Simona Pagliuca

Côme Bommier

David Michonneau

Veronique Meignin

Maud Salmona

Marie Robin

Pedro Henrique Prata

Aliénor Xhaard

Flore Sicre De Fontbrune

Linda Feghoul

Nathalie Dhedin

Régis Peffault De Latour

Sophie Caillat-Zucman

Jérôme Le Goff

Gérard Socié

MD G Socie

Epstein-Barr Virus-Associated Post-Transplantation Lymphoproliferative Disease in Patients Who Received Anti-CD20 after Hematopoietic Stem Cell Transplantation

Keywords: PTLD, EBV infection, anti CD20 therapy, HSCT

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Although there has been an improvement of supportive strategies of infectious complications in allogeneic stem cell transplantation (HSCT), Epstein-Barr virus (EBV)-related diseases remain a persistent issue associated with significant morbidity and mortality rate. [START_REF] Arnaout | Complications of allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Tomblyn | Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective[END_REF][START_REF] Styczynski | Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines[END_REF] Post-transplant lymphoproliferative disease (PTLD) represents the most fearsome complication associated with EBV infection in patients undergoing HSCT. The overall incidence of EBV-related PTLD varies among transplant centers: ranging between 1-14%, depending on donor type, intensity of conditioning regimen, use of T-cell depletion, and stem cell sources. [START_REF] Styczynski | Response to Rituximab-Based Therapy and Risk Factor Analysis in Epstein Barr Virus-Related Lymphoproliferative Disorder After Hematopoietic Stem Cell Transplant in Children and Adults: A Study From the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation[END_REF] Before the era of pre-emptive strategies based on anti-CD20 targeting, the incidence of early and late EBV-related PTLD was very high. [START_REF] Curtis | Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study[END_REF] The European Conference on Infections in Leukemia (ECIL) recently published guidelines for prevention and treatment of EBV-related diseases after HSCT, including PTLD. [START_REF] Styczynski | Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines[END_REF] Assessment of pre-transplant EBV serology status and prospective monitoring of EBV-DNAemia by quantitative polymerase chain reaction (PCR) are highly recommended after high-risk allogeneic transplantations. [START_REF] Styczynski | Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines[END_REF] Significant (or high-load) DNAemia is defined as EBV >10,000 copies/mL of plasma or whole blood. [START_REF] Styczynski | Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines[END_REF] Persisting DNAemia can be associated with EBV-driven manifestations, such as EBV-driven organ disease or EBV-PTLD. Accepted histopathological criteria of EBV-PTLD are: the presence of lymphoid infiltrates, with or without disruption of underlying cellular architecture and evidence of EBV infection in many cells by in-situ hybridization (EBER-ISH). [START_REF] Jaffe | Iatrogenic Immunodeficiency-Associated Lymphoproliferative Disorders[END_REF] The World Health Organization (WHO) classification recognizes four types of morphological lesions: polyclonal early lesions (non-destructive lymphoid hyperplasia), polymorphic, monomorphic (B-cell or T/NK-cell) and classical Hodgkin lymphoma-type PTLD. [START_REF] Swerdlow | The 2016 revision of the World Health Organization classification of lymphoid neoplasms[END_REF] Three main therapeutic approaches have been proposed for the prevention and treatment of EBV-PTLD: withdrawal of immunosuppression, 8 administration of rituximab [START_REF] García-Cadenas | Impact of Epstein Barr virus-related complications after high-risk allo-SCT in the era of pre-emptive rituximab[END_REF][START_REF] Styczynski | Response to Rituximab-Based Therapy and Risk Factor Analysis in Epstein Barr Virus-Related Lymphoproliferative Disorder After Hematopoietic Stem Cell Transplant in Children and Adults: A Study From the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation[END_REF][START_REF] Van Der Velden | Reduced PTLD-related mortality in patients experiencing EBV infection following allo-SCT after the introduction of a protocol incorporating pre-emptive rituximab[END_REF][START_REF] Ahmad | Preemptive management of Epstein-Barr virus reactivation after hematopoietic stem-cell transplantation[END_REF][START_REF] Worth | Pre-emptive rituximab based on viraemia and T cell reconstitution: a highly effective strategy for the prevention of Epstein-Barr virusassociated lymphoproliferative disease following stem cell transplantation[END_REF] and use of EBV-specific cytotoxic T lymphocytes (CTLs). [START_REF] Heslop | Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients[END_REF] Rituximab therapy seems to be the most effective strategy in the pre-emptive (decreasing the risk of PTLD transformation) and treatment settings. CTLs are not accessible to most transplant centers and the reduction of immunosuppression alone has shown limited efficacy and is not always possible in the context of graft-versus-host disease (GVHD). Therefore, prompt tapering of immunosuppression when possible in combination with pre-emptive rituximab treatment has been the mainstay in patients with EBV infection. [START_REF] Styczynski | Response to Rituximab-Based Therapy and Risk Factor Analysis in Epstein Barr Virus-Related Lymphoproliferative Disorder After Hematopoietic Stem Cell Transplant in Children and Adults: A Study From the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation[END_REF] New approaches to monitoring and pre-emptive treatment of EBV-DNAemia have reduced the incidence and improved outcomes of these patients. [START_REF] Dierickx | How I treat posttransplant lymphoproliferative disorders[END_REF][START_REF] Cesaro | A prospective study on modulation of immunosuppression for Epstein-Barr virus reactivation in pediatric patients who underwent unrelated hematopoietic stem-cell transplantation[END_REF][START_REF] García-Cadenas | Impact of Epstein Barr virus-related complications after high-risk allo-SCT in the era of pre-emptive rituximab[END_REF] However, the mortality rate associated with this complication remains largely unsatisfactory [START_REF] Styczynski | Response to Rituximab-Based Therapy and Risk Factor Analysis in Epstein Barr Virus-Related Lymphoproliferative Disorder After Hematopoietic Stem Cell Transplant in Children and Adults: A Study From the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation[END_REF] and risk factors of EBV-PTLD transformation in patients already diagnosed with EBV-DNAemia and receiving preemptive rituximab remain poorly defined.

In this single center retrospective study, we analyzed the incidence, outcomes of EBV infection and risk factors associated with PTLD transformation despite the use of preemptive rituximab in patients after allogeneic HSCT.

PATIENTS AND METHODS

Patient population

We retrospectively analyzed all consecutive patients receiving at last 1 course of preemptive rituximab treatment for significant EBV DNAemia after HSCT in our center between 2010 and 2017.

The review of medical records was approved by the institutional ethical committee in agreement with the Helsinki Declaration of 1975, revised in 2008. [START_REF] Food | Human subject protection; foreign clinical studies not conducted under an investigational new drug application. Final rule[END_REF] Data collection was based on the European Society of Blood and Marrow Transplantation (EBMT) registry via the central data processing system "Project Manager Internet Server" (ProMISe). All patients had been regularly followed until April 2018 (or death). Pertinent clinical data including age, gender, disease diagnosis, comorbidities, type of transplant, human leukocyte antigen (HLA) matching, conditioning regimen, GVHD prophylaxis, acute and chronic GVHD, and other clinical complications were collected. Additional biological and clinical data such as EBV DNA viral load in whole blood, inflammatory markers, symptoms at onset of EBV infection and PTLD transformation, histopathological characteristics, phenotypic characterization of lymphocyte subsets at the onset of EBV infection, as well as number of courses of rituximab, and further treatment of PTLD were also retrospectively collected. Objective clinical data, including ancillary testing, laboratory results, and medical complications, and medication profiles, were abstracted through standardized chart review after each visit.

