataxia-telangiectasia (A-T) cells. Thus, pharmacological inhibition of ATM kinase is expected to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiotherapy and other DNA double-strand breaks-inducing chemotherapy. Herein, we report a discovery of a potent, selective, orally bioavailable, and brain penetrable ATM inhibitor WSD0628, as a radiosensitizer for GBM and metastatic CNS tumors with IC50 against ATM < 1nM with high selectivity ( >400 folds) for ATR and DNA-PK. WSD0628 is highly selective over other kinases. In-vitro MDCKII transfected cells and Caco-2 assays have shown that WSD0628 is highly permeable and not a substrate of P-gp or BCRP, two main efflux transporters expressed on human BBB. Preclinical CNS PK studies in rat and mouse confirmed good brain penetration of WSD0628 with Kp,uu,brain and Kp,uu,csf > 0.3. Significant prolongation of overall survival for mice bearing GBM PDX intracranial model was achieved by treatment with WSD0628 (5mpk, QD) combo with radiation. Moreover, WSD0628 shows low PK variation liability without aldehyde oxidase (AO) metabolism, low hERG liability ( >30 uM), and good safety window based on DRF studies. Taken together, our data provide a good rationale for WSD0628 to be developed toward clinic combo with radiation for the treatment of patients with GBM and cancers with CNS metastasis.

NL-201, a De Novo Agonist of IL-2 and IL-15 Receptors, Demonstrates Synergistic Antitumor Activity with Anti-PD-1 Checkpoint Inhibitor Therapy in a Preclinical Non-Hodgkin Lymphoma Model

NL-201 is a potent, selective, and long-acting computationally designed alpha-independent agonist of the IL-2 and IL-15 receptors that is being developed as an immunotherapy for cancer. NL-201 binds to the beta and gamma subunits, selectively stimulating dose-dependent expansion and tumor infiltration of cytotoxic CD8+ T cells and natural killer (NK) cells, thereby enhancing the immune response to the tumor. Absence of binding to the IL-2 alpha subunit reduces the undesirable effects of traditional IL-2 therapies, such as vascular leak syndrome and expansion of immunosuppressive regulatory T cells. In this abstract, we demonstrate that NL-201, alone or in combination, demonstrates robust antitumor activity in preclinical models of non-Hodgkin lymphoma (NHL).We have previously demonstrated that NL-201 has marked antitumor activity in multiple syngeneic tumor models, including the A20 lymphoma model. These observations in lymphoma have been extended to explore the effects of NL-201 in combination with anti-mPD-1 checkpoint inhibitor therapy in vivo. In this model, NL-201 and anti-mPD-1 demonstrated tumor growth inhibition and increased median survival (21 days each vs 17 days as observed in control) when given alone. In combination, NL-201 and anti-mPD-1 resulted in increased antitumor activity and significant prolongation of survival (>51 days). We have also demonstrated that NL-201 does not directly induce signaling or cell death in B cell−derived NHL, suggesting that the observed antitumor activity is due to activation of non-malignant host immune cells.Additional in vitro and in vivo NHL models are being tested to enhance understanding of the interaction between NL-201 and other approved therapies within the hematopoietic tumor microenvironment. These data will be used to design future clinical trials of NL-201 in novel regimens to treat hematological malignancies. DisclosuresHuard: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Tatalick: Neoleukin Therapeutics, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Independent paid nonclinical consultant for Neoleukin. Walkey: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Swanson: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Preclinical Evaluation of a Novel Orally Bioavailable Menin-MLL Interaction Inhibitor, DSP-5336, for the Treatment of Acute Leukemia Patients with MLL-Rearrangement or NPM1 Mutation

Background: The 11q23 abnormalities involving mixed lineage leukemia (MLL) gene are frequently found in adult and pediatric patients with acute leukemia. MLL rearrangements (MLL-r) are often associated with a poor prognosis and show poor response to currently available therapies, thus developing more effective therapy is urgently required. The leukemogenic activity of MLL fusion proteins, the products of the chimeric genes of MLL and its fusion partners generated by MLL-r, is critically dependent on direct interaction with MENIN, the product of the MEN1 gene. Interaction of MLL fusion proteins with MENIN plays an important role to enhance the proliferation and to block the differentiation of hematopoietic cells by maintaining high expression of hematopoietic stem cell program genes, such as HOXA9 and MEIS1. It has also been reported that the MENIN interaction with wild-type MLL, is required to induce HOXA9 and MEIS1 expression and also crucial for the development of acute leukemia with nucleophosmin (NPM1) mutations. Therefore, the MENIN-MLL interaction inhibitor is expected to have anti-leukemogenic activity against acute leukemia with MLL-r or NPM1 mutation by suppressing the expression of HOXA9 and MEIS1 and inducing terminal differentiation.Results: We generated DSP-5336, a novel, potent, and orally bioavailable MENIN-MLL interaction inhibitor for the treatment of acute leukemia patients with MLL-r or NPM1 mutation. DSP-5336 directly bound to the MENIN protein (Kd = 6.0 nM) and inhibited the MENIN-MLL interaction (IC 50 = 1.4 ± 0.058 nM). DSP-5336 selectively inhibited the cell growth of human leukemia cell lines including MV-4-11, MOLM-13, KOPN-8, and OCI-AML3 (IC 50 = 10, 15, 31 and 15 nM, respectively). These DSP-5336-sensitive cell lines possess a MLL-r or NPM1 mutation. On the other hand, DSP-5336 did not affect the cell growth of human leukemia cell lines such as HL-60, MOLT-4, and Reh (IC 50 > 10 μM), which do not have MLL-r or NPM1 mutations. In a mouse xenograft model using MV-4-11 cells, which express MLL-AF4, DSP-5336 exhibited a significant antitumor activity at the doses of 25 mg/kg and 50 mg/kg, administered twice daily (BID) for 20 days. There were no dose related changes in general condition or body weight. The effects of DSP-5336 on the expression of MENIN-MLL-regulated genes and differentiation marker genes were evaluated using MV-4-11 cells as pharmacodynamics markers in vitro and in vivo. In both cases, DSP-5336 significantly reduced the gene expression of MEIS1 and HOXA9, representative leukemic genes regulated by the MENIN-MLL complex. On the contrary, DSP-5336 significantly increased the gene expression level of ITGAM, a terminal differentiation marker.The efficacy of DSP-5336 was further assessed in acute leukemia patient samples and in mouse AML models. DSP-5336 strongly inhibited blast colony formation and changed the gene expression of the pharmacodynamics markers (HOXA9, MEIS1 and ITGAM ) in an AML patient sample carrying the MLL-AF6 fusion. In patient-derived xenograft (PDX) model with NPM1 mutation, human CD45 positive cells in peripheral blood progressively decreased during and beyond the 28 day period of DSP-5336 administration at doses of 25, 50, and 100 mg/kg BID and achieved a complete remission with no relapse at the doses of 50 and 100 mg/kg BID. At these three dose levels, DSP-5336 also induced a significant prolongation of survival compared to the vehicle control. Similarly, in a PDX model with MLL-AF4, DSP-5336 induced complete remission and significant prolongation of survival at the doses of 100 mg/kg BID compared to the vehicle control. In mouse AML models wherein MLL-ENL- or MLL-AF10-transduced bone marrow cells are transplanted in syngeneic mice, DSP-5336 induced a significant prolongation of survival at the doses of 200 mg/kg once daily (QD) compared to the vehicle control and the standard chemo therapy (cytarabine+daunorubicin) group.Summary: DSP-5336 has a potential as an antitumor drug that provides survival advantages in acute leukemia patients with MLL rearrangement or NPM1 mutation. Currently, a Phase 1/2 clinical study of DSP-5336 is planned in AML and ALL patients. DisclosuresEguchi: Sumitomo Dainippon Pharma: Current Employment. Shimizu: Sumitomo Dainippon Pharma: Current Employment. Kato: Sumitomo Dainippon Pharma: Current Employment. Furuta: Sumitomo Dainippon Pharma: Current Employment. Kamioka: Sumitomo Dainippon Pharma: Current Employment. Ban: Sumitomo Dainippon Pharma: Current Employment. Ymamoto: Sumitomo Dainippon Pharma: Current Employment. Yokoyama: Sumitomo Dainippon Pharma: Research Funding. Kitabayashi: Sumitomo Dainippon Pharma: Research Funding.

