In a context where injection of antigen (Ag)-specific T cells probably represents the future of leukemia immunotherapy, identification of optimal target Ags is crucial. We therefore sought to discover a reliable marker for selection of the most potent Ags. To this end, (1) we immunized mice against 8 individual Ags: 4 minor histocompatibility Ags (miHAs) and 4 leukemia-associated Ags (LAAs) that were overexpressed on leukemic relative to normal thymocytes; (2) we assessed their ability to reject EL4 leukemic cells; and (3) we correlated the properties of our Ags (and their cognate T cells) with their ability to induce protective antileukemic responses. Overall, individual miHAs instigated more potent antileukemic responses than LAAs. Three features had no influence on the ability of primed T cells to reject leukemic cells: (1) MHC-peptide affinity; (2) the stability of MHC-peptide complexes; and (3) epitope density at the surface of leukemic cells, as assessed using mass spectrometry. The cardinal feature of successful Ags is that they were recognized by high-avidity CD8 T cells that proliferated extensively in vivo. Our work suggests that in vitro evaluation of functional avidity represents the best criterion for selection of Ags, which should be prioritized in clinical trials of leukemia immunotherapy.

Purpose:The follow-up of Bosniak IIF renal cysts is associated with significant costs, radiation, and anxiety. Recent studies have suggested a risk of malignancy and upgrading lower than previously reported. We aimed to determine their clinical outcomes and to evaluate the impact of the 2019 Bosniak classification on the diagnosis of such lesions.Materials and Methods:We identified all radiology reports with the diagnosis of a Bosniak IIF cyst at our institution between January 2000 and December 2018. Imaging was reviewed to confirm the diagnosis and determine progression based on the 2005 Bosniak classification. Radiological and clinical characteristics were established, and the 2019 Bosniak criteria were retrospectively applied.Results:Out of 252 cysts reviewed, 55 (22%) were reclassified as Bosniak II upon revision using the 2005 Bosniak classification. A total of 181 Bosniak IIF cysts were included for final analysis. The median imaging follow-up was 50 months. Four (2.2%) cysts progressed to Bosniak III or IV. Five (2.8%) patients underwent surgical interventions, with only 1 malignant pathology being reported. No malignant progression was observed after 36 months. When applied to our cohort, the 2019 Bosniak classification would have led to a 76% decrease in Bosniak IIF diagnoses, with no increase in Bosniak III or IV diagnoses, and identical classification of the confirmed malignant pathology.Conclusions:Upgrading and malignancy rates among Bosniak IIF cysts was markedly lower than traditionally reported. No patient had a significant progression beyond 36 months. More than 20% of Bosniak IIF cysts were initially overdiagnosed. The 2019 Bosniak classification may help to reduce the overdiagnosis of Bosniak IIF lesions requiring follow-up.

Neuroblastoma (NB) is an aggressive childhood cancer that represents the leading cause of cancer deaths in children. Despite aggressive therapy, more than half of the children with advanced NB usually die because of uncontrolled metastatic disease. In order to develop new therapeutic strategies to limit NB's metastatic potential, it is crucial to identify key molecular targets governing the invasive process. The study of Cancer stem cells (CSCs) may be of interest in this regard because after chemotherapy, CSCs persist in tumors and cause relapse and metastasis. We had recently demonstrated that CD133 allowed to detect CSCs in NB. Membrane type-1 matrix metalloproteinase (MT1-MMP) is important in the metastatic process and its expression was correlated with unfavourable outcome in NBs. Our preliminary data had indicated that MT1-MMP was highly expressed in CD133high NB. The purpose of this study is to characterize the interaction between CD133 and MT1-MMP in NB and determinate the role of MT1-MMP in pro-invasive properties of CSCs. We constructed paraffin-embedded blocks of tissue microarrays (TMA) from 235 patients. In vitro experiments were performed on four established NB cell lines (SK-N-DZ, SK-N-FI and SK-N-SH and SJNB-10). We performed immunohistochemical studies on paraffin-embedded TMA sections with two antibodies (CD133, MT1-MMP). To verify correlation of expression of MT1-MMP and CD133, we realized western blot and immunofluorescence (IF) of both proteins in non treated versus treated NB cell lines. CD133high NB cells were isolated by flow cytometry. CD133high and CD133low NB cells were grown within a 3D collagen matrix and the cell migration assay was tested in collagen-coated transwells. All experiments were performed with or without an anti-MT1-MMP neutralizing antibody or GM6001, a broad-spectrum MMP inhibitor. To assess the interaction between CD133 and MT1-MMP, lysates were subjected to immunoprecipitation (IP) using an anti-CD133 antibody, followed by immunodetection with an anti-MT1-MMP antibody. Finally, we determined which domain of MT1-MMP is involved in its interaction with CD133. Different dominant negative mutants of MT1-MMP (catalytically inactive E240A, cytoplasmic domain-deleted CΔ20 and non-phosphorylatable Y573F) were transfected into NB cells followed by an IP. There is a correlation between MT1-MMP and CD133 expressions in tumors of patients and in cell lines. Cells selected after chemotherapy express both CD133 and MT1-MMP. CD133high cells presented higher migration and invasion properties than CD133low which were MT1-MMP dependent. IF and IP showed a colocalization and interaction between CD133 and MT1-MMP. The cytoplasmic domain of MT1-MMP seems to be responsible for the interaction with CD133. These results contribute to a better understanding CSCs properties in NB and may be of great interest to improve new therapeutic strategies. Citation Format: Assila Belounis, Carine Nyalendo, Sonia Cournoyer, Sarah Hadj-Mimoune, Alexandre Benoit, Elliot Lasalle, Jonathan Girard, Mona Beaunoyer, Pierre Teira, Hervé Sartelet. Regulation of the pro-invasive properties of neuroblastoma-stem cells by membrane type-1 matrix metalloproteinase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2013-3774

