[Unrelated donor selection for allogeneic hematopoietic stem cell transplantation: Guidelines from the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)]. / Choix d'un donneur non apparenté en vue d'une allogreffe de cellules souches hématopoïétiques : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

The selection of a donor is an essential element in allogeneic hematopoietic stem cell transplantation. In the absence of an HLA-matched related donor, the selection of an unrelated donor is considered, and is currently the most common type of allogenic donor used in practice. Many criteria are considered for the selection when multiple donors are available, particularly in case of partial match. The aim of this workshop is to assist in the selection of an unrelated donor, in keeping with recent data from the literature.

DNA methylation profiling in Trisomy 21 females with and without breast cancer.

Background: Down Syndrome (DS) is the most common chromosome anomaly in humans and occurs due to an extra copy of chromosome 21. The malignancy profile in DS is unique, since DS patients have a low risk of developing solid tumors such as breast cancer however they are at higher risk of developing acute myeloid leukemia and acute lymphoblastic leukemia. Methods: In this study, we investigated DNA methylation signatures and epigenetic aging in DS individuals with and without breast cancer. We analyzed DNA methylation patterns in Trisomy 21 (T21) individuals without breast cancer (T21-BCF) and DS individuals with breast cancer (T21-BC), using the Infinium Methylation EPIC BeadChip array. Results: Our results revealed several differentially methylated sites and regions in the T21-BC patients that were associated with changes in gene expression. The differentially methylated CpG sites were enriched for processes related to serine-type peptidase activity, epithelial cell development, GTPase activity, bicellular tight junction, Ras protein signal transduction, etc. On the other hand, the epigenetic age acceleration analysis showed no difference between T21-BC and T21-BCF patients. Conclusions: This is the first study to investigate DNA methylation changes in Down syndrome women with and without breast cancer and it could help shed light on factors that protect against breast cancer in DS.

Autoimmunity in Down's syndrome via cytokines, CD4 T cells and CD11c+ B cells.

Down's syndrome (DS) presents with a constellation of cardiac, neurocognitive and growth impairments. Individuals with DS are also prone to severe infections and autoimmunity including thyroiditis, type 1 diabetes, coeliac disease and alopecia areata1,2. Here, to investigate the mechanisms underlying autoimmune susceptibility, we mapped the soluble and cellular immune landscape of individuals with DS. We found a persistent elevation of up to 22 cytokines at steady state (at levels often exceeding those in patients with acute infection) and detected basal cellular activation: chronic IL-6 signalling in CD4 T cells and a high proportion of plasmablasts and CD11c+TbethighCD21low B cells (Tbet is also known as TBX21). This subset is known to be autoimmune-prone and displayed even greater autoreactive features in DS including receptors with fewer non-reference nucleotides and higher IGHV4-34 utilization. In vitro, incubation of naive B cells in the plasma of individuals with DS or with IL-6-activated T cells resulted in increased plasmablast differentiation compared with control plasma or unstimulated T cells, respectively. Finally, we detected 365 auto-antibodies in the plasma of individuals with DS, which targeted the gastrointestinal tract, the pancreas, the thyroid, the central nervous system, and the immune system itself. Together, these data point to an autoimmunity-prone state in DS, in which a steady-state cytokinopathy, hyperactivated CD4 T cells and ongoing B cell activation all contribute to a breach in immune tolerance. Our findings also open therapeutic paths, as we demonstrate that T cell activation is resolved not only with broad immunosuppressants such as Jak inhibitors, but also with the more tailored approach of IL-6 inhibition.

UM171 Expansion of Cord Blood Improves Donor Availability and HLA Matching For All Patients, Including Minorities.