Preclinical Evidence for the Efficacy of CD79b Immunotherapy in B Cell Precursor Acute Lymphoblastic Leukemia

Patients with B cell precursor acute lymphoblastic leukemia (BCP-ALL) have a favorable prognosis, yet current treatment protocols are based on intensive cytotoxic chemotherapy and therapy options are limited when patients relapse. Novel immunotherapy approaches are therefore needed. We recently reported that the preB cell receptor signaling unit CD79a (known as Igα) is crucial for BCP-ALL engraftment in vivo, particularly in the central nervous system (CNS) employing BCR-ABL + and E2A-PBX1 + patient derived xenograft (PDX) models (Lenk et al., Communications Biology, 2021). CD79a forms a heterodimer with CD79b (known as Igß), which is also expressed on the surface of mature B cells as part of the B cell antigen receptor. Accordingly, the CD79b antibody drug conjugate (ADC) Polatuzumab Vedotin (PolVed) has shown therapeutic efficacy in the treatment of refractory/relapsed (r/r) diffuse large B cell lymphoma. Moreover, CD79a/CD79b may also be present on the cell surface at the pro/pre B cell stage. We therefore hypothesize that CD79b can serve as a therapeutic target in BCP-ALL.First, to substantiate that CD79b is important for BCP-ALL engraftment in vivo, a murine/murine transplantation model was applied. B cell precursors were isolated from mice harboring CD79b with a non-functional signaling domain (CD79b-ITAM-KO) or wildtype mice, malignantly transformed with a BCR-ABL fusion construct and transplanted into NSG-mice. Whereas control cells caused overt leukemia in all animals within 25 days, no animal injected with CD79b-ITAM-KO cells had developed leukemia by the time of sacrifice at 162 days (P<0.001).To investigate the frequency of surface (s)CD79b expression in BCP-ALL patients, we measured sCD79b levels via flow cytometry in diagnostic samples of pediatric BCP-ALL patients with different cytogenetic backgrounds. We detected sCD79b-positivity (defined as ≥10% sCD79b + BCP-ALL cells) in 23/94 patients including BCR-ABL +, E2A-PBX1 +, MLL-rearranged (MLLr), TEL-AML1 + and B-other BCP-ALL patients indicating a population of sCD79b + patients within different cytogenetic BCP-ALL subgroups (Figure 1a).To validate CD79b as a therapeutic target, we applied an unconjugated monoclonal CD79b antibody (CD79b-mAB) to PDX mice bearing either pediatric BCR-ABL + or E2A-PBX1 + PDX samples with high sCD79b expression (49.6% sCD79b + cells and 37.7% sCD79b + cells, respectively). Treatment was initiated one day after BCP-ALL injection, modelling a minimal residual disease (MRD) situation. Therapy with CD79b-mAB (1mg/kg) resulted in a mild reduction of leukemia burden in the spleen (SP) and bone marrow (BM) of PDX mice and a significant reduction of CNS-involvement in both PDX-models as compared to control treated mice (P<0.05 and P<0.01, respectively). To test the hypothesis that treatment of BCP-ALL with a CD79b-ADC outperforms CD79b-mAB, we applied PolVed (1mg/kg) in NSG mice bearing the same E2A-PBX1 + and BCR-ABL + PDX cells. PolVed therapy resulted in a significant reduction of BCP-ALL engraftment in SP, BM and CNS (P<0.01, respectively) and a significant survival prolongation compared to control treated mice in both models (P<0.01, respectively). Of note, 4/5 PolVed-treated E2A-PBX1-PDX animals were free of leukemia by the time of sacrifice (236 days) (Figure 1B).To test the efficacy of PolVed on different BCP-ALL subgroups, we conducted a preclinical phase II-like PDX study, using sCD79b high and sCD79b low PDX samples (defined by sCD79b-expression above or below the median) (5E2A-PBX1 +, 3 BCR-ABL +, 2 MLLr, 1 E2A-HLF + and 1 ETV6-NTRK3 +). Two NSG mice per patient were injected with PDX cells, randomly assigned into treatment groups and PolVed therapy was initiated when 1% PDX-cells were detected in the peripheral blood, modelling an overt leukemia situation. sCD79b low PDX mice did not respond to PolVed treatment (Figure 1c), but we detected a response to therapy and a significant survival prolongation in 5/6 sCD79b high PDX samples irrespective of the cytogenetic background (P<0.05) (Figure 1d).Taken together, our data indicate that a subgroup of BCP-ALL patients is sCD79 + positive and may respond to PolVed treatment. Therefore, we suggest CD79b as a novel therapeutic target in BCP-ALL and propose PolVed as a potential therapeutic agent in r/r disease.*LL and DW contributed equally to this work [Display omitted] DisclosuresLenk: OSE Immunotherapeutics: Research Funding. Richter: HTG Molecular Diagnostics, Inc.: Current Employment, Research Funding. Schrappe: SHIRE: Other: research support; JazzPharma: Honoraria, Other: research support; Servier: Honoraria, Other: research support; SigmaTau: Other: research support; Novartis: Honoraria; JazzPharma: Honoraria; Novartis: Honoraria, Other: research support; Servier: Honoraria; Amgen: Other: research support. Cario: Novartis: Other: Lecture Fee. Brüggemann: Incyte: Other: Advisory Board; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Schewe: Jazz Pharmaceuticals: Other: Advisory Board; SOBI: Other: Advisory Board; Bayer: Other: Advisory Board; OSE Immunotherapeutics: Research Funding.