Transplant procedures

HLA-typing using high-resolution methods were used to select donors for allogeneic HSCT. Donor types included matched related donor (MRD), matched (MUD) and mismatched unrelated donor (MMUD), and haploidentical donors. Stem cell sources were peripheral blood (PB), bone marrow (BM), and cord blood (CB). Myeloablative conditioning regimens (MAC) included busulfan (3.2 mg/kg/day for 4 days) combined with cyclophosphamide (60 mg/kg/day for 2 days) [START_REF] Angelucci | Allogeneic bone marrow transplantation for hematological malignancies following therapy with high doses of busulphan and cyclophosphamide[END_REF] or with fludarabine (30 mg/m2/day for 5 days) or total body irradiation (TBI) of 1200 cGy combined with cyclophosphamide (60 mg/kg/day for 2 days) [START_REF] Fière | Adult acute lymphoblastic leukemia: a multicentric randomized trial testing bone marrow transplantation as postremission therapy. The French Group on Therapy for Adult Acute Lymphoblastic Leukemia[END_REF] , or busulfan (3.2 mg/kg/day for 2-3 days) combined with Thiotepa (5 mg/kg/day for 2 days) and fludarabine (40 mg/m 2 /day for 4 days). [START_REF] Eder | Thiotepa-based versus total body irradiation-based myeloablative conditioning prior to allogeneic stem cell transplantation for acute myeloid leukaemia in first complete remission: a retrospective analysis from the Acute Leukemia Working Party of the Euro[END_REF] Reduced-intensity regimens (RIC) included Fludarabine-based protocols, according to the disease and age of the recipient. Standard protocols of immunosuppression including cyclosporine and short-term methotrexate or cyclosporine with mycophenolic acid were used for GvHD prophylaxis. In addition, recipients of unrelated donor transplants received rabbit anti-thymocyte globulin or anti-lymphocyte globulin. [START_REF] Storek | Rabbit Anti-T Cell Globulin in Allogeneic Hematopoietic Cell Transplantation[END_REF]

EBV monitoring and EBV-PTLD diagnosis

Patients' EBV viral load was routinely monitored twice weekly by real-time quantitative PCR, as previously described [START_REF] Salmona | Automated quantification of Epstein-Barr Virus in whole blood of hematopoietic stem cell transplant patients using the Abbott m2000 system[END_REF] until infection or EBV disease resolution and according to patients' risk profile. [START_REF] Styczynski | Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines[END_REF] Of notice, a change in EBV load determination took place during the study period to standardize molecular quantification of EBV to requirements of the WHO Expert Committee on Biological Standardization (Abbott RealTime EBV assay, Abbott Molecular Inc., Des Plaines, IL, USA). A conversion factor was applied to convert results expressed in copies/milliliters (ml) to international units (IU)/ml. [START_REF] Salmona | Automated quantification of Epstein-Barr Virus in whole blood of hematopoietic stem cell transplant patients using the Abbott m2000 system[END_REF] EBV infection (or reactivation) was defined as any positive detection of EBV DNA in blood. Significant EBV-DNAemia was characterized by an increase of EBV load above the threshold of 4.5 log10 copies/ml or 3.8 log10 IU/ml. For standardization, after conversion, all results were expressed in log10 IU/ml.

To rule out that patients with EBV-reactivation did not already had developed PTLD, a careful clinical examination, laboratory assessment (including a complete hematological and biochemical profile, a serum immunoglobulin quantification and a lymphocyte immunophenotyping) and CT-scan were performed.

Together with significant EBV DNAemia, proven EBV-PTLD diagnosis was based on radiological and histological findings according to the 2018 revision of the WHO classification of lymphoid neoplasms. [START_REF] Swerdlow | The 2016 revision of the World Health Organization classification of lymphoid neoplasms[END_REF] EBV-encoded RNA in situ hybridization (EBER-ISH) was performed in all cases to confirm the presence of EBV infection. Diagnosis of probable EBV-PTLD was established when the EBV-DNAemia was associated with lymphadenopathy, hepatosplenomegaly or any other organ involvement on computed tomography (CT) findings, without tissue biopsy and in the absence of other documented cause.

Pre-emptive therapy and treatment of PTLD

Rituximab was administered pre-emptively to all patients with significant EBV DNAemia at a dose of 375 mg/m2 weekly until EBV load fell below 2.5 log10 IU/ml. Patients with rituximab-refractory EBV-DNAemia and therefore developing PTLD, received either an intensification of rituximab treatment (1000 mg total dose weekly until resolution of EBV-DNAemia and of clinical signs) associated with reduction of immunosuppression whenever feasible, or further systemic treatment such as cyclophosphamide, etoposide, doxorubicin, brentuximab or donor cellular products (donor lymphocytes or CD34+-selected stem cell boost infusions).

Statistical analysis

Data are presented as counts and percentage or median and range or inter-quartile range (IQR). Kaplan-Meier estimates [START_REF] Kaplan | Nonparametric Estimation from Incomplete Observations[END_REF] were used to determine the unadjusted probability of overall survival (OS), through 2-year post-transplant, with differences between the curves determined using log-rank tests, for univariate comparisons. OS was defined as the time from transplantation to death for any cause. In the case of a non-event, observations were censored at the time of last follow-up. Cumulative incidence functions (CIF) of acute and chronic GVHD, relapse and PTLD were calculated in a competing risk setting, where death was considered the competing event. [START_REF] Gray | A Class of K-Sample Tests for Comparing the Cumulative Incidence of a Competing Risk[END_REF][START_REF] Ruan | Analyses of cumulative incidence functions via non-parametric multiple imputation[END_REF] Predictive analyses for PTLD were based on the proportional hazard model for subdistribution of competing risk. A set of independent predictors was constructed with a stepwise backward procedure. All predictors with a P-value below 0.10 were considered and sequentially removed if the P-value in the multivariable model was above 0.05. Lymphocyte subgroups were analyzed as continuous and categorized variables, with cut-offs at the 50th percentile of the sample distribution (i.e. two equal size groups). Fisher's exact test was used for categorical variables and Kruskal-Wallis test for continuous variables. Groups were compared with the Mann-Whitney U non-parametric method or with Student's t-test. All tests were two-sided and P-values <0.05 were considered significant.

Statistical analyses were performed using R version 3.5.1.

RESULTS

Baseline characteristics

Among 1024 allogeneic HSCT performed at Saint Louis Hospital in Paris between 2010 and 2017, 208 patients presented a significant EBV DNAemia> 3.8 log10 IU/ml in plasma following allogeneic HSCT. Patient characteristics and features of EBV infection are described in table 1 and 2, respectively.