Introduction. Neuroblastoma is the most common and deadly extracranial solid tumour of childhood. Unlike localized neuroblastomas which have generally good prognosis, metastatic stages are associated with poor outcome. In this context, increasing evidences suggest that neuroblastoma may contain tumor-initiating cells (TICs) that cause relapse and metastasis. Our recent work indicates that CD133, a putative marker of TICs in various cancers, is associated with chemoresistance and poor outcome in neuroblastoma. In addition, we and others have demonstrated that membrane-type 1 matrix metalloproteinase (MT1-MMP) is associated with neuroblastoma progression and metastasis. Purpose. The purpose of this study was to characterize the tumor-initiating properties of CD133high neuroblastoma cells and determinate the role of MT1-MMP in pro-invasive properties of these cells. Methods/Results. Following CD133high cells selection from neuroblastoma cell lines (SK-N-DZ, SK-N-FI, SK-N-SH and SJ-NB10) with FACSAria cell sorter, we have examined their tumor-initiating features with neurosphere-forming assay in serum-free medium, colony-forming assay in soft agar and orthotopic transplantation into the adrenal gland (major site of primary neuroblastoma) of severe immunocompromised NOD/SCID/IL2Rγ- mice (NSG). Contrary to their counterpart CD133low, CD133high cells formed more colonies in soft agar and develop more neurospheres. Interestingly, orthotopic transplantation of as few as 500 CD133high cells into NSG mice resulted in tumor formation into the adrenal gland and dissemination to liver, lungs, brain and bone marrow, while matched control CD133low presented no tumor, suggesting that CD133high cells are TICs in neuroblastoma. Further western blot analyses of neuroblastoma cell lines revealed that CD133 expression correlated with that of MT1-MMP. In addition, CD133high cells growth within three-dimensional type I collagen matrices was markedly higher than CD133low cells and was significantly reduced by anti-MT1-MMP neutralizing antibodies. In the same vein, we performed cell migration assay in Boyden chamber and observed that CD133high cells exhibit more migratory abilities that CD133low cells, this migration being inhibited by anti-MT1-MMP neutralizing antibodies. Conclusions. Altogether, these findings strongly suggest that CD133-expressing TICs of neuroblastoma exhibit high pro-invasive capacities, requiring the involvement of MT1-MMP. Targeting pro-invasive capacities of neuroblastoma TICs with MMPs-activaed pro-drugs in combination with conventional therapy, represents an attractive therapeutic strategy aimed at eliminating both neuroblastoma TICs and the bulk of the tumour. Acknowledgment. Fondation Centre de cancérologie Charles-Bruneau. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2011-4344

The main barrier in allogeneic hematopoietic cell transplantation is the risk of developing graft-versus-host disease. This can be prevented by the injection of CD8 T cells targeted to leukemia associated antigens (LAAs) or minor histocompatibility antigens (MiHAs). Several studies in humans have established the value of LAAs and MiHAs as target for immunotherapy on solid tumors and leukemia, respectively. Importantly, their therapeutic efficacy has never been assessed on a per antigen basis. Thus, our goal is to directly compare the therapeutic efficacy of T-cells targeted to LAAs or MiHAs expressed on EL4 cells. We selected 4 MiHAs and 4 LAAs and confirmed their immunogenicity by in vitro cytotoxicity assays. We evaluated the anti-leukemic activity of antigen-specific CD8 T cells in vivo. Notably, mice immunized against MiHAs showed enhanced survival compared to mice immunized against LAAs. We found that decreased survival of EL4 bearing mice cannot be explained by weaker MHC I/Peptide interactions. Interestingly, we showed that T cells specific for 4 LAAs and 1 MiHA are undetectable by flow cytometry and that the abundance of tetramer positive cells correlates strongly with survival of EL4 bearing mice. We propose that CD8 T cells, mainly targeted to LAAs, bind weakly to their MHC I/Peptide complexes, leading to decreased immunogenicity. We believe that the insights gained from our studies will serve as guide for selecting the best antigens for future clinical trials.