Cord blood (CB) stem cell transplantation offers a greater tolerance to HLA mismatches compared to adult-derived stem cell transplants (i.e., bone marrow or peripheral blood stem cells). Indeed, 4/6 or 5/8 HLA-matched CB transplantations are regularly performed for patients lacking a matched unrelated donor. Unfortunately, most banked CB units contain a stem cell dose that is too small to treat adult patients, resulting in only 4% to 5% of available CB units offering an adequate cell dose for prompt engraftment for adult patients. Ex vivo stem cell expansion appears to be an attractive strategy to circumvent this cell dose issue, while also enabling the selection of better HLA-matched CB units. In this study, we retrospectively performed HLA matching simulations to assess how the minimal cell content requirements associated with UM171 CB expansion may improve usability of existing CB unit inventories and donor availability for patients of different races and ethnicities. We analyzed a dataset of 58,971 adults for whom a donor search was initiated through the National Marrow Donor Program Be The Match registry against 142,942 CB units from major U.S. public CB banks listed on the Be The Match registry. Our results show that by enabling selection of smaller CB units, UM171-expanded CB transplantation increases donor availability from 72% to 84% for all patients compared to single unmanipulated CB transplantation. Furthermore, the low cell dose criteria for UM171-expanded CB also increases donor availability compared to double CB transplantation, while enabling better HLA matching between donor and recipient. UM171 expanded CB appears particularly beneficial for racial and ethnic minority patients as CB availability increases from 53% to 78% for African Americans, from 66% to 85% for Hispanics, and from 68% to 84% for Asians and Pacific Islanders, compared to single unmanipulated CB transplantation. In addition, UM171 expansion dramatically improves usability of CB units currently in inventories, as only 4.3% and 0.6% of banked CBs have sufficient cell doses for a 70 kg and 100 kg patient, respectively. UM171 raises this proportion to 53.8% and 20.2%, respectively, making CB banks potentially more cost effective. In conclusion, UM171 expansion allows the use of smaller CB units while also improving access to transplantation for racial and ethnic minorities.

LRP-1 Matricellular Receptor Involvement in Triple Negative Breast Cancer Tumor Angiogenesis.

BACKGROUND: LRP-1 is a multifunctional scavenger receptor belonging to the LDLR family. Due to its capacity to control pericellular levels of various growth factors and proteases, LRP-1 plays a crucial role in membrane proteome dynamics, which appears decisive for tumor progression. METHODS: LRP-1 involvement in a TNBC model was assessed using an RNA interference strategy in MDA-MB-231 cells. In vivo, tumorigenic and angiogenic effects of LRP-1-repressed cells were evaluated using an orthotopic xenograft model and two angiogenic assays (Matrigel® plugs, CAM). DCE-MRI, FMT, and IHC were used to complete a tumor longitudinal follow-up and obtain morphological and functional vascular information. In vitro, HUVECs' angiogenic potential was evaluated using a tumor secretome, subjected to a proteomic analysis to highlight LRP-1-dependant signaling pathways. RESULTS: LRP-1 repression in MDA-MB-231 tumors led to a 60% growth delay because of, inter alia, morphological and functional vascular differences, confirmed by angiogenic models. In vitro, the LRP-1-repressed cells secretome restrained HUVECs' angiogenic capabilities. A proteomics analysis revealed that LRP-1 supports tumor growth and angiogenesis by regulating TGF-ß signaling and plasminogen/plasmin system. CONCLUSIONS: LRP-1, by its wide spectrum of interactions, emerges as an important matricellular player in the control of cancer-signaling events such as angiogenesis, by supporting tumor vascular morphology and functionality.

Transcriptomic study in women with trisomy 21 identifies a possible role of the GTPases of the immunity-associated proteins (GIMAP) in the protection of breast cancer.