The median age of the 208 patients with EBV reactivation was 42.52 years (8.35-74.77), and the median follow-up for surviving patients was 47.33 (3.18-126.20) months. Diagnosis of EBV infection was made within a median of 35 days (range 6-713) after HSCT. Transplant indication was mainly acute leukemia (51%), while only 9% of the patients underwent HSCT for lymphoma. Conditioning regimens were more frequently of reduced intensity (57%) and fludarabine-based (57%). Most patients received an anti-T-cell serotherapy (80%) for GVHD prophylaxis. The majority of patients were transplanted with a donor matched for gender, ABO group, and cytomegalovirus (CMV) and EBV serology. Main stem cell source was PB (81%), whereas BM and CB were used respectively in 17% and 2% of cases.

EBV-PTLD cumulative incidence and subgroup description

Thirteen patients developed an EBV-related PTLD despite a pre-emptive anti-B cell therapy with rituximab. Calculated cumulative incidence was 6.3% (95% Confident Interval (CI) 3.5-10.1%, Figure 1). Table 4 summarizes characteristics of patients diagnosed with a PTLD. The median age of patients with PTLD was 40.51 years (17.4-74.7), and their median follow up was 37.85 (24.85-52.59) months.

Patients were transplanted mainly from a non-relative donor (seven from a Mismatched, five from a matched unrelated donor) and only one patient received a graft from an HLAidentical sibling donor. Chimerism analysis showed a donor reconstitution in ten patients, a mixed chimerism in two cases and an autologous reconstitution in one patient. Seven patients had acute GVHD and nine were receiving a corticosteroid treatment at PTLD onset. An extra-nodal involvement was seen in ten patients with central nervous system involvement in two cases. At PTLD presentation, four patients (30%) had fever >38.5°Celsius, five (38%) had lymphadenopathy. Elevated lactate dehydrogenase levels (LDH >1.5x the upper limit of normal [ULN, 480 U/L]) and elevated ferritin (>3000 mg/L) serum levels, thrombocytopenia (<100x10 9 platelets/L), anemia (hemoglobin < 10 g/dl), lymphopenia (<0.5 x10 9 /L), but not neutropenia, were frequent at disease onset. The median number of rituximab injection was five (2-11). EBV-DNAemia became negative within one month in 5 out of 13 patients. PTLD diagnosis was histologically proven in ten patients. Biopsies showed a monomorphic PTLD in six cases and a polymorphic subtype in four patients. Immunohistochemistry analysis showed CD20 positivity in all patients with available histological samples. In three patients, the diagnosis was highly probable basing on CTfindings and tumor cytology. Patients developed PTLD within a median of 85 days after transplant (27-140) and 34 days (9-105) after EBV reactivation. Rituximab was initiated after a median of 14 days (3-32) after the onset of EBV infection. Median number or rituximab courses before PTLD transformation was 2 (1-4). In all patients developing a PTLD, immunosuppressive drugs were drastically withdrawn. In three patients the intensification of rituximab treatment, together with a reduction of immunosuppression, allowed achieving a complete response (CR) of PTLD. Five patients received other treatments including etoposide, cyclophosphamide, doxorubicin, intrathecal methotrexate, and brentuximab. In the other five patients, no further treatment was possible because of poor performance status. Donor lymphocyte infusion was performed in one case and one patient received a CD34-selected stem cell boost in the context of poor graft function. Overall response rate of all treatments for PTLD was 46%: six patients experienced a complete remission, but two of them died because of other complications (one for interstitial pneumonia and one for invasive aspergillosis). Overall, nine patients with PTLD diagnosis died for a mortality rate of 69%.

OS and cumulative incidence of relapse and of acute and chronic GVHD

Two-year OS of the entire cohort was 62.8 (95%CI [56.4-69.9]) (Figure 2 A). CIF of relapse from the original disease was 12.7% (95%CI [8.6-17.7]) at one year and 16.5 (95%CI [11.7-22.0]) at 2 years. The CIF of acute grade II-IV GVHD at day +100 was 59.9 (95%CI [52.9-66.2]) whereas that of grade III-IV acute GVHD was 25.1% (95%CI [19.4-31.2]). CIF of chronic GVHD was 34.7 (95%CI [28.2-41.2]) at 1 year and 41.4 (95%CI [34.5-48.2]) at 2 years (Figure 2-B, -C, andD).

Factors affecting overall survival in EBV infected transplanted patients

Univariable analysis showed several risk factors affecting OS of transplanted patients diagnosed with EBV infection: age>42 years (HR 2.69 [95%CI 1.69-4.28], p<0.001), diagnosis of myelodysplastic syndrome and myeloproliferative neoplasm (MDS and MPN) (HR 2.37 [95%CI 1.47-3.81], p<0.001), presence of grade III-IV acute GVHD (HR 2.42 [95%CI 1.56-3.77], p<0.001). Absence of T-depleted therapy, RIC or MAC without TBI regimens, EBV viral load clearance within 1 month, and EBV serological status other than donor positive/recipient positive (D+/R+) represented protective factors for survival. In a multivariable model, only age, EBV serological status and presence of grade III-IV GVHD influenced OS. EBV clearance was withdrawn from the model because of a violation of assumption of proportional hazard. (Table 3 and Figure 3).

Risk factor analysis of PTLD transformation

The univariable analysis identified 3 risk factors associated with PTLD incidence despite preemptive rituximab: donor/recipient sex mismatch (HR 1.559, p=0. 039), presence of HLADRB11:01 allele (HR 4.850, p=0.004), EBV viral load clearance after 1 rituximab course (H3 6.556, p=0.001) (table 4).

In multivariable model EBV clearance after 1 rituximab course was removed because of a violation of assumption of proportionality basing on the scaled Schoenfeld's residuals. [START_REF] Schoenfeld | Chi-Squared Goodness-of-Fit Tests for the Proportional Hazards Regression Model[END_REF] Significant risk factors associated with PTLD incidence in multivariable analysis were: HSCT from an unrelated donor (HR 2.11 [95%CI 1.00-4.45], p=0.05), recipient HLA DRB1*11:01 (HR 4.85 [95%CI 1.57-14.97], p=0.006), fever at onset of EBV infection (HR 6.12 [95%CI 1.74-21.58], p=0.005), and sex-mismatched HSCT (HR 4.69 [95%CI 1.35-16.22] p=0.015) (Figure 4).

A risk factor index was created and figure 5 illustrates the cumulative incidence of PTLD according to the number of risk factors of EBV-infected transplanted patients. At day +180 from transplant, incidence of PTLD was very low in case of 0-1 or 2 risk factors (1.9%, [95%CI 0.4-6.1%] and 4.6 [95%CI 1.5 -10.5] respectively) and very high in case of contemporary presence of 3-4 risk factors (43.8 [95%CI 18.7 -66.5]).