CCS1477, a Novel p300/CBP Bromodomain Inhibitor, Enhances Efficacy of Azacitidine and Venetoclax in Pre-Clinical Models of Acute Myeloid Leukaemia and Lymphoma

E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferases that co-activate key oncogenes such as MYC and IRF4 which are relevant in a number of haematological malignancies. Here we describe the effects of CCS1477, a new orally available inhibitor of the p300/CBP bromodomains, given alone or in combination with azactidine or venetoclax in pre-clinical models of AML and B-cell lymphoma.As a monotherapy, daily oral dosing with CCS1477 (5, 10 and 20mg/kg) caused a significant dose-dependent reduction in tumour growth in a MOLM-16 xenograft model of AML, with regressions observed at the highest and well tolerated dose of 20mg/kg/qd. The inhibition of tumour growth during drug treatment was accompanied by a significant reduction in tumour expression of MYC by qPCR. In murine retroviral transduction and transplantation models of human AML initiated by either MLL-AF10 or MLL-AF9, daily monotherapy by oral gavage with CCS1477 at 30mg/kg for 42 days, caused a highly significant prolongation of survival. All seven control treated mice died of AML within 160 days whereas only one of seven CCS1477 treated mice had died in each model by 300 days.In the MOLM-16 xenograft model, a sub-maximal dose of CCS1477 (10mg/kg) demonstrated superior tumour growth inhibition by comparison with azacitidine (0.5mg/kg i.p) and with significant combination benefit when these two agents were combined. In this same model venetoclax (50 or 100 mg/kg/qd oral) had no effect on tumour growth, indicating that MOLM-16 tumours are intrinsically resistant. CCS1477 dosed daily at 10mg/kg restored sensitivity to venetoclax as evidenced by significantly greater tumour growth inhibition when compared with CCS1477 given alone. In a venetoclax-sensitive xenograft model of AML (MV-4-11), CCS1477 (10mg/kg) and venetoclax (100mg/kg) resulted in a similar and partial reduction in tumour growth over a 21 day dosing period. When venetoclax and CCS1477 were combined there was a significant combination benefit compared to either drug given alone. The DOHH-2 xenograft model of B-cell lymphoma was also sensitive to venetoclax (50mg/kg) and with a similar impact on tumour growth compared with CCS1477 (10mg/kg). In this model the combination of CCS1477 and venetoclax was more profound and caused complete tumour stasis during the dosing period.These data support the clinical testing of CCS1477 in combination with azacitidine and/or venetoclax in AML and B-cell lymphomas. CCS1477 is currently in Phase I/II clinical trials in AML, Non-Hodgkin lymphoma (including B-cell lymphoma) and multiple myeloma. (NCT04068597). DisclosuresBrooks: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Knurowski: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Hughes: CellCentric Ltd: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Clegg: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. West: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Pegg: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Somervaille: Novartis: Consultancy, Honoraria.

PiggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells

Promising progress has been made in adoptive transfer of allogeneic natural killer (NK) cells to treat relapsed or refractory acute myeloid leukemia (AML). In this regard, chimeric antigen receptor (CAR)-modification of NK cells is considered as a compelling approach to augment the specificity and cytotoxicity of NK cells against AML. Using a non-viral piggyBac transposon technology and human peripheral blood-derived primary NK cells, we generated CAR-NK cells to target NKG2D ligands and demonstrated their in vitro activity in lysing cancer cells expressing the ligands and in vivo efficacy in inhibiting tumor growth in a xenograft KG-1 AML model. We further generated CAR-NK cells co-expressing transgenes for the NKG2D CAR and interleukin-15 (IL-15). The ectopic expression of IL-15 improved the in vitro and in vivo persistence of NKG2D CAR-NK cells, leading to enhanced in vivo tumor control and significant prolongation of mouse survival in the KG-1 AML model. Collectively, our findings demonstrate the ectopic expression of IL-15 as an important means to improve the antileukemic activity of NKG2D CAR-NK cells. Our study further illustrates the feasibility of using the piggyBac non-viral platform as an efficient and cost-effective way for CAR-NK cell manufacturing.

