ALBERT

Description: A key feature when designing DNA targeting tools and especially nucleases is specificity. The ability to control and tune this important parameter represents an invaluable advance to the development of such molecular scissors. Here, we identified and characterized new non-conventional RVDs (ncRVDs) that possess novel intrinsic targeting specificity features. We further report a strategy to control TALEN targeting based on the exclusion capacities of ncRVDs (discrimination between different nucleotides). By implementing such ncRVDs, we demonstrated in living cells the possibility to efficiently promote TALEN-mediated processing of a target in the HBB locus and alleviate undesired off-site cleavage. We anticipate that this method can greatly benefit to designer nucleases, especially for therapeutic applications and synthetic biology. Scientific Reports 5 doi: 10.1038/srep08150

Description: The last few years have seen the increasing development of new DNA targeting molecular tools and strategies for precise genome editing. However, opportunities subsist to either improve or expand the current toolbox and further broaden the scope of possible biotechnological applications. Here we report the discovery and the characterization of BurrH, a new modular DNA binding protein from Burkholderia rhizoxinica that is composed of highly polymorphic DNA targeting modules. We also engineered this scaffold to create a new class of designer nucleases that can be used to efficiently induce in vivo targeted mutagenesis and targeted gene insertion at a desired locus. Scientific Reports 4 doi: 10.1038/srep03831

Description: Article Diatoms are photosynthetic microalgae with underutilized biotechnological potential. Here, the authors carry out targeted gene modifications of lipid metabolism genes in the diatom, Phaeodactylum tricornutum , resulting in a strain that exhibits a 45-fold increase in triacylglycerol accumulation. Nature Communications doi: 10.1038/ncomms4831 Authors: Fayza Daboussi, Sophie Leduc, Alan Maréchal, Gwendoline Dubois, Valérie Guyot, Christophe Perez-Michaut, Alberto Amato, Angela Falciatore, Alexandre Juillerat, Marine Beurdeley, Daniel F. Voytas, Laurent Cavarec, Philippe Duchateau

Description: Key Points Matching for MICA significantly reduces the incidence of acute and chronic GVHD in otherwise HLA 10/10-matched unrelated-donor HCT. Our results formally define MICA as a novel major histocompatibility complex-encoded human transplantation antigen.

Description: Hematopoietic-cell transplantation (HCT) from HLA-mismatched unrelated donors can cure life-threatening blood disorders but its success is limited by graft-versus-host disease (GVHD). HLA-B leaders encode methionine (M) or threonine (T) at position 2 and give rise to TT, MT or MM genotypes. The dimorphic HLA-B leader informs GVHD risk in HLA-B-mismatched HCT. If the leader influences outcome in other HLA-mismatched transplant settings, the success of HCT could be improved for future patients. We determined leader genotypes for 11872 patients transplanted between 1988 and 2016 from unrelated donors with one HLA-A -B,-C,-DRB1 or -DQB1 mismatch. Multivariate regression methods were used to evaluate risks associated with patient leader genotype according to the mismatched HLA locus and with HLA-A,-B,-C,-DRB1 or -DQB1 mismatching according to patient leader genotype. The impact of the patient leader genotype on acute GVHD and mortality varied across different mismatched HLA loci. Non-relapse mortality was higher among HLA-DQB1-mismatched MM patients compared to HLA-DQB1-mismatched TT patients (hazard ratio 1.35; P = .01). Grades III-IV GVHD risk was higher among HLA-DRB1-mismatched MM or MT patients compared to HLA-DRB1-mismatched TT patients (odds ratio 2.52 and 1.51, respectively). Patients tolerated a single HLA-DQB1 mismatch better than mismatches at other loci. Outcome after HLA-mismatched transplantation depends on the HLA-B leader dimorphism and the mismatched HLA locus. The patient's leader variant provides new information on the limits of HLA mismatching. The success of HLA-mismatched unrelated transplantation might be enhanced through the judicious selection of mismatched donors for a patient's given leader genotype.