Analysis of lymphocyte subsets

Supplementary Figures 1 (a-e) and 2 show the distribution of CD4+, CD8+, B and NK cells and a heatmap of the absolute number of all the other lymphocytes subsets at the onset of EBV infection, in patients with resolved EBV infection and in patients developing PTLD. The absolute number of T-lymphocytes was significantly higher in patients resolving EBV, whereas NK cells were higher in patients developing PTLD (p=0.016 and p=0.049 respectively, not confirmed in multivariable analysis).

DISCUSSION

In the present study, we analyzed outcomes and risk factors associated with PTLD transformation in patients developing an EBV infection after HSCT despite pre-emptive administration of anti-CD20 therapy.

We were able to show that after pre-emptive rituximab, the cumulative incidence of PTLD after EBV infection was 6.3%, which is very close to previously reported incidence rates of PTLD. [START_REF] Uhlin | Risk factors for Epstein-Barr virus-related posttransplant lymphoproliferative disease after allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Sanz | EBV-associated post-transplant lymphoproliferative disorder after umbilical cord blood transplantation in adults with hematological diseases[END_REF] In multivariate analyses PTLD was associated with several risk factors: HSCT in unrelated and sex-mismatched context, recipient HLA allele DRB1*11:01 and fever at diagnosis of EBV infection. Presence of more than two risk factors was strongly associated with risk of developing PTLD (more than 40% at six months post-HSCT).

Sex-mismatched HSCT, especially HSCT of male recipients with female donors, is well known to be associated with a higher incidence of GVHD and inferior survival. [START_REF] Nakasone | Risks and benefits of sex-mismatched hematopoietic cell transplantation differ according to conditioning strategy[END_REF][START_REF] Randolph | Female donors contribute to a selective graft-versus-leukemia effect in male recipients of HLA-matched, related hematopoietic stem cell transplants[END_REF] Immune response against minor histocompatibility antigens encoded on the Y-chromosome of a male recipient (H-Y antigens) can be at the basis of this adverse effect. [START_REF] Popli | Clinical impact of H-Y alloimmunity[END_REF] H-Y antigens have been shown to elicit a coordinated B-cell and T-cell response. [START_REF] Zorn | Minor Histocompatibility Antigen DBY Elicits a Coordinated B and T Cell Response after Allogeneic Stem Cell Transplantation[END_REF] In our cohort a sex-mismatched transplant was a factor associated with a higher risk of developing PTLD.

In multivariable analysis HLA DRB1*11:01 allele was an independent predictive factor of EBV-PTLD. Supplementary figure 4 shows a dot chart including the allele frequency of more representative class I and class II HLA. DRB1*1101 was the most frequent HLA allele in PTLD group (found in heterozygosis in 6 of 13 patients, 46%) patients. Possible associations between certain HLA types and the risk of developing PTLD have been reported in other studies, especially in PTLD occurring in solid organ transplant recipients. [START_REF] Subklewe | Association of Human Leukocyte Antigen Haplotypes with Posttransplant Lymphoproliferative Disease After Solid Organ Transplantation[END_REF][START_REF] Pourfarziani | Associations of Human Leukocyte Antigen (HLA) haplotypes with risk of developing lymphoproliferative disorders after renal transplantation[END_REF][START_REF] Lustberg | Human Leukocyte Antigen Type and Posttransplant Lymphoproliferative Disorder[END_REF][START_REF] Reshef | Association of HLA Polymorphisms with Post-transplant Lymphoproliferative Disorder in Solid-Organ Transplant Recipients: Association of HLA Polymorphisms with PTLD[END_REF] An association has also been found between some HLA class II molecules and other EBV related malignancies. [START_REF] Rubicz | A Genome-Wide Integrative Genomic Study Localizes Genetic Factors Influencing Antibodies against Epstein-Barr Virus Nuclear Antigen 1 (EBNA-1)[END_REF] The most likely hypothesis is that HLA variants may affect the success of Tcell surveillance for EBV and thereby influence a transplant recipient's predisposition to PTLD. It has been shown that HLA-DR mediates interaction between EBV envelope glycoprotein gp42 and B cell participating to the viral envelope fusion necessary for virion entry in B cells. [START_REF] Molesworth | Epstein-Barr virus gH is essential for penetration of B cells but also plays a role in attachment of virus to epithelial cells[END_REF] The possibility that EBV might actively evade CD4+T lymphocytes through some HLA-DR polymorphisms, has been suggested in some in vitro systems in which lytically infected B cells shed significant amounts of a soluble form of gp42 that can bind to mature HLA class II at the cell surface, protecting those cells from CD4+ recognition. [START_REF] Ressing | Interference with T cell receptor-HLA-DR interactions by Epstein-Barr virus gp42 results in reduced T helper cell recognition[END_REF] Other proteins, playing a key role in reactivation and viral genome persistence (such as Zta and EBV nuclear antigens [EBNAs], also interact with HLA-DR to limit viral antigen presentation. [START_REF] Li | Down-Regulation of MHC Class II Expression through Inhibition of CIITA Transcription by Lytic Transactivator Zta during Epstein-Barr Virus Reactivation[END_REF][START_REF] Keating | The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules[END_REF] Epitopes selected according to HLA-DR polymorphisms may trigger different patterns of immune responses. [START_REF] Li | Down-Regulation of MHC Class II Expression through Inhibition of CIITA Transcription by Lytic Transactivator Zta during Epstein-Barr Virus Reactivation[END_REF] The best-characterized components of these mechanisms are specific cytotoxic T cells (CTL) directed against viral gene products of the latent state, which include EBNAs the 1 to 6 and the latent membrane proteins (LMP) 1 and 2. [START_REF] Bogedain | Specific cytotoxic T lymphocytes recognize the immediate-early transactivator Zta of Epstein-Barr virus[END_REF] It has been also reported that the lytic cycle of EBV correlates with the diminution of specific major histocompatibility complex (MHC) class I and class II molecules and vice versa the downregulation of surface MHC class I and II expression is maintained throughout the lytic cycle of EBV, with a significant effect on Ag presentation. [START_REF] Keating | The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules[END_REF][START_REF] Tschochner | Identifying Patient-Specific Epstein-Barr Nuclear Antigen-1 Genetic Variation and Potential Autoreactive Targets Relevant to Multiple Sclerosis Pathogenesis[END_REF] We can speculate that such mechanisms can be at the basis of HLA DRB1*11:01-associated risk of PTLD transformation after HSCT, but this obviously need confirmation from other studies.

Hyperthermia at the moment of EBV infection onset was the most clinically relevant risk factor associated with PTLD transformation (HR 6.12, p=0.005) in our cohort. This finding is not surprising if we consider fever as a clinical feature consistent with probable or proven EBV disease. [START_REF] Kinch | Long-term outcome of Epstein-Barr virus DNAemia and PTLD with the use of preemptive rituximab following allogeneic HSCT[END_REF] However, despite the presence of fever in 4 out of 13 patients further developing PTLD, none of those patients presented with clinical signs of lymphoproliferative disease at the beginning of EBV-DNAemia. In our study, fever at the onset of EBV-DNAemia also impacted on survival outcome in univariable and in multivariable analysis.