Multimodal, interdisciplinary therapeutic concepts for liver metastasized colorectal cancer

About half of all patients with colorectal carcinoma (CRC) develop metastases mainly in the liver during the course of their disease. Metastatic disease is associated with a low 5-year overall survival rate of only 5-7 %, particularly when there is no possibility of local treatment. However, if there is an opportunity to resect the metastases, especially isolated liver metastases, the chance of long-term survival is approximately 15-27 % after both primary resection or secondary resection after neoadjuvant pretreatment. Overall, long-term survival of patients with metastatic CRC has improved significantly in recent years due to a combination of modern systemic therapies, advanced liver surgery and local ablative procedures.Of note, for the vast majority of patients, metastatic resection does not mean cure, but a significant prolongation of overall survival with a good quality of life. Chemotherapy-free intervals after metastasis resection maintain quality of life and can help to reduce toxicity.In this review, we would like to present the "toolbox" for the multidisciplinary treatment of metastatic CRC and give recommendations how the individual modalities should be optimally used, considering tumor-specific characteristics and patient preferences.

230 Preclinical efficacy of CLEC-1 antagonist as novel myeloid immune checkpoint therapy for oncology

BackgroundC-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) tempers T cell’s responses in vivo by limiting antigen cross-presentation by cDC1. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment, in particular tumor-associated macrophages.MethodsMacrophages were generated from monocytes of healthy volunteers for phagocytosis assays. MC38 and Hepa 1.6 murine tumor cells were implanted in Clec1a KO or KI mice for immunotherapeutic treatment evaluation.ResultsUsing newly developed anti-human CLEC-1 monoclonal antibodies (mAbs), we found that antagonist anti-CLEC-1 mAbs with the capacity to block CLEC-1/CLEC-1Ligand interaction, as opposed to non-antagonist CLEC-1 mAbs, increase the phagocytosis of CLEC-1Ligand-positive human tumor cells by human macrophages, in particular when opsonized by tumor-associated antigen mAbs (Rituximab, Cetuximab, Trastuzumab) or with anti-CD47 mAb (Magrolimab). In-vivo, CLEC-1 knock-out (KO) mice (n=19) display significant prolonged survival in monotherapy as compared to wild-type littermates (n=12) in an orthotopic hepatocellular carcinoma (HCC) model and anti-tumor memory responses was demonstrated by tumor rechallenge in cured mice. CLEC1 KO mice also illustrate significant eradication of MC38 colorectal tumors in combination with chemotherapy promoting CLEC-1Ligand expression by tumor cells (n=16 with Gemcitabine or n=11 with Cyclophosphamide). HCC tumor microenvironment analysis after 2 weeks of tumor implantation shows significantly higher number of CD8+ and memory CD8+ T cells with reduced PD1 expression in CLEC1 KO animals (n=16 versus n=12 for KO vs WT mice respectively). Finally, we recently generated human CLEC-1 knock-in mice expressing the extracellular human CLEC1 domain fused to the intracellular mouse CLEC1 tail and confirmed preclinical efficacy in vivo with anti-human CLEC1 antagonist mAb in monotherapy in the orthotopic HCC model.ConclusionsThese data illustrate that CLEC-1 inhibition represents a novel therapeutic target for immuno-oncology modifying T cell immune responses and tumor cell phagocytosis by macrophages.

The Mandible Ameliorates Facial Allograft Rejection and Is Associated with the Development of Regulatory T Cells and Mixed Chimerism.

Vascularized composite allografts contain various tissue components and possess relative antigenicity, eliciting different degrees of alloimmune responses. To investigate the strategies for achieving facial allograft tolerance, we established a mouse hemiface transplant model, including the skin, muscle, mandible, mucosa, and vessels. However, the immunomodulatory effects of the mandible on facial allografts remain unclear. To understand the effects of the mandible on facial allograft survival, we compared the diversities of different facial allograft-elicited alloimmunity between a facial osteomyocutaneous allograft (OMC), including skin, muscle, oral mucosa, and vessels, and especially the mandible, and a myocutaneous allograft (MC) including the skin, muscle, oral mucosa, and vessels, but not the mandible. The different facial allografts of a BALB/c donor were transplanted into a heterotopic neck defect on fully major histocompatibility complex-mismatched C57BL/6 mice. The allogeneic OMC (Allo-OMC) group exhibited significant prolongation of facial allograft survival compared to the allogeneic MC group, both in the presence and absence of FK506 immunosuppressive drugs. With the use of FK506 monotherapy (2 mg/kg) for 21 days, the allo-OMC group, including the mandible, showed prolongation of facial allograft survival of up to 65 days, whereas the myocutaneous allograft, without the mandible, only survived for 34 days. The Allo-OMC group also displayed decreased lymphocyte infiltration into the facial allograft. Both groups showed similar percentages of B cells, T cells, natural killer cells, macrophages, and dendritic cells in the blood, spleen, and lymph nodes. However, a decrease in pro-inflammatory T helper 1 cells and an increase in anti-inflammatory regulatory T cells were observed in the blood and lymph nodes of the Allo-OMC group. Significantly increased percentages of donor immune cells were also observed in three lymphoid organs of the Allo-OMC group, suggesting mixed chimerism induction. These results indicated that the mandible has the potential to induce anti-inflammatory effects and mixed chimerism for prolonging facial allograft survival. The immunomodulatory understanding of the mandible could contribute to reducing the use of immunosuppressive regimens in clinical face allotransplantation including the mandible.

Prognostic impact of bone invasion in canine oral malignant melanoma treated by surgery and anti-CSPG4 vaccination: A retrospective study on 68 cases (2010-2020).

Prognosis of canine oral malignant melanoma encompasses clinical, histological and immunohistochemical parameters. The aim of this study was to evaluate the prognostic impact of bone invasion in oral canine melanoma. Sixty‐eight dogs bearing oral melanoma staged II and III that underwent surgery and anti‐CSPG4 electrovaccination, with available histological data and a minimum follow up of minimum 1 year, were retrospectively selected. Bone invasion was detected on imaging and/or histology. Median survival time of dogs with evidence of bone invasion (group 1) was 397 days and significantly shorter compared with dogs with oral melanomas not invading the bone (group 2, 1063 days). Dogs with tumours localised at the level of the cheek, lip, tongue and soft palate (soft tissue ‐ group 3) lived significantly longer compared with dogs having tumours within the gingiva of the maxilla or mandible (hard tissue ‐ group 4) with a median survival time of 1063 and 470 days, respectively. Within group 4, the subgroup of dogs with tumours not invading the bone (group 5) showed a significant prolonged survival time (972 days) in comparison with dogs of group 1 (bone invasion group). Similar results were obtained for the disease‐free intervals amongst the different groups. Statistical analysis showed that Ki67 and mitotic count were correlated with shorter survival in patients of group 1 (with bone invasion). Bone invasion should always be assessed since it appears to be a negative prognostic factor.