Description: Background: Graft-versus-host disease (GVHD) is a major cause of mortality after unrelated hematopoietic stem cell transplantations (HSCT). Despite the development of modern immunosuppressive strategies, a nearly perfectly controlled compatibility of the classical HLA genes (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) and availability of numerous so-called minor histocompatibility antigens (e.g. HY or HA-1), its incidence remains largely unexplained to date. MIC genes (MHC class I chain-related) - a distinct lineage of MHC class I genes – are promising candidates to explain, at least partially, the incidence of GVHD in HLA-matched transplantations. MICA and MICB are highly polymorphic (100 alleles for MICA and 40 for MICB) and encode functional cell-surface glycoproteins up-regulated by cell stress. They interact with NKG2D, an activating receptor expressed on the surface of cytotoxic αβ CD8+ and γδ T lymphocytes and natural killer cells. MIC genes are already known to have a HLA-independent effect on solid graft outcomes and may play a similar role in HSCT by triggering GVHD. Objective: The objective of the present study was to determine the impact of donor/patient matching at the MICA and MICB loci on the incidence of GVHD in patients undergoing unrelated HSCT. Methods: We retrospectively analyzed a multicenter cohort of 1072 unrelated transplantations performed between 1996 and 2013. All donor-recipient pairs were fully typed at high resolution for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 and were matched for ten of ten HLA alleles (HLA 10/10 matched). High resolution genotyping of MICA and MICB was performed by sequenced-based typing in order to define matching grades between donors and patients. The endpoints of the study were acute and chronic GVHD. Apart from HLA-DPB1 matching, statistical models were adjusted for major clinical variables which have been shown to be associated with outcome (patient’s age, patient’s and donor’s sex, patient’s and donor’s serological status for cytomegalovirus, year of transplantation, time to transplantation, transplantation center, source of stem cells, conditioning regimen, GVHD prophylaxis, treatment with anti-thymocyte globulin, disease category and severity at transplantation). Results: Of the 1072 transplantations, 134 (12.5 %) and 380 (35.4 %) were mismatched at the MICA and MICB locus, respectively. Both MICA and MICB mismatches were significantly associated with an increased incidence of severe acute GVHD (grades III-IV) in univariate and multivariate models (multivariate model: HR = 2.32, 95 % CI = 1.84-2.92; p=0.0003 for MICA and HR = 1.49, 95 % CI = 1.24-1.79; p=0.03 for MICB). At day 100 post-HSCT severe acute GVHD incidences in mismatched vs. matched transplantations were 19.62 % vs. 15.08 % and 20.00 % vs. 14.84 % for MICA and MICB, respectively (Figure 1). Chronic GVHD was associated with MICA and MICB mismatches in univariate analysis (HR = 1.55, 95 % CI = 1.27-1.89; p=0.029 for MICA and HR=1.38, 95 % CI = 1.19-1.62; p=0.03 for MICB), but showed only a trend for association in multivariate models. Figure 1 Estimated cumulative incidence curves of grades III–IV acute GVHD according to MICA (panel A) and MICB (panel B) matching status. The solid and dashed lines represent MIC matched and mismatched grafts, respectively. The Fine and Gray model was used with relapse and death considered as competing risks. Figure 1. Estimated cumulative incidence curves of grades III–IV acute GVHD according to MICA (panel A) and MICB (panel B) matching status. The solid and dashed lines represent MIC matched and mismatched grafts, respectively. The Fine and Gray model was used with relapse and death considered as competing risks. Conclusion: To date this is the largest reported MICA and MICB sequence analysis whether in HSCT or solid organ transplantation. Inclusion of MICA and MICB typing in the donor selection process may be a practical clinical strategy for lowering the risks of severe acute GVHD after unrelated HSCT. Disclosures No relevant conflicts of interest to declare.