Unrelated donor, T-cell depletion, donor positive and recipient negative EBV serological status, RIC regimens, grade II-IV acute GVHD and splenectomy, represent several risk factors identified for EBV-related PTLD in general transplanted population. [START_REF] Styczynski | Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines[END_REF][START_REF] Uhlin | Risk factors for Epstein-Barr virus-related posttransplant lymphoproliferative disease after allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Styczynski | Outcome of treatment of Epstein-Barr virusrelated post-transplant lymphoproliferative disorder in hematopoietic stem cell recipients: a comprehensive review of reported cases[END_REF] . In our cohort of patients already infected with EBV and already receiving anti-CD20 pre-emptive treatment, T cell depletion, reduced intensity conditioning regimens and EBV serological status did not represent parameters predisposing evolution towards PTLD. Almost 80% of patients received a T-cell depleted treatment before transplant and all patients with PTLD had received ATG or ALG (thus it was impossible to include this factor in multivariable analysis). ATG may therefore be considered a risk factor for EBV DNAemia but not a risk factor for PTLD. Clearance of EBV-DNAemia after one month represents an independent factor impacting OS and PTLD incidence in the univariable analysis, but its consideration in the multivariable model violated the assumption of proportionality. [START_REF] Grambsch | Proportional hazards tests and diagnostics based on weighted residuals[END_REF] Indeed, PTLD transformation is per se strongly associated with EBV-DNAemia persistence, but statistically, the relative effect of this covariate on the hazard function seems to change over the time, probably due to the change in detection methods during the study period (see patients and methods).

Presence of a monoclonal immunoglobulin was not a risk factor of PTLD transformation in our study because it was also present frequently in patients with EBV-DNAemia solely.

Interestingly, immunohistochemistry analysis of available samples showed a CD-20 positivity in all patients diagnosed with PTLD. This is an important finding, which leads to the speculation that the resistance to rituximab treatment in our cohort is merely clinical and should be considered as a "breakthrough" mechanism, occurring with the tumor mass progression and EBV proliferation under immunosuppression, rather than a change in molecular pathways of infected and transformed B-cells. Undoubtedly, these results must be confirmed with further invitro or invivo studies aiming to elucidate the mechanisms of resistance to anti-CD20 therapy in this particular setting.

EBV-PTLD incidence in our study is quite low if we consider that analysis has been conducted on a potentially high-risk population. We can argue that this result depends on the preemptive rituximab therapy that is considered the best way to optimize outcomes in HSCT patients with significant EBV DNAemia. [START_REF] Styczynski | Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines[END_REF] The overall survival rate of patients not developing PTLD was nearly 70% at 3 years that is similar to a survival rate expected in an HSCT population. [START_REF] Gratwohl | Economics and Outcome After Hematopoietic Stem Cell Transplantation: A Retrospective Cohort Study[END_REF] Therefore we can assume that EBV infection after HSCT, when occurred in absence of risk factors of PTLD transformation, is not associated with poor outcomes. However, in our analysis, PTLD occurrence is still associated with a high mortality rate (close to 70%), even if the overall response rate of patients developing an EBV-PTLD was nearly 50%.

To date, no standard therapy has been accepted for rituximab-resistant EBV-PTLD. In the setting of rituximab failure, second-line therapy options include cellular therapy (DLI or CTLs) or rituximab associated with chemotherapy. Unselected DLI from an EBV-positive donor is employed to restore broad T-cell reactivity, including EBV-specific responses; however, this procedure can be associated with severe GVHD. CHOP-like [START_REF] Trappe | Sequential treatment with rituximab followed by CHOP chemotherapy in adult B-cell post-transplant lymphoproliferative disorder (PTLD): the prospective international multicentre phase 2 PTLD-1 trial[END_REF] or dose-adjusted-EPOCH-R [START_REF] Destefano | DA-EPOCH-R for post-transplant lymphoproliferative disorders[END_REF] regimens have been proposed especially in the context of solid-organ PTLD. However, in the setting of HSCT chemotherapy regimens are difficult to manage. Brentuximab has been reported as a possible agent for PTLD with evidence of CD30+ expression. [START_REF] Choi | T Cell PTLD Successfully Treated With Single-Agent Brentuximab Vedotin First-Line Therapy[END_REF][START_REF] Gandhi | Brentuximab Vedotin (BV) Plus Rituximab (R) As Frontline Therapy for Patients (Pts) with Epstein Barr Virus (EBV)+ and/or CD30+ Lymphoma: Phase I Results of an Ongoing Phase I-II Study[END_REF][START_REF] Jacobsen | Brentuximab vedotin demonstrates objective responses in a phase 2 study of relapsed/refractory DLBCL with variable CD30 expression[END_REF] In our series only 4 patients with PTLD are alive at last follow-up time: 1 after a chemotherapy regimen including etoposide and high dose of intrathecal methotrexate, 3 without receiving secondline chemotherapy, but with an intensification of rituximab treatment and drastic reduction of immunosuppressive therapy. Patients receiving other chemotherapy regimens (CHOPlike) died with persistent disease. Brentuximab was associated with chemotherapy in 2 cases without success. One patient received DLI, and one other received a CD34+ selected boost in a context of poor graft function. These patients experienced a complete remission, but they died some months later because of other infectious complications (Table 5). Our group already showed that pre-emptive rituximab is an important strategy to decrease risk of EBVrelated PTLD, however, the associated prolonged and profound B-cell deficiency is responsible for an increased bacterial infection and mortality risk. [START_REF] Petropoulou | Lymphoblastic Leukemia ; AML : Acute Myelogenous Leukemia ; MDS : Myelodysplastic syndrome ; MPN : Myeloproliferative neoplasms ; CLL: Chronic Lymphocytic Leukemia; BMF: Bone Marrow Failure; MAC: Myeloablative;conditioning; RIC: Reduced intensity conditioning ; TBI: Total body irradiation[END_REF] The retrospective nature and the limited number of PTLD cases are definitive drawbacks of this study and need further validation on larger cohorts. Despite these limitations, we believe that our results may be useful for a better management of this post-transplant complication and implementation of pre-emptive strategies (such as the escalation of rituximab doses or virus-specific T-cell products) for selected patients presenting with multiple risk factors.

Figure 1 :

 1 Figure 1:

Figure 3 :

 3 Figure 3:

Figure 1 : 2 :

 12 Figure1: Cumulative incidence of PTLD in transplanted patients with significant EBV-DNAemia receiving Rituximab pre-emptive therapy Figure2: Outcomes of patients receiving at least 1 course of rituximab for an significant EBV-DNAemia A) Overall survival B) Cumulative incidence of relapse C) Cumulative incidence of grade III-IV acute GVHD D) Cumulative incidence of extensive chronic GVHD. Figure3: Forest Plot Multivariable Analysis of factors influencing OS. Figure4: Forest Plot Multivariable Analysis Risk Factors of CIF of PTLD. Figure5: PTLD cumulative incidence in transplanted patients with significant EBV-DNAemia receiving rituximab pre-emptive therapy according to number of risk factors: 1) HSCT from an unrelated donor, 2) Recipient HLA DRB1*11:01, 3) Fever at diagnosis of EBV infection, 4) Sex-mismatched HSCT.