Pre-Therapeutic VEGF Level in Plasma Is a Prognostic Bio-Marker in Head and Neck Squamous Cell Carcinoma (HNSCC)

Simple SummaryIn the context of a growing variety in treatment strategies for patients with cancer, especially approaches based on antiangiogenetic pathways, we aimed to identify a useful biomarker for patients with head and neck squamous cell carcinoma (HNSCC). Our experimental results detected vascular endothelial growth factor (VEGF) in patients’ pre-therapeutic plasma, and not serum, which serves as a suitable biomarker for outcome prognostication. Results were validated in an independent cohort, confirming VEGF as an independent predictor (Pi) of outcomes in HNSCC patients. Therefore, pre-therapeutic VEGF in plasma may be an attractive biomarker in future HNSCC studies.Vascular endothelial growth factor (VEGF) is centrally involved in cancer angiogenesis. We hypothesized that pre-therapeutic VEGF levels in serum and plasma differ in their potential as biomarkers for outcomes in head and neck squamous cell carcinoma (HNSCC) patients. As prospectively defined in the study protocols of TRANSCAN-DietINT and NICEI-CIH, we measured VEGF in pretreatment serum and plasma of 75 HNSCC test cohort (TC) patients. We analyzed the prognostic value of VEGF concentrations in serum (VEGFSerum) and plasma (VEGFPlasma) for event-free survival (EFS) utilizing receiver-operating characteristics (ROC). Mean VEGF concentrations in plasma (34.6, 95% CI 26.0–43.3 ng/L) were significantly lower (p = 3.35 × 10−18) than in serum (214.8, 95% CI 179.6–250.0 ng/L) but, based on ROC (area under the curve, AUCPlasma = 0.707, 95% CI 0.573–0.840; p = 0.006 versus AUCSerum = 0.665, 95% CI 0.528–0.801; p = 0.030), superiorly correlated with event-free survival (EFS) of TC patients. Youden indices revealed optimum binary classification with VEGFPlasma 26 ng/L and VEGFSerum 264 ng/L. Kaplan–Meier plots demonstrated superiority of VEGFPlasma in discriminating patients regarding outcome. Patients with VEGFPlasma < 26 ng/L had superior nodal (NC), local (LC) and loco-regional control (LRC) leading to significant prolonged progression-free survival (PFS) and EFS. We successfully validated VEGFPlasma according the cut-off <26 ng/L as predictive for superior outcome in an independent validation cohort (iVC) of 104 HNSCC patients from the studies DeLOS-II and LIFE and found better outcomes including prolonged tumor-specific (TSS) and overall survival (OS). Outcomes in TC and iVC combined again was related to VEGFPlasma, and multivariate Cox regression revealed that VEGFPlasma was an independent outcome predictor. In HNSCC, pre-therapeutic VEGFPlasma is prognostic for outcomes.

It’s not ‘just a tube of blood’: principles of protocol development, sample collection, staffing and budget considerations for blood-based biomarkers in immunotherapy studies

Immunotherapy for cancer is now a standard pillar in the armamentarium of treatments for many cancers. Immune checkpoint inhibitors, in particular, have resulted in significant therapeutic benefit and prolongation of...

Abstract 1463: ATP-site inhibitor olverembatinib, HQP1351, enhanced the effect of allosteric inhibitor on the resistance conferred by the compound mutations of BCR-ABL

Abstract Treatment with tyrosine kinase inhibitors (TKIs) directed against the ATP-binding site of BCR-ABL promotes recovery of Ph+ leukemia. However, emergence of gatekeeper mutation T315I and compound mutants confer resistance to these TKIs. The allosteric inhibitor, asciminib, effectively inhibits BCR-ABL kinase through binding to the myristoyl-binding site. Combining asciminib with ponatinib can overcome only a subset of the resistance caused by BCR-ABL compound mutants. Olverembatinib (HQP1351) is a new generation TKI targeting BCR-ABL and currently in development for r/r CML. The purpose of this study is to evaluate whether a novel combination of olverembatinib and asciminib, targeting both ATP pocket and allosteric region of BCR-ABL protein, can promote the inhibitory effect on the kinase harboring compound mutations. A series of cell lines expressing BCR-ABL single or compound mutations were constructed based on BaF3 murine pro-B cell line. Effect of olverembatinib as a single agent or in combination with asciminib were analyzed using antiproliferation assay, Western blot and FACS assays in vitro. In vivo efficacy was evaluated using the syngeneic mouse model derived from BaF3 cells with T315I or compound mutations. The cell based antiproliferation studies demonstrated superior activity of olverembatinib toward BCR-ABL single or compound mutations with IC50 values ranging between 6-300 nM. In particular, olverembatinib was more effective than ponatinib, asciminib and other TKIs against those compound mutations. Moreover, the combination of olverembatinib with asciminib was highly effective against BCR-ABL compound mutations, especially those containing T315I. In vivo studies further revealed that co-administration of olverembatinib with asciminib resulted in significant prolongation of survival compared to single agents. Importantly, the anti-tumor effect was more potent than that of ponatinib plus asciminib in models harboring compound mutations. In terms of mechanism, the combined treatment synergistically downregulated phosphorylation of BCR-ABL and the downstream proteins CRKL and STAT5, and augmented cleavage of Caspase-3 and PARP-1, thus triggered apoptosis and subsequently enhanced the antitumor effects. Our results demonstrated that the combination of ATP binding site inhibitor olverembatinib and allosteric inhibitor have synergistic anti-tumor effect on tumor cells harboring single or compound mutations in BCR-ABL. This novel strategy may help to overcome the secondary compound mutations post the treatment with TKIs. Citation Format: Guangfeng Wang, Jing Lv, Chunyang Tang, Ping Min, Li Rui, Fei Zhang, Lvcheng Wang, Yangfeng Ge, Kejie Lian, Eric Liang, Dajun Yang, Yifan Zhai. ATP-site inhibitor olverembatinib, HQP1351, enhanced the effect of allosteric inhibitor on the resistance conferred by the compound mutations of BCR-ABL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1463.