BACKGROUND: People with trisomy 21 (T21) are predisposed to developing hematological tumors, but have significantly lower-than-expected age-adjusted incidence rates of having a solid tumor. MATERIAL AND METHODS: To identify novel genetic factors implicated in the lower breast cancer (BC) frequency observed in women with T21 than in the general population, we compared the transcriptome pattern of women with a homogeneous T21, aged more than 30 years, with or without BC, and tumoral BC tissue of control women with a normal karyotype from the study of Varley et al. (2014). RESULTS: Differential analysis of gene expression between the 15 women in the T21 without BC group and BC patients in the other groups (two women with T21 and fifteen control women, respectively) revealed 154 differentially expressed genes, of which 63 were found to have similar expression profile (up- or downregulated). Of those 63 genes, four were in the same family, namely GIMAP4, GIMAP6, GIMAP7 and GIMAP8, and were strongly upregulated in the T21 without BC group compared to the other groups. A significant decrease in mRNA levels of these genes in BC tissues compared to non-tumor breast tissues was also noted. CONCLUSION: We found that the expression of some GIMAPs is significantly higher in women with T21 without BC than in patients with sporadic BC. Our findings support the hypothesis that GIMAPs may play a tumor-suppressive role against BC, and open the possibility that they may also have the same role for other solid tumors in T21 patients. The search for new prognostic factors and hopefully new therapeutic or preventive strategies against BC are discussed.

Major multilevel molecular divergence between THP-1 cells from different biorepositories.

The THP-1 cell line is broadly used as a model for acute myeloid leukemia (AML) with MLL fusion and to study monocyte differentiation and function. We studied THP-1 cells obtained from two major biorepositories. The two cell lines were closely related with a percentage match of short tandem repeat (STR) profiles ranging from 93.75% to 100%, depending on the algorithm used. Nevertheless, we found that the two cell lines presented discordant HLA type, cytogenetic aberrations and AML-related gene expression (including critical targets of MLL fusion). These discrepancies resulted mainly from loss of heterozygosity (LOH) involving five chromosomal regions. In view of their aberrant expression of key "leukemia" genes (e.g., LIN28B, MEIS1 and SPARC), we argue that one of the THP-1 cell lines may not be a reliable model for studying leukemia. Their defective expression of HLA molecules and abnormal adhesion properties is also a caveat for studies of antigen presentation. In a more general perspective, our findings show that seemingly minor discrepancies in STR profiles among cell lines may be the sign of major genetic drift, of sufficient magnitude to affect the reliability of cell line-based research.

Quantitative characterization of single-cell adhesion properties by atomic force microscopy using protein-functionalized microbeads.

A method was developed to characterize the adhesion properties of single cells by using protein-functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin-functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM-based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.

A gentle approach to investigate the influence of LRP-1 silencing on the migratory behavior of breast cancer cells by atomic force microscopy and dynamic cell studies.

The aim of the study was to get more insight into the role of LRP-1 in the mechanism of tumor progression in triple negative breast cancer. Atomic force microscopy, videomicroscopy, confocal microscopy and Rho-GTPAse activity assay were used on MDA-MB-231 and LRP-1-silenced cells. Silencing of LRP-1 in MDA-MB-231 cells was shown to led to a dramatic increase in the Young's modulus in parallel to a spectacular drop in membrane extension dynamics as well as a decrease in the cells migration abilities on both collagen I and fibronectin substrates. These results were perfectly correlated to a corresponding change in cell morphology and spreading capacity as well as in Rho-GTPases activity. By a multi-technique approach, it was demonstrated that LRP-1 played a crucial role in the migration of MDA-MB-231 cells by modulating the membrane extension dynamic. The originality of this AFM investigation lies in the non-invasive aspect of the measurements.

Gynecologic follow up of 129 women on dialysis and after kidney transplantation: a retrospective cohort study.