Figure 3 :

 3 Figure1: Cumulative incidence of PTLD in transplanted patients with significant EBV-DNAemia receiving Rituximab pre-emptive therapy Figure2: Outcomes of patients receiving at least 1 course of rituximab for an significant EBV-DNAemia A) Overall survival B) Cumulative incidence of relapse C) Cumulative incidence of grade III-IV acute GVHD D) Cumulative incidence of extensive chronic GVHD. Figure3: Forest Plot Multivariable Analysis of factors influencing OS. Figure4: Forest Plot Multivariable Analysis Risk Factors of CIF of PTLD. Figure5: PTLD cumulative incidence in transplanted patients with significant EBV-DNAemia receiving rituximab pre-emptive therapy according to number of risk factors: 1) HSCT from an unrelated donor, 2) Recipient HLA DRB1*11:01, 3) Fever at diagnosis of EBV infection, 4) Sex-mismatched HSCT.

Figure 4 :

 4 Figure1: Cumulative incidence of PTLD in transplanted patients with significant EBV-DNAemia receiving Rituximab pre-emptive therapy Figure2: Outcomes of patients receiving at least 1 course of rituximab for an significant EBV-DNAemia A) Overall survival B) Cumulative incidence of relapse C) Cumulative incidence of grade III-IV acute GVHD D) Cumulative incidence of extensive chronic GVHD. Figure3: Forest Plot Multivariable Analysis of factors influencing OS. Figure4: Forest Plot Multivariable Analysis Risk Factors of CIF of PTLD. Figure5: PTLD cumulative incidence in transplanted patients with significant EBV-DNAemia receiving rituximab pre-emptive therapy according to number of risk factors: 1) HSCT from an unrelated donor, 2) Recipient HLA DRB1*11:01, 3) Fever at diagnosis of EBV infection, 4) Sex-mismatched HSCT.

Figure 5 :

 5 Figure1: Cumulative incidence of PTLD in transplanted patients with significant EBV-DNAemia receiving Rituximab pre-emptive therapy Figure2: Outcomes of patients receiving at least 1 course of rituximab for an significant EBV-DNAemia A) Overall survival B) Cumulative incidence of relapse C) Cumulative incidence of grade III-IV acute GVHD D) Cumulative incidence of extensive chronic GVHD. Figure3: Forest Plot Multivariable Analysis of factors influencing OS. Figure4: Forest Plot Multivariable Analysis Risk Factors of CIF of PTLD. Figure5: PTLD cumulative incidence in transplanted patients with significant EBV-DNAemia receiving rituximab pre-emptive therapy according to number of risk factors: 1) HSCT from an unrelated donor, 2) Recipient HLA DRB1*11:01, 3) Fever at diagnosis of EBV infection, 4) Sex-mismatched HSCT.

Table 1 : Patient characteristics Frequency (%), median (range)

 1

		All patients	PTLD+	PTLD-
		208	13	195
	Age in years, median (range)	42.52 (8.35-74.77)	40.51 (17.49-74.77)	42.52 (8.35-69.13)
	Follow-up, months (range)	47,33(3.18-126.20)	37.85 (24.85-52.59)	47.80 (3.18-126.20)
	SEX
	Male	123 (58%)	5 (38%)	118 (60%)
	Female	84 (42%)	8 (62%)	76 (40%)
	Diagnosis
	AML	61 (30%)	1 (8%)	60 (31%)
	ALL	43 (21%)	4 (31%)	39 (20%)
	MPN; CML	32 (15%)	2 (14%)	30 (15%)
	MDS	20 (10%)	1 (8%)	19 (10%)
	Lymphoma and CLL	19 (9%)	1 (8%)	18 (9%)
	BMF	24 (11%)	4 (31%)	20 (10%)
	Hemoglobinopathy	5 (2%)		5 (3%)
	MM and Plasma Cell disorders	4 (2%)		4 (2%)
	Conditioning regimen
	MAC with TBI	46 (22%)	5 (38%)	41 (21%)
	MAC without TBI	44 (21%)	1 (8%)	43 (22%)
	RIC	118 (57%)	7 (54%)	111 (57%)
	Busulfan contained regimen	102 (49%)	3 (23%)	99 (50%)
	Fludarabine contained regimen	118 (57%)	7 (54%)	111 (57%)
	Cyclophosphamide contained	113 (54%)	10 (77%)	103 (53%)
	regimen
	Splenectomy before conditioning	7 (13%)	0	7 (14%)
	regimen
	T-cell depleting therapy
	Thymoglobuline	124 (60%)	10 (77%)	114 (59%)
	Lymphoglobuline	43 (20%)	3 (23%)	40 (20%)
	Alemtuzumab	3 (1%)	0	3 (2%)
	GVHD prophylaxis
	CSA-MTX	90 (43%)	6 (46%)	84 (43%)
	CSA-MMF	112 (54%)	7 (54%)	105 (54%)
	PTCy and CSA-MMF	6 (3%)		6 (3%)
	Type of donor
	MRD	48 (23%)	1 (8%)	47 (24%)
	MUD	59 (28%)	5 (38%)	54 (27%)
	MMUD	95 (46%)	7 (54%)	88 (45%)
	Haploidentical	6 (3%)		6 (3%)
	Stem cell source
	PB	169 (81%)	9 (70%)	160 (82%)
	BM	35 (17%)	2 (15%)	33 (17%)

Table 2 :

 2

	EBV Infection Characteristics
		All patients	PTLD+	PTLD-
	Time between transplant and EBV infection (days, range)	35 (6-713)	35 (18-118)	36 (6-713)
	EBV maximal viral load (PCR median log UI/ml, IQR1st and 3rd )	4.68 (4.31-5.144)	5.55 (4.98-6.05)	4.64 (4.30-5.08)
	EBV viral load before rituximab treatment (PCR median log UI/ml, IQR1st and 3rd ) 4.55 (4.19-5.05)		5.37 (4.95-6.05)	4.53 (4.17-5.02)
	Time between EBV infection and PTLD transformation (median, range)		34 (9-105)
	Time between transplant and PTLD transformation (days, range)		85 (27-140)
	Time between EBV infection and rituximab initiating (days, range)	14 (0-387)	14 (3-32)	14 (0-133)
	Time between rituximab initiating and PTLD transformation (days, range)		19 (2-73)
	EBV viral load after 1st course of rituximab (PCR median log, IQR1st and 3rd )	2.8 (0-4.17)	4.66 (3.95-5.10)	2.68 (0-2.42)
	Fever*	38 (18%)	4 (30%)	34 (17%)
	Adenopathy*	10 (5%)	5 (45%)	5 (2%)
	CRP (mg/L, median IQR1st and 3rd, normal level <2 mg/L)*	3.0 (2.0-12.0)	7 (2-22.5)	3 (2-10)
	LDH (U/L, median IQR1st and 3rd, normal range 240 -480 U/L)*	604 (399-702)	662 (452-819)	525 (400-688)
	Ferritine (μg/L, median IQR1st and 3rd, normal range 13-150 μg/L)*	2406 (1723-4953)	3092 (2390-5706)	2379 (1684-4930)
	Hemoglobin (g/dL, median IQR1st and 3rd, normal range 12 -16 g/dL))*	10 (9.2-11.1)	9.2 (8.7-10.3)	10 (9.3-11.1)
	Gammaglobulin (g/dL, median IQR1st and 3rd)*	5.40 (3.75-7.9)	5.40 (4.40-5.70)	5.40 (3.75-7.55)
	Presence of monoclonal gamma protein*	32 (15%)	3 (23%)	29 (14%)
	Lymphocytopenia (<0.5 x 10 9 /L)*	88 (50%)	7 ( 63%)	81 (44%)
	Neutropenia (<1.0 x 10 9 /L)*	17 (9%)	1 (9%)	16 (9%)
	Thrombocytopenia (<100 x 10 9 /L)*	115 (61%)	8 (72%)	107 (61%)
	Median number of rituximab injections	2 (1-11)	5 (2-11)	2 (1-6)
	Negative EBV viral load after 1 month of treatment (Nb of patients, %)	184 (88%)	5 (38%)	179 (91%)