Clinical BET Inhibitor RG6146 Induces Intrinsic Apoptosis and Down-Regulates PD-L1 Expression and Induces Potent Anti-Tumor Activity in Models of Hematological Malignancy

Bromodomain and extra terminal (BET) family of bromodomains BET proteins are epigenetic ‘readers’ that bind acetylated histone residues and transcription factors at the chromatin interface and drive the expression of oncogenic transcription programs. BET inhibition using small molecules is an anti-cancer strategy being evaluated in early phase clinical trials. RG6146 is a novel small molecule, non-covalent inhibitor of (BET) family of bromodomains and potently inhibits BRD2, BRD3, BRD4 and BRDT. RG6146 is in clinical development for hematological malignancies including diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM) and solid tumors. In this study, we comprehensively examined the pre-clinical activity of RG6146 in genetically distinct models of hematological malignancy.To understand the effects of BET inhibition on myc-levels and viability hematological malignancies, a panel of human MM and DLBCL cell lines were treated with RG6146 for 24-72 hours. Treatment with RG6146 potently down-regulated c-myc levels that was concomitant with induction of apoptosis and loss of viability.To elucidate additional Myc-independent effects of RG6146, we used the Eμ- Myc model of aggressive B-cell lymphoma where the expression of transgenic Myc is insensitive to BET inhibition. Recapitulating the biological activity of prototypical BET inhibitor JQ1, RG6146 potently activated the intrinsic apoptotic cascade independently of p53 mutational status. Moreover, ectopic Bcl-2 overexpression or genetic loss of Bim were sufficient to attenuate RG6146-induced apoptosis. In the setting of ectopic Bcl-2 expression and protection from apoptosis, RG6146 impairs cell cycle progression leading to G1 cell cycle arrest.We next compared the transcriptional consequences of RG6146 or JQ1 treatment in cell derived from the Eμ- Myc model using RNA sequencing. Acute BET inhibition following 2 hours treatment with JQ1 or RG6146 was associated with a highly concordant modulation of global transcription. Accordingly, gene ontology analysis revealed that identical biological pathways were affected by either inhibitor. As a bona fide BET inhibitor target gene, we demonstrated rapid suppression of Il7r mRNA by qPCR and subsequent loss of cell surface CD127 expression by flow cytometry following exposures to RG6146.Finally, we demonstrate single-agent activity of RG6146 in Eμ- Myc lymphoma-bearing syngeneic recipient mice. Administration of a single-dose of RG6146 significantly depleted tumor cells in the lymph node, spleen, and peripheral blood within 24 hours. Chronic RG6146 therapy was well tolerated in vivo and led to dose-dependent reduction in tumor burden and significant prolongation of survival. In this setting, we also demonstrated that RG6146 therapy suppressed the BRD4-dependent expression of immune checkpoint ligand PD-L1 on Eμ- Myc lymphomas.Taken together, these data indicate that RG6146 is an on-target BET bromodomain inhibitor with potent anti-cancer activity. We demonstrated disruption of the intrinsic apoptotic pathway was sufficient to reduce the activity of RG6146. Moreover, we have previously demonstrated that acquired resistance to BET inhibition is associated with elevated expression of pro-survival Bcl-2 and cell surface CD20 expression. We are now investigating the efficacy RG6146 in combination with BH3 mimetics or monoclonal antibodies. Finally, we are investigating the ability of RG6146 to modulate the immunogenicity of tumors and augment anti-tumor immunity. DisclosuresWellinger:Roche: Employment. Rueflibrasse:Roche: Employment.

ALKBH5-mediated m6A-demethylation of USP1 regulated T-cell acute lymphoblastic leukemia cell glucocorticoid resistance by Aurora B.

Recent studies evidence that ubiquitin-specific proteases (USPs) are associated with the occurrence and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL). N6 -methyladenosine (m6A) demethylase AlkB homolog 5 (ALKBH5) exerts a carcinogenic effect in human cancers and improves the mRNA stability of USPs. Whether ubiquitin-specific protease 1 (USP1) controls chemoresistance of T-ALL is unknown. Our study demonstrated that USP1 expression was upregulated in glucocorticoid (GC)-resistant T-ALL patients and cells (CEM-C1). High expression of USP1 was correlated to the poor prognosis in T-ALL patients. Silencing USP1 increased CEM-C1 cell sensitivity to dexamethasone (Dex), reduced cell invasion, promoted cell apoptosis, and ameliorated glucocorticoid receptor (GR) expression. USP1 mediated T-ALL chemoresistance by interacting with and deubiquitination of Aurora B. Overexpression of USP1 reversed the amelioration effect of Aurora B inhibitor on CEM-C1 cell resistance to Dex. Mechanistically, ALKBH5 enhanced USP1 expression by reducing m6A level and mRNA stability in USP1 mRNA transcript. Downregulation of ALKBH5 reduced the levels of USP1 and Aurora B, facilitated CEM-C1 cell sensitivity to Dex, apoptosis, and GR expression, suppressed cell invasion. However, overexpression of USP1 reversed all the effects of ALKBH5 on CEM-C1 cells. In vivo results showed that tail vein injection of sh-USP1 resulted in a significant prolongation of mouse survival, suppressed tumor growth, maintained the normal weight of mice, reduced USP1 expression and facilitated GR expression. In conclusion, inhibition of ALKBH5-mediated m6A modification decreased USP1 expression and downregulation of USP1 ameliorated GC resistance of T-ALL through suppressing Aurora B expression and elevating GR level.