OBJECTIVE: To describe the gynecologic issues and follow-up in our referral center of women on dialysis and after kidney transplantation. STUDY DESIGN: This retrospective cohort study included 129 dialysed women among whom 102 had had transplants. Data on menstrual pattern, pregnancies, contraception, and cervical cytology were retrieved from patients' files. RESULTS: The follow-up started at age 41.6±14.2 years and lasted for 9.5±10.2 years. Of the women, 78.7% had regular menses before dialysis, decreasing to 30.6% on dialysis (p<0.001), when 43.1% were amenorrheic (p<0.001). After transplantation, more patients had regular menstruation and fewer were amenorrheic (respectively 57.1% and 23.1%, p<0.001). On dialysis and after transplantation, 25% and 30.5% of patients suffered from metrorrhagia (compared to 17.1% before, p<0.01). Concerning pregnancies, rates of spontaneous abortions (33.3%, p=0.01), intrauterine growth retardation (28.5%, p<0.001) and prematurity (23.8%, p=0.008) were significantly higher after transplantation than before dialysis. Prescriptions for the combined contraceptive pill and intrauterine device decreased whereas chlormadinone acetate was widely used: it treated metrorrhagia and relieved mastodynia in 80% and 12% of the cases. Smear tests showed more inflammation (33% vs 0.8%, p<0.05), condylomas (13.6% vs 3.1%, p=0.005) and intraepithelial neoplasias (12.6% vs 2.3%, p=0.003) among patients after renal graft than before dialysis. CONCLUSION: Women on dialysis and after kidney transplantation suffered more from irregular menses and metrorrhagia which was improved by chlormadinone acetate. We noted high rates of obstetrical complications and abnormal smear tests. Consequently, this population must have close follow-up to identify and treat gynecologic issues.

Leukemic stem cell persistence in chronic myeloid leukemia patients with sustained undetectable molecular residual disease.

Sustained undetectable molecular residual disease (UMRD) is obtained in a minority of patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors. It remains unclear whether these patients are definitively cured of their leukemia or whether leukemic stem cells (LSCs) persist in their BM. We have evaluated the presence of BCR-ABL-expressing marrow LSCs in 6 patients with chronic myeloid leukemia with sustained UMRD induced by IFN-α (n = 3), imatinib mesylate after IFN-α failure (n = 2), and dasatinib after imatinib intolerance (n = 1). Purified CD34(+) cells were used for clonogenic and long-term culture-initiating cell assays performed on classic or HOXB4-expressing MS-5 feeders. Using this strategy, we identified BCR-ABL-expressing LSCs in all patients. Interestingly, long-term culture-initiating cell assays with MS-5/HOXB4 stromal feeders increased detected numbers of LSCs in 3 patients. The relation between LSC persistency and a potential risk of disease relapse for patients with durable UMRD (on or off tyrosine kinase inhibitor therapy) warrants further investigation.

Two host factors regulate persistence of H7-specific T cells injected in tumor-bearing mice.

BACKGROUND: Injection of CD8 T cells primed against immunodominant minor histocompatibility antigens (MiHA) such as H7(a) can eradicate leukemia and solid tumors. To understand why MiHA-targeted T cells have such a potent antitumor effect it is essential to evaluate their in vivo behavior. In the present work, we therefore addressed two specific questions: what is the proliferative dynamics of H7(a)-specifc T cells in tumors, and do H7(a)-specific T cells persist long-term after adoptive transfer? METHODOLOGY/PRINCIPAL FINDINGS: By day 3 after adoptive transfer, we observed a selective infiltration of melanomas by anti-H7(a) T cells. Over the next five days, anti-H7(a) T cells expanded massively in the tumor but not in the spleen. Thus, by day 8 after injection, anti-H7(a) T cells in the tumor had undergone more cell divisions than those in the spleen. These data strongly suggest that anti-H7(a) T cells proliferate preferentially and extensively in the tumors. We also found that two host factors regulated long-term persistence of anti-H7(a) memory T cells: thymic function and expression of H7(a) by host cells. On day 100, anti-H7(a) memory T cells were abundant in euthymic H7(a)-negative (B10.H7(b)) mice, present in low numbers in thymectomized H7(a)-positive (B10) hosts, and undetectable in euthymic H7(a)-positive recipients. CONCLUSIONS/SIGNIFICANCE: Although in general the tumor environment is not propitious to T-cell invasion and expansion, the present work shows that this limitation may be overcome by adoptive transfer of primed CD8 T cells targeted to an immunodominant MiHA (here H7(a)). At least in some cases, prolonged persistence of adoptively transferred T cells may be valuable for prevention of late cancer relapse in adoptive hosts. Our findings therefore suggest that it may be advantageous to target MiHAs with a restricted tissue distribution in order to promote persistence of memory T cells and thereby minimize the risk of cancer recurrence.