INTRODUCTION AND OBJECTIVE:The follow-up of Bosniak BIIF renal cysts is associated with significant costs, radiation, and anxiety. Recent studies suggest a risk of malignancy and upgrading lower than previously reported. New radiological definitions of the Bosniak categories have been introduced in 2019. We aimed to determine the radiological and clinical evolution of BIIF cysts diagnosed at our institution and to establish the impact of the 2019 Bosniak classification on the diagnosis of such lesions.METHODS: We identified all radiology reports with the diagnosis of a BIIF cyst at our institution between January 2000 and December 2018. Diagnostic and follow-up imaging was reviewed by trained radiologists to confirm the diagnosis and determine progression. Radiological and clinical characteristics were established, and the 2019 Bosniak criteria were retrospectively applied.RESULTS: Out of 252 cysts initially reviewed, 55 (22%) were re-classified as BII upon revision. A total of 181 BIIF cysts were included for final analysis. The median imaging follow-up was 50 months. Only 4 (2%) cysts progressed to BIII or BIV. Five (3%) patients underwent surgical interventions, with only one malignant pathology being reported. No patient had a radiological progression without a confirmed benign pathology beyond 36 months. When applied to our cohort, the 2019 Bosniak classification would have led to a 76% decrease in BIIF diagnoses, with no increase in BIII or BIIV diagnoses, and identical classification of the confirmed malignant pathology.CONCLUSIONS: The rate of upgrading and malignancy among BIIF renal cysts was markedly lower than traditionally reported. No patient had a significant progression beyond 36 months. More than 20% of BIIF cysts were initially overdiagnosed by radiologists. The 2019 Bosniak classification may help to reduce the overdiagnosis of BIIF lesions requiring follow-up, avoiding important costs/harm to patients.

3016 Allogeneic hematopoietic cell transplantation is the most widely used form of adoptive T-cell cancer immunotherapy (ATCI) but its efficacy is limited by the fact that donor lymphocytes are neither selected nor primed (activated) prior to transfer. This can lead to tolerization of donor lymphocytes by tumor cells and causes graft-versus-host disease in 60% of recipients. These two caveats can be circumvented by the injection of primed antigen-specific CD8 T-cells targeted to tumor associated antigens (TAAs) or minor histocompatibility antigens (MiHAs). Several studies in humans have established the value of TAAs and MiHAs in therapies against solid tumors and leukemia, respectively. Importantly, the value of TAAs and MiHAs as targets for antigen-specific cancer immunotherapy has never been assessed against the same tumor. Therefore, our main goal is to directly compare the therapeutic efficacy of T-cells targeted to TAAs vs. MiHAs. More specifically, we want to evaluate the in vivo anti-leukemic potential of MiHA- vs. TAA-primed CD8 T-cells and identify the mechanisms responsible for the differential anti-leukemic activity of antigen-specific T-cells. We elected to work with 8 antigens known to be expressed on a mouse lymphoblastoma cell line (EL4 cells). Our panel includes 4 MiHAs of known sequence. We also selected 4 TAAs, whose sequences were elucidated by our group using a high-throughput mass spectrometry based approach (Fortier M.H. et al. 2008, J. Exp. Med). In vitro cytotoxicity assays revealed a functional CTL response against all of our antigens, thereby confirming their immunogenicity. We first evaluated the anti-leukemic activity of antigen-specific CD8 T cells in vivo. Mice were immunized twice with peptide pulsed dendritic cells and challenged with 5 × 105 EL4 cells 7 days after the last immunization. We observed an enhanced survival rate for mice immunized against MiHAs as opposed to mice immunized against TAAs. Indeed, while none of the mice immunized against TAAs survived the EL4 challenge, mice immunized against 3 out of the 4 MiHAs demonstrated either full survival (2 MiHAs) or partial survival with a delayed tumor onset. To better understand the mechanisms responsible for the differential anti-leukemic activity of antigen-specific T-cells, we assessed the quality of binding of the 8 antigens to the MHC I molecule. More specifically, we measured the half-life of MHC I/peptide complexes at the cell surface and performed a peptide binding competition assay to evaluate the affinity of our 8 antigens for MHC I molecules. Notably, MiHAs and TAAs showed comparable half-life of MHC I/peptide complexes at the cell surface and binding affinities. We conclude that differential MHC I/peptide interactions are not responsible for the differential anti-leukemic activity of MiHA- vs. TAA-primed CD8 T-cells. We then investigated the frequency of antigen-specific CD8 T-cells in immunized mice using MHC class I tetramers. Our preliminary results show that CD8 T-cells specific for 4 of our antigens (3 TAAs and 1 MiHA) are undetectable by flow cytometry. Interestingly, we found a strong correlation between the lack of tetramer staining and lack of survival of EL4 bearing mice. This suggests that CD8 T-cells targeting TAAs bind weakly to their respective MHC I/peptide complexes, which could indicate a weaker immunogenicity compared to MiHAs. It will be interesting to investigate whether and how other mechanisms of immunogenicity, such as TCR avidity and T-cell frequency, can influence the outcome of antigen-specific leukemia immunotherapy. Our studies will provide the first direct comparison of the anti-leukemic potential of MiHA- vs. TAA-primed CD8 T-cells. We believe that these crucial informations will serve as guide for selecting the best antigens for antigen-specific ATCI in future clinical trials. Disclosures: No relevant conflicts of interest to declare.