Description: Background: Matching for all alleles of the HLA-A, -B, -C, and -DRB1 loci (8/8 match) is associated with the highest overall survival (OS) rates after unrelated donor (URD) hematopoietic stem cell transplantation (HSCT). In Europe, patients (pts) are also matched at the HLA-DQB1 loci (10/10 match) with no overall evidence of improved OS. HLA-DPB1 mismatching has been associated with a higher risk of acute graft-versus-host disease (aGvHD) and a decreased risk of relapse. A detrimental role of additional HLA-DQB1, HLA-DPB1 and HLA-DRB3/4/5 mismatches (MM) has been recently identified on OS but only in 7/8 HLA-A, -B, -C, and -DRB1 loci URDs HSCT. We investigated the impact of HLA-DPB1 and HLA-DRB3/4/5 MM on outcomes in a large cohort of 10/10 matched URD HSCTs. Patients and methods: 2,393 pts who received an initial HSCT from a 10/10 matched URD in 35 French centers were included between January 2000 and October 2012. Informed consent was obtained in accordance with the Declaration of Helsinki. High-resolution typing was performed for HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 and -DRB3/4/5 loci for all donor/recipient pairs. Clinical data were obtained through ProMISe (Project Manager Internet Server), an internet-based system shared by all French transplantation centers. Impact of HLA-DRB3/4/5 and -DPB1 MM was quantitatively and qualitatively evaluated. For quantitative evaluation, patients were classified into 5 different groups according to their global matching level for the 7 considered loci (14/14, 13/14, 12/14, 11/14, 10/14). For qualitative evaluation, MM type and directionality (GvH and host versus graft (HvG) directions) were evaluated. HLA-DPB1 MM were classified as permissive or non-permissive (K Fleischhauer, Lancet Oncol. 2012). The primary composite endpoint for the analysis was GvHD-free and relapse-free survival (GRFS). We defined early GRFS as being alive at 3 months after HSCT with no previous grade III-IV aGvHD and no relapse and late GRFS as being alive 〉3 months with no previous grade III-IV aGvH, no relapse and no moderate or severe chronic GvHD (cGvHD). Late GRFS was evaluated at months 6, 12, 24 and 36 after HSCT. Acute GvHD, cGvHD, relapse and OS were also studied. Models were adjusted for HSCT period, disease risk, age, sex matching, stem cell source, and conditioning regimen. Results: Table 1showspopulation characteristics and distribution of cumulative MM. The median follow-up was 59 months. Compared to 14/14 pairs, quantitative evaluation showed a significantly lower early GRFS for pts who received a 10-11/14 (Hazard Ratio HR 2.0, 95% CI 1.4 to 2.9, p=0.0003) or a 12/14 URD HSCT (HR 1.4, 95% CI 1.1 to 1.8, p=0.01). This was related to a significantly increased risk of grade III-IV aGVHD associated with 10-11/14 (HR 2.3, 95% CI 1.5 to 3.7, p=0.0004) and 12/14 pairs (HR 1.7, 95% CI 1.2 to 2.4, p=0.003). 10-11/14 pairs were also associated with a higher risk of death at 3 months (HR 2, 95% CI 1.1 to 3.6, p=0.024). Qualitatively, in pts matched for HLA-DRB3/4/5 but MM for HLA-DPB1 (n=1846, 77.1%), 2 HLA-DPB1 MM and non-permissive HLA-DPB1 MM were associated with a lower early GRFS (HR 1.4, 95% CI 1.1 to 1.9, p=0.01 and HR 1.3, 95% CI 1.0 to 1.7, p=0.02 respectively) due to an increased risk of aGvHD (HR 1.7, 95% CI 1.2 to 2.5, p=0.002 and HR 1.50, 95% CI 1.08 to 2.08, p= 0.017 respectively). Outcomes were not influenced by either GvHD or HvG mismatch directions. Late GRFS analyses once adjusted were not significantly different according to MM numbers, type and directions. Only 19 pairs (0.8%) were DPB1 matched and DRB3/4/5 mismatched (high linkage disequilibrium between DRB3/4/5 and DRB1); HLA-DRB3/4/5 MM could thus not be qualitatively analyzed. Conclusion: MultipleHLA-DPB1 and HLA-DRB3/4/5 MM have an early impact after 10/10 matched URDs HSCT. The best outcomes are seen in 13 and 14/14 pairs. Early GRFS is significantly impacted by 10-11/14, 12/14, 2 DPB1 MM as well as non-permissive HLA-DPB1 MM URD HSCT which is related to an increased risk of grade III-IV aGvHD. There is a significantly increased risk of mortality for 10-11/14 pairs at 3 months. Prospective evaluation of matching for HLA-DPB1 and HLA-DRB3/4/5 is warranted to reduce early post-HSCT toxicity in donor-recipient 10/10 matched pairs. Disclosures Michallet: Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria. Peffault De Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