The effect of covalent cross-links between the membrane components of microcapsules on the dissemination of encapsulated malignant cells.

Stem cells and immortalized cells have considerable therapeutic potential but present risks of malignant transformation. Cell microencapsulation allows transplantation without immunosuppression. We have developed a method for microencapsulating living cells within covalently cross-linked membranes that are chemically and mechanically extremely resistant. We provide herein direct evidence that these microcapsules can prevent malignant cell dissemination. When 20,000 or more nonencapsulated EL-4 thymoma cells were implanted intraperitoneally in mice, all recipients died with widespread metastasis within 26.3+/-1.0 days. All recipients of 250,000 EL-4 cells microencapsulated in covalently cross-linked membranes were living and disease-free, 150 days post-implantation. Encapsulation in standard microcapsules only slightly delayed the recipient death. Pancreatic islets transplanted using either type of microcapsule presented similar survival. We conclude that microencapsulation in covalently cross-linked membranes prevents malignant cell dissemination.

T cells targeted against a single minor histocompatibility antigen can cure solid tumors.

T cells responsive to minor histocompatibility (H) antigens are extremely effective in curing leukemia but it remains unknown whether they can eradicate solid tumors. We report that injection of CD8(+) T cells primed against the immunodominant H7(a) minor H antigen can cure established melanomas in mice. Tumor rejection was initiated by preferential extravasation at the tumor site of interferon (IFN)-gamma-producing H7(a)-specific T cells. Intratumoral release of IFN-gamma had two crucial effects: inhibition of tumor angiogenesis and upregulation of major histocompatibility complex (MHC) class I expression on tumor cells. Despite ubiquitous expression of H7(a), dissemination of a few H7(a)-specific T cells in extralymphoid organs caused neither graft-versus-host disease (GVHD) nor vitiligo because host nonhematopoietic cells were protected by their low expression of MHC class I. Our preclinical model yields unique insights into how minor H antigen-based immunotherapy could be used to treat human solid tumors.

Tissue distribution of target antigen has a decisive influence on the outcome of adoptive cancer immunotherapy.

Adoptive transfer of allogeneic T cells has unmatched efficacy to eradicate leukemic cells. We therefore sought to evaluate in kinetic terms interactions between T cells and allogeneic leukemic cells. T cells primed against the model B6(dom1) minor histocompatibility antigen were adoptively transferred in irradiated B10 (B6(dom1)-positive) and congenic B10.H7(b) (B6(dom1)-negative) recipients, some of which were also injected with EL4 leukemia/lymphoma cells (B6(dom1)-positive). A key finding was that the tissue distribution of the target epitope dramatically influenced the outcome of adoptive cancer immunotherapy. Widespread expression of B6(dom1) in B10 recipients induced apoptosis and dysfunction of antigen-specific T cells. Furthermore, in leukemic B10 and B10.H7(b) hosts, a massive accumulation of effector/memory B6(dom1)-specific T cells was detected in the bone marrow, the main site of EL4 cell growth. The accumulation of effector/memory cells in recipient bone marrow was EL4 dependent, and its kinetics was different from that observed in recipient spleen. We conclude that strategies must be devised to prevent apoptosis of adoptively transferred T cells confronted with a high antigen load and that local monitoring of the immune response at the site of tumor growth may be mandatory for a meaningful assessment of the efficacy of adoptive immunotherapy.