  Number of events: 13; Global p-value (Log-Rank): 0.0014497; AIC 120.12; Concordance Index: 0.78 + + + + + + + ++ + + + + + ++ + ++ + + +++ + + + + + ++ +++ + + ++ +++ + + + + + +++ ++ ++ ++ + + + + + + +++ + + ++ + + + + + ++ ++ ++ ++ ++ +++ + + + +

	Figure 4: Figure 5
		Cumulative incidence of PTLD according risk factors
		Variable	Strata +	0-1 risk factor	+	2 risk factors	N +	Hazard ratio 3-4 risk factors	p
		Variable						N Hazard ratio	p
		1.00
		0.75
		Unrelated_donor				161	2.11 (1.00, 4.45)	0.051
	CI of PTLD	0.50		++		++			+	75 (1.64, 4.62)	<0.001
		Sex_mismatched_HSCT			161	4.69 (1.35, 16.22)	0.015
		0.25	++
			p < 0.0001						Reference
								+ +++ ++ + + +	++
		Presence_of_HLADRB1_1101_allele 0.00 0 360	161 720	0.26 (0.08, 0.83) 4.85 (1.57, 14.97) 1080	0.006 0.02
				Days after transplant
									2.48 (1.52, 4.05)	<0.001
		Fever_at_onset_of_EBV_infection	161	6.12 (1.74, 21.58)	0.005
									1.79 (1.04, 3.07)	0.04
									0.10.2 0.5 1 2 1 2 5 10 20
									29

Authorship and Disclosures

Conception and design: GS, SP, CB Data collection and assembly: CB, SP Data analysis and interpretation: SP Manuscript writing: SP, CB & GS Final approval of manuscript: All authors Conflict-of-interest disclosure:

The authors declare no competing financial interests. ABO


M Me ´tairie
email: marie_metairie@hotmail.fr

D Korb

C Morin

T Schmitz

O Sibony

E ´s

en cas de grossesse ge ´mellaire §

Keywords:

E ´valuer l'efficacite ´du cerclage indique ´sur des ante ´ce ´dents en cas de grossesses ge ´mellaires. Me´thode. -Il s'agit d'une e ´tude re ´trospective de cohorte de type expose ´es-non expose ´es, mene ´e entre 2002 et 2017. Les patientes incluses pre ´sentaient une grossesse ge ´mellaire avec au moins un ante ´ce ´dent de fausse couche tardive (FCT) ou d'accouchement pre ´mature ´(AP) avant 34 SA. Deux groupes e ´taient compare ´s : « cerclage pre ´ventif » et « absence de cerclage pre ´ventif ». Le crite `re de jugement principal e ´tait le taux de pre ´maturite ´avant 34 SA. Les caracte ´ristiques des femmes et de la grossesse ont e ´te de ´crites et la survenue de pre ´maturite ´avant 34 SA a e ´te ´compare ´e apre `s ajustement sur des facteurs de confusion. Re´sultats. -Parmi les 1972 patientes pre ´sentant une grossesse ge ´mellaire et ayant accouche ´a `Robert-Debre ´entre 2002 et 2017, 69 (3,5 %) femmes avaient au moins un ante ´ce ´dent de FCT ou d'AP avant 34 SA.

Parmi elles, 20 (29 %) patientes ont e ´te ´incluses dans le groupe « cerclage pre ´ventif » et 49 (71 %) dans le groupe « absence de cerclage pre ´ventif ». Les femmes du groupe « cerclage pre ´ventif » avaient des ante ´ce ´dents plus se ´ve `res. Le taux d'accouchements pre ´mature ´s avant 34 SA n'e ´tait pas diffe ´rent entre les deux groupes (45,0 % versus 44,9 %, p = 0,99, OR brut : 1,00 (0,35-2,83), OR ajuste ´: 1,06 (0,33-3,44)). Conclusion. -Le taux de pre ´maturite ´avant 34 SA en cas de grossesse ge ´mellaire est similaire en cas de re ´alisation ou non d'un cerclage pre ´ventif chez les femmes pre ´sentant des ante ´ce ´dents de FCT ou d'AP. L'absence d'efficacite ´du cerclage doit e ˆtre toutefois nuance ´e par le fait que les femmes du groupe cerclage avaient des ante ´ce ´dents obste ´tricaux plus se ´ve `res.

Objectives. -Evaluate the effectiveness of preventive cervical cerclage for twin pregnancy with obstetrical history. Methods. -Through this retrospective cohort study, subjects exposed between 2002 and 2017 were compared with unexposed ones. All patients who had twin pregnancy with at least one previous late pregnancy loss or prematurity before 34 SA were included. Two groups were compared: ''preventive cerclage'' versus ''no preventive cerclage''. The outcome was the prematurity before 34 gestation weeks (GW) rate.

Results. -Among 1972 twin pregnancies registered between 2002 and 2017, 69 (3.5%) patients with at least one previous late pregnancy loss or prematurity before 34 GW, were part of the study. There were 20 (29.0%) women in the group ''preventive cerclage'' and 49 (71.0%) women in the group ''no preventive cerclage''. Women in the ''preventive cerclage'' group had poorer obstetrical history. The rate of § Travail pre ´sente ´au congre `s de la SFMP a `Lyon le 19/10/2017. § § Travail pre ´sente ´au congre `s du CNGOF a `Lille le 06/12/2017.