Description: Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) from unrelated donors has been increasingly used worldwide in the last decade in patients with hematological malignancies when HLA-identical sibling donors are unavailable. Identification of the HLA locus matching at the allele level is important in optimizing transplantation outcomes by minimizing non-relapse mortality (NRM) as well as in enhancing the graft-versus-leukemia effect. It has been demonstrated that patients with HSC donors matched on HLA-A, -B, -C, -DRB1, and -DQB1 alleles can have different outcomes if considering matching on HLA-DPB1 allele. HLA-DPB1 mismatches based on T-cell-epitope groups could identify mismatches that might be tolerated (permissive) and those that would negatively impact transplantation outcomes (non-permissive). We conducted this study to evaluate the impact of HLA matching degree between patient and HSC donor including HLA-DPB1, taking into account the other impacting variables in the allo-HSCT settings. Material and methods A total of 235 patients who received allo-HSCT at our center between January 2005 and December 2014 with a full donor/recipient HLA class I and II locus available data were included, 131 (56%) were males, the median age at allo-HSCT was 50 years (range: 18-69). There was 123 (53%) acute leukemia (93 AML, 30 ALL), 24 (10%) MDS, 35 (15%) multiple myeloma, 20 (8%) NHL, 7 (3%) Hodgkin's disease, 10 (4%) myeloproliferative neoplasms, 13 (6%) CML, and 3 (1%) CLL. One hundred and nineteen patients (51%) received myeloablative conditioning (MAC) and 116 (49%) received reduced intensity conditioning (RIC). Disease status at allo-HSCT was complete remission (CR) in 144 (61%) patients and less than CR in 91 (39%). HSC donor was 10/10 HLA matched unrelated (MUD) for 162 (69%) (80 PBSC and 93 BM), among them 21 (9%) were matched for HLA-DPB1, 41 (18%) had permissive mismatch and 100 (42%) had non-permissive mismatch; while 73 (31%) had 9/10 HLA mismatched donor MMUD (48 PBSC and 25 BM), among them, 7 (3%) were matched for HLA-DPB1, 12 (5%) had permissive mismatch and 54 (23%) had non-permissive mismatch; 110 (47%) were ABO compatible, 58 (24%) had minor incompatibility and 67 (29%) had major incompatibility. For sex mismatching, in 33 (14%) cases, it was female donor to a male patient. Results After a median follow-up for surviving patients of 29 months (range: 4-108), patients with 10/10 HLA MUD had better overall survival (OS) than those with 9/10 MMUD without considering the HLA-DPB1 matching, with 2 years OS probability of 51% vs 35% respectively (p=0.09), which was reflected by a lower NRM at 2 years of 29% vs 42% (p=0.07). When considering the HLA-DPB1 matching, we found comparable outcomes in terms of OS and NRM for: 1) 10/10 HLA MUD - DPB1 matched vs 10/10 HLA MUD - DPB1 permissive mismatched, 2) 10/10 HLA MUD - non-permissive DPB1 mismatched vs 9/10 HLA MMUD - DPB1 matched, 3) 9/10 HLA MMUD - DPB1 matched vs 9/10 HLA MMUD - DPB1 permissive mismatched; all these 3 groups were not significantly different between each other expect for a last group which included 9/10 HLA MMUD with non-permissive DPB1 mismatch, this group had worse OS and NRM compared to all others with 2 years rates of 34% vs 49% (p=0.05) and 47% vs 29% (p=0.04) respectively. In multivariate analysis, patient age (〉50 years), disease status (less than CR), HLA matching (9/10 