Aberrant Hedgehog Signaling in Myelodysplastic Syndrome Accelerates Leukemic Transformation Via Smoothened-Dependent and Smoothened-Independent Gli1 Activation

Introduction: Myelodysplastic syndromes (MDS) are malignant hematopoietic stem cell disorders with increased risk of transformation to secondary acute myeloid leukemia (sAML) that carries uniformly dismal prognosis. The exact mechanisms driving this process remain obscure. Aberrant Hedgehog (Hh) signaling has been demonstrated in a number of solid tumors and hematologic malignancies. An emerging body of literature suggests the importance of Hh signaling in leukemic transformation of MDS. Moreover, a recent Phase II clinical trial using Smoothened (SMO) inhibitor with low-dose chemotherapy resulted in significant prolongation of overall survival (Lancet et al. Blood 2016). Thus, we sought to examine the clinical impact of aberrant Hh activation in MDS and further explore the mechanism of Hh-driven leukemic transformation.Methods/Results:To determine the pathway activity, we quantified the level of GLI1 in CD34+ cells from patients with MDS and healthy controls in published databases (N=17 and N=159, respectively; GSE58831) and while absent in controls, GLI1 was significantly upregulated in MDS patients (p<0.05). Analysis of serial samples revealed Hh activation at disease progression in 67% of patients (4/6). In addition, GLI1 levels were significantly higher in poor-risk AML with complex cytogenetics comparing to favorable risk disease (p<0.001, TCGA).To further evaluate the impact of of Hh activation in MDS progression, we crossed Nup98-HoxD13 (NHD13) mice with mice conditionally expressing the constitutively active mutant of Smoothened (SmoM2). Double transgenic NHD13/SmoM2 mice succumbed to aggressive myeloid leukemia (median survival of NHD13/SmoM2 and NHD13 controls, 3 months vs. 13 months, respectively, p<0.0001) characterized by leukocytosis (p<0.05), splenomegaly (p<0.0001) and significant expansion of immature myeloid progenitors in peripheral blood (p<0.0001) and bone marrow (p<0.0001). Analysis of hematopoietic stem/progenitor (HSPC) compartment revealed expansion of phenotypic granulocyte macrophage progenitors (GMP) (6244 vs. 131487 mean GMPs/femur , in NHD13 v. NHD13/SmoM2 respectively, p<0.05). To determine whether Hh activation resulted in aberrant self-renewal in GMP compartment, we carried out an in vivo repopulation assay. While NHD13 GMPs showed only transient engraftment, characteristic of non-self-renewing progenitors, NHD13/SmoM2 GMPs robustly engrafted and significantly expanded over time, and all recipient animals developed aggressive leukemia within 2-3 months. The engraftment was sustained through secondary transplantation. Our results suggest that Hh activation resulted in abnormal self-renewal in normally non-self-renewing progenitors.To delineate the mechanism leading to aberrant activation of self-renewal we compared the transcriptome of GMPs isolated from NHD13/SmoM2 and NHD13 controls. In addition to expected Hh activation, Gene Set Enrichment Analysis (GSEA) showed enrichment in gene signatures associated with other developmental pathways such as Wnt or TGF-β signaling as well as EWS/FLI1-protooncogen signature. Interestingly, these pathways have been found to act via direct activation of GLI1 .Using human AML (TF-1) and MDS (MDS-L) cell lines we confirmed that GLI1 may be activated in the absence of SMO. GLI1 knock-down and/or inhibition with GANT61 resulted in growth arrest (p<0.05, both) and significantly lower clonogenic potential in vitro (p<0.05, both) more so than upstream SMO inhibition.Conclusions: Our data suggest that aberrant Hh/GLI1 signaling is frequent in MDS and marks disease progression and leukemic transformation. In some cases, GLI1 can be activated in a SMO-independent manner and therapeutic strategies focused on targeting downstream Gli transcription factors may be more effective than currently used Smo inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Targeting the DNA Damage Response Pathway in MLL- rearranged B-Acute Lymphoblastic Leukemias

Infant B-acute lymphoblastic leukemias (B-ALL) that harbor MLL-AF4 rearrangements are associated with a poor prognosis (event-free survival (EFS) of 35%-50%), especially when compared to non-MLL-rearranged (MLL - r ) childhood ALL (EFS > 85%). Thus, effective therapies that ensure long-term durable responses are of extreme interest. Recently, the targeting of the DNA damage response pathway (DDR) has been shown to be cytotoxic to MLL-r acute myeloid leukemia (AML) cells with oncogene driven replicative stress. In order to determine if targeting of the DDR pathway would be efficacious in MLL-r B-ALLs, we tested a panel of human AML and ALL cell lines and found that all B-ALL cell lines (those with and without MLL-r ) were highly sensitive to ATR kinase inhibition. At 72 hours, IC50s for MLL- r B-ALL cell lines (RS4;11, SEMK2, Hb11;19) were 2-3 fold lower than MLL-AF9 AML cell lines (Molm13, THP-1, Nomo1). Inhibition of ATR kinase induced double stranded DNA damage (as assessed by the increase in phospho-γH2AX levels), apoptosis and loss of the G2-M cell cycle checkpoint in a dose dependent manner in all cells treated, with B-ALL cells exhibiting these effects more rapidly and at lower doses.In order to better understand the mechanism behind the increased sensitivity of MLL -r B-ALLs to ATR inhibition, we generated a highly aggressive, serially transplantable B-ALL by the retroviral overexpression of activating N-RasG12D mutant in bone marrow of an inducible knock-in MLL-Af4 murine model. Recipient mice injected with Mll-Af4/N-RasG12D pre-leukemic bone marrow cells developed an acute B-ALL (B220+CD43+IgM-) with short latency to development of disease (median 35 days). Furthermore, the resultant primary B-ALL was serially transplantable, with accelerated latency to secondary and tertiary disease. Multiple sequencing studies have indicated that while MLL -r B-ALL have relatively few additional somatic mutations, activating mutants of RAS seem to co-occur at a frequency close to 50%. Moreover, among MLL -r infant leukemias, those harboring mutant RAS mutations have even worse overall prognosis, further emphasizing the utility of our model.We were able to detect increased basal levels of DNA damage and replicative stress in our Mll-Af4/NrasG12D pre-leukemic cells and sensitivity to a small molecule inhibitor of ATR, AZ20. In vitro and in vivo treatment with AZ20 resulted in increased leukemia cytotoxicity. However, tertiary B-ALL transplanted mice eventually succumbed to disease following prolonged AZ20 treatment in vivo . As our model includes an activating mutation in N-Ras , we hypothesized that cells would be sensitive to the combination with small molecule inhibitors of downstream effectors of Ras. The combination of AZ20 and MEK inhibitor GSK212 (Trametinib) increased cytotoxicity and cell cycle arrest in vitro at concentrations well below the IC50, as compared to single agent treatment. In vivo combination treatment of not only our mouse model but also of patient derived xenografts, harboring both mutations, exhibited decreased leukemia burden and significant prolonged survival compared to either AZ20 or Trametinib alone. These data suggest a potent combination therapy for the treatment of this subgroup of poor prognosis MLL-AF4 patients with RAS mutations.To try to gain some further mechanistic insight, we conducted gene set enrichment analysis of RNA-sequencing of primary Mll-Af4/N-RasG12D leukemias and normal pro-B cells. Our analyses revealed significant negative correlations with gene signatures involving the G2/M checkpoint and ATR activation in response to replication stress, suggesting derangements in DDR pathways. We validated these observations with quantitative RT-PCR of ATR pathway members in murine mouse models of MLL-r AML and B-ALL. Our studies suggest that the observed sensitivity to ATR inhibition correlates to the overall expression level of ATR and ATR pathway members. We observed similar patterns of sensitivity and expression in the human cell lines we tested, as well as in an analysis of published gene expression data of infant and pediatric B-ALL patients. Altogether, our data indicates the inhibition of ATR in cells with oncogene driven replicative stress and abrogated ATR and ATR pathway expression, as part of an effective combination therapeutic strategy in this subtype of acute leukemia. DisclosuresArmstrong:C4 Therapeutics: Consultancy; Imago: Consultancy; Epizyme: Consultancy; Cyteir: Consultancy.