HLA MUD non-permissive DPB1) and sex mismatching (female donor to male patient) were significantly impacting OS and NRM. We included all these variables in a risk score: age 〈 50 years= 0, 〉 50 years= 1; CR= 0, less than CR= 1; HLA 10/10 (matched on DPB1) or HLA 10/10 with permissive MM on DPB1= 0; HLA 10/10 with non-permissive MM on DPB1 or HLA 9/10 (matched on DPB1 or with permissive MM on DPB1)=1; HLA 9/10 with non-permissive MM on DPB1=2; for sex matching, female donor to male patient=1, all other= 0. The risk score distinguished low risk patients (total score=0), intermediate (total score=1 or 2) and high risk (total score 〉2) with 2 years OS and NRM rates of 66%, 52%, 30% (p=0.003) and 22%, 29% 48% (p=0.004) respectively. Conclusion MMUD with non-permissive T-cell-epitope mismatch at HLA-DPB1 should be avoided due to increased rates of NRM. The risk score combining HLA matching with age, disease status and sex matching is very helpful for daily clinical practice offering patients better treatment strategy. Figure 1. Figure 1. Disclosures Nicolini: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ariad Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 2371 Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) appears to offer a potential advantage in terms of event free survival (EFS) and overall survival (OS) in patients with acute leukemia (AL) with bad prognostic factors. The main issue is to find a donor; usually, a patient has 30% chance to find an HLA identical sibling donor (SD) and approximately 40% chance to find a suitable unrelated donor (UD) from international registries. Unfortunately, 40% of registered patients relapse or die before finding a donor. Aim: Our objective was to evaluate the outcome (OS & EFS) in AL patients, whether they had an HLA identical SD, an UD or no donor (ND) after registration on France Greffe de Moelle (FGM) registry, either transplanted later or not. Our secondary objective was to evaluate the impact of interval between, diagnosis and allo-HSCT, donor finding and allo-HSCT, interval registration-allo-HSCT, on OS and EFS. Methods: We have analyzed 251 AL patients diagnosed between January 2000 and December 2008, for whom a search for a donor was initiated for a required allo-HSCT. There were 117 (47%) males and 134 females with a median age at diagnosis of 40 years [16-66], 177 (71%) were AML (prognosis according to cytogenetics & molecular markers: 59 were good, 97 poor and 21 not done) 75 were ALL (prognosis according to cytogenetics & molecular markers: 33 were good, 16 poor and 26 not done). Seventy six (30%) patients had an available SD and received allo-HSCT within a median time of 3.5 months (0.5 – 43) (59 AML & 17 ALL), and 38 (15%) with SD but were not transplanted due to early relapse and/or death. For patients with no available SD, a registration on FGM registry was done. One hundred thirty seven patients were registered after a median interval of 2.3 months (0.4-135) from diagnosis, 33 (13%) patients (24 AML & 9 ALL) did not find any donor, have not been transplanted and they received the standard of care. One hundred and four (41%) patients (62 AML & 42ALL) found an UD or cord blood cell (CBC) unit after a median registration time of 1.6 months (0.3 – 26): 86 with UD of which only 60 have been transplanted within a median time of 2.3 months (0.4 – 14), 18 with CBU of which only 17 were transplanted. Among transplanted patients, 113 (74%) were in complete response (CR) at transplantation, and 40 were in less than CR. Fifty (33%) received peripheral blood (PBSC) (23 UD & 27 SD), 86 (57%) received bone marrow (BM) (37 UD & 49 SD) and 17 (10%) CBC units. For conditioning, 56 (37%) were reduced intensity (29 SD & 27 UD) and 96 standard (47SD & 50 UD). For HLA, there were 45 HLA 10/10, (27BM & 18 PBSC), 14 HLA 9/10 (9BM 5PBSC) 1 HLA 8/10 (BM) and for CBC 14 HLA 4/6 and 3 HLA 5/6. Results: After a median follow-up of 25 