The Clinical Outcomes of Lenalidomide and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma: A Nationwide Registry Study of the Korean Multiple Myeloma Working Party (KMMWP)

Background : Lenalidomide and dexamethasone (RD) is a standard of care for relapsed/refractory multiple myeloma (RRMM), but there is limited reported data on its efficacy and safety in the real clinical practice. The purpose of this study was estimate necessity of RD chemotherapy for improving survival in RRMM in real clinical fields.Methods : Data from patients at twenty two university hospitals in South Korea between December 2012 and Oct 2016 were collected retrospectively. Clinical and laboratory data were collected retrospectively by review of medical records. The study protocol was approved by the institutional review board of each participating hospital. Patient characteristics were described at the status before lenalidomide treatment start except for cytogenetics and international staging systems, which were described from the data established at the time of diagnosis. The progression free survival (PFS) was defined duration from the date of starting RD chemotherapy to the date of disease progression, relapse, or death from any causes. OS was calculated from the start of lenalidomide treatment to the date of death or the date of last follow-up. Cox proportional hazard regression model was used for a multivariate analysis for PFS and OS. Prognostic factors significant in univariate analysis for PFS or OS were entered for multivariate analysis.Results : The median age of the 527 patients was 66 years (range, 30-109 years) and the male to female ratio was 1.22:1.0. Patients previously were exposed to bortezomib only (40.0 %), both thalidomide and bortezomib (40.1%), and patients who had prior autologous stem cell transplantation were 37.2 %. Median number of cycles of RD were 6 (range, 1-37). The response rates were following: CR or stringent CR (sCR) in 84 (15.4%), VGPR in 106 (20.1%),PR in 166 (31.5%), MR in 24 (4.6%) and < MR in 101 (19.2%). Overall response rates (≥PR (partial response)) rate was 74.0 %. The differences of 2-year PFS of patients with achieved VGPR or more than VGPR and less than VGPR were 47.9% vs 30.2%, p<0.001. The 2-year OS were 66.4% vs 43.1%, p<0.001. The differences of 2-year PFS of patients treated with RD less than 6 cycles and 6 cycles or more than 6 cycles were 16.6% vs 53.4%, p<0.001. The 2-year OS were 26.2% vs 70.3%, p<0.001. In multivariate analysis, these two prognostic factors were shown significant prolonged survival (PFS; p=0.031 and p<0.001, OS; 0.017 and p<0.001). Adverse events of more than grade 2 were reported. Hematologic toxicities including cytopenia was observed only in 9.7 % of patients, infections including pneumonia in 15.1%, fatigue in 14.8%, skin rash in 7.0% and thrombosis in 3.0 %.Conclusions : Our study confirms that RD is effective and safe in RRMM. It produces prolonged survival especially in patients who received more than 6 cycles and achieved more than PR response. Toxicities from RD were tolerable for RRMM patients. DisclosuresNo relevant conflicts of interest to declare.

Molecular follow-up of first-line treatment by osimertinib in lung cancer: Importance of using appropriate tools for detecting EGFR resistance mutation C797S

The C797S mutation encoded by EGFR exon 20 is classically observed as a tertiary event in EGFR-mutant non-small-cell lung carcinoma (NSCLC) primarily treated by first generation tyrosine kinase inhibitors (TKI) and secondarily treated by third-generation TKI, such as osimertinib, if the EGFR-T790M resistance mutation is detected. Recently, significant prolonged progression free survival has been observed following first-line osimertinib, in EGFR-mutant NSLC. While mechanisms of molecular resistance to first-generation TKI have been well studied, little is known about resistance induced by primary third-generation TKI treatments. We report the case of a 65 year-old female treated by first-line osimertinib for a multimetastatic exon 19-EGFR-mutant NSCLC. EGFR-C797S resistance mutation and PIK3CA mutation were detected together with the remaining EGFR-exon 19 deletion. This observation provides insights of acquired resistance to first line-osimertinib. It also highlights the importance of making molecular platforms which perform routine EGFR testing in lung cancer aware of the kind of therapeutic protocols given to the patient. Indeed, for rapid results or low-costs procedures, some targeted methods specifically targeting T790M may be used at relapse and may overlook alterations such as C797S or PIK3CA mutations. Targeted next generation sequencing is therefore a recommended option.