months (0.2- 234), the median OS was 78 months (51 – 133) for transplanted patients with SD (3years OS: 68%), it was 33 months (27 – 47) for transplanted patients with UD (3years OS: 44%), 21 months (15 – 37) for not transplanted patients with available SD or UD (3years OS: 34%) and finally it was 31 months (23-221) for patients with ND (3years OS: 45%). The median EFS for the same groups was 38 months (23 – 133), 24 months (17 – 36), 15 months (11-24) and 23 months (14 – 48) respectively. The only factors negatively affecting OS in multivariate analysis (studying age, sex, AL type, cytogenetics, donor or no donor, transplanted or not, UD or SD) were age and allo-transplanted from UD. When adjusting only on transplanted patients, taking into account in addition to previous factors, the time between diagnosis-registration, time between registration-allo-HSCT, disease status at allo-HSCT, stem cell source, conditioning; three significant factors affected OS: disease status (

Description: Introduction Since recently, HLA typing of CP-CML patients (pts) ≤65 years old was still recommended in at diagnosis and treated by Imatinib (IM) first-line, especially in case of baseline warnings (Baccarani et al. 2006/2009/2013), in the aim of performing allo-transplant if TKI failure occurs. Additionally, HLA molecules are processing BCR-ABL peptides at the surface of leukemic cells and trigger immune response, exploitable in autologous (Bocchia Lancet 2005) and allogeneic (Fefer NEJM 1979; Kolb Blood 1995) settings, including in residual disease conditions. The performance of antigen processing might differ according to the type of HLA molecules involved, but this remains largely debatable. Aims 1) To analyse the frequency of HLA subtypes in CP-CML pts as compared to those of the general population. 2) To investigate if IM first-line response differ according to the HLA subtypes, suggesting an involvement of the autologous immune system in the response observed. Methods We retrospectively analyzed a cohort of newly diagnosed CP-CML pts treated with IM first-line 400 mg alone and HLA typed (generic and/or allelic) in our center between years 2000 and2018. All pts were followed according to the ELN recommendations 2006/2010/2013. Clinical data were extracted from medical files, and responses were analysed with standard methods. Molecular results were ELN standardised since 2003, and were expressed as BCR-ABLIS in %. Overall survival (OS), progression-free survival (PFS), failure-free survival (FFS: progression to advanced phases death, loss of CHR, CCyR, or MMR, discontinuation of IM for toxicity, primary cytogenetic resistance) were analysed since IM initiation in intention-to-treat. Results Eighty-three pts have been included, with a median age of 47.5 (11-64.8) years at diagnosis, with 58% males and 42% females. Sokal score (n=80) was low in 44%, intermediate in 42% and high in 14%. ACA were present at diagnosis in 7% of pts (NA in 3), 2 pts had a cryptic Ph, and 2 a variant Ph. Major BCR transcripts were found in 96.4% pts, and atypical transcripts in 3.6%. We compared the frequencies of HLA A, B, C and DR in our 83 CP-CML to those of 5,225 unrelated bone marrow donors (UBMD) typed in the registry of the Rhône-Alpes region (35 million inhabitants). We could demonstrate that the HLA B13, B28, B51, B53 were significantly over-represented in the CP-CML population vs UBMD (4.24 vs 2.02%; 3.63 vs 2.48%, 11.5 vs 8.53%, 1.82 vs 0.88, overall p value = 0.007, Monte-Carlo test) as well as C05 (13.75 vs 8.39%, p=0.044) respectively. In addition, HLA B27, B45 and B55 were underrepresented in CP-CML pts vs UBMD (0.60 vs 3.43%, 0 vs 0.6%, 0.6 vs 1.66%, p=0.007, Monte-Carlo test). There was no difference for HLA A, and DR antigens. All pts got IM (Glivec®) 400 mg daily since diagnosis. The median follow-up after IM initiation was 111.5 (7.6-203) months. CHR was reached after a median of 1 (0-3.5) month,