Introduction

Le taux de grossesses ge ´mellaires a augmente ´ces dernie `res anne ´es. Il est estime ´a `1,8 % en France. Le taux de pre ´maturite ´en cas de grossesse ge ´mellaire est de 47,5 %, alors qu'il est de 6,0 % pour les singletons [START_REF]Enque ˆtes Nationales Pe ´rinatales[END_REF]. Par ailleurs, les ante ´ce ´dents de fausse couche tardive ou d'accouchement pre ´mature ´sont des facteurs de risque de pre ´maturite

´. Ainsi, les grossesses ge ´mellaires avec ante ´ce ´dent de fausse couche tardive et/ou d'accouchement pre ´mature ´sont a `haut risque d'accouchement pre ´mature ´, du fait de la pre ´sence de deux facteurs de risque majeurs [START_REF] Easter | Obstetric History and Likelihood of Preterm Birth of Twins[END_REF].

En cas de grossesse ge ´mellaire sans ante ´ce ´dents obste ´tricaux, plusieurs moyens de pre ´vention de la pre ´maturite ´ont e ´te ´e ´tudie

´, notamment le pessaire [START_REF] Goya | Cervical pessary to prevent preterm birth in women with twin gestation and sonographic short cervix: a multicenter randomized controlled trial (PECEP-Twins)[END_REF][START_REF] Nicolaides | Cervical pessary placement for prevention of preterm birth in unselected twin pregnancies: a randomized controlled trial[END_REF][START_REF] Berghella | Prevention of preterm birth with pessary in twins (PoPPT): a randomized controlled trial: prevention of preterm birth with pessary in twins[END_REF], la progeste ´rone [START_REF] Schuit | Effectiveness of progestogens to improve perinatal outcome in twin pregnancies: an individual participant data meta-analysis[END_REF][START_REF] Romero | Vaginal progesterone decreases preterm birth and neonatal morbidity and mortality in women with a twin gestation and a short cervix: an updated meta-analysis of individual patient data: Vaginal progesterone decreases preterm birth in twin gestation with short cervix[END_REF] ou la surveillance cervicale e ´choguide ´e avec cerclage e ´cho-indique ´ [START_REF] Gordon | Transvaginal cervical length scans to prevent prematurity in twins: a randomized controlled trial[END_REF][START_REF] Saccone | Cerclage for short cervix in twin pregnancies: systematic review and meta-analysis of randomized trials using individual patient-level data[END_REF][START_REF] Houlihan | Cervical cerclage for preterm birth prevention in twin gestation with short cervix: a retrospective cohort study[END_REF]. Ces derniers ont pour la plupart e ´te ´e ´tudie ´s dans une population de grossesses ge ´mellaires asymptomatiques avec de ´couverte e ´chographique de col court. Aucune e ´tude n'a mis en e ´vidence une efficacite ´supe ´rieure d'une de ces me ´thodes pour pre ´venir la pre ´maturite ´en cas de grossesse ge ´mellaire.

Tre `s peu d'e ´tudes se sont inte ´resse ´es aux moyens pre ´ventifs en cas de grossesse ge ´mellaire et d'ante ´ce ´dents obste ´tricaux. En cas de grossesse monofoetale avec ante ´ce ´dents obste ´tricaux, le cerclage semble e ˆtre efficace dans la pre ´vention de la pre ´maturite én cas ante ´ce ´dents re ´pe ´te ´s. Se pose la question d'appliquer les indications de cerclage des singletons aux grossesses ge ´mellaires. Une seule analyse ancienne datant de 1993 issue d'un essai randomise ´a e ´tudie ´en sous-groupe le cerclage pre ´ventif des grossesses ge ´mellaires avec ante ´ce ´dent de fausse couche tardive et/ou d'accouchement pre ´mature ´ [START_REF] Party On Cervical Cerclage | Final report of the Medical Research Council/Royal College of Obstetricians and Gynaecologists Multicentre Randomised Trial of Cervical Cerclage[END_REF]. Elle n'a pas montre ´de diffe ´rence significative sur le taux d'accouchement avant 34 SA. Cependant, cette e ´tude avait des limites, notamment un tre `s faible nombre de cas de grossesses ge ´mellaires.

L'objectif de cette e ´tude e ´tait d'e ´valuer l'efficacite ´du cerclage pre ´ventif indique ´sur des ante ´ce ´dents obste ´tricaux de fausse couche tardive ou d'accouchement pre ´mature ´en cas de grossesse ge ´mellaire.

Me ´thodes

Il s'agissait d'une e ´tude re ´trospective de cohorte hospitalie `re de type expose ´es-non expose ´es.

Toutes les patientes pre ´sentant une grossesse ge ´mellaire ayant accouche ´, dans une maternite ´universitaire de type 3, entre 2002 et 2017 et ayant au moins un ante ´ce ´dent de fausse couche tardive et/ ou d'accouchement pre ´mature ´spontane ´avant 34 SA ont e ´te íncluses (n = 113) (Fig. 1). Ont e ´te ´exclues les patientes ayant des ante ´ce ´dents de pre ´maturite ´induite (n = 44). Deux groupes d'e ´tude ont e ´te ´de ´finis : les patientes ayant e ´te ´cercle ´es pre ´ventivement (n = 20) et les patientes n'ayant pas e ´te ´cercle ´es pre ´ventivement (n = 49). Les patientes qui n'e ´taient pas cercle ´es n'ont pas eu de surveillance particulie `re, notamment pas de surveillance cervicale e ´chographique syste ´matique.

La de ´cision du cerclage relevait d'une discussion colle ´giale en staff, en tenant compte des ante ´ce ´dents des patientes et de leur histoire obste ´tricale. Le cerclage e ´tait re ´alise ´selon la technique de Mac Donald. Dans notre centre, la pratique du cerclage re ´pond a des indications larges avec re ´alisation d'un cerclage pre ´ventif de `s un ante ´ce ´dent de fausse couche tardive ou d'accouchement pre ´mature ´spontane ´si l'histoire clinique est e ´vocatrice d'une be ´ance cervicale. Le cerclage est re ´alise ´apre `s l'e ´chographie du premier trimestre. Sa pratique est associe ´e a `du repos au lit non strict et a `un arre ˆt de travail mais aucun traitement comple ´mentaire n'est prescrit par ailleurs (comme la progeste ´rone). La suite de la prise en charge consiste en une surveillance clinique mensuelle associe ´e a `un pre ´le `vement vaginal et traitement antibiotique si pre ´sence de Gardnerella Vaginalis.

prematurity before 34 GW was not significantly different between these both groups (45.0% versus 44.9%; P = 0.99, crude OR: 1.00 (0.35-2.83), adjusted OR: 1.06 (0.33-3.44)).

Conclusions. -The prematurity rate before 34 GW, in twin pregnancies with a previous late pregnancy loss or preterm birth, is not different with or without preventive cervical cerclage.

C 2018 Elsevier Masson SAS. All rights reserved. Les donne ´es de cette e ´tude sont issues de la base de donne ´es informatise ´e du service, remplie prospectivement a `chaque consultation, a `chaque passage aux urgences, a `chaque hospitalisation et en salle de travail. L'informatisation de ces donne ´es e ´tait faite via le logiciel de la maternite ´par le professionnel de sante ŕesponsable de la patiente, sage-femme ou me ´decin. L'exactitude des donne ´es a e ´te ´ve ´rifie ´e lors du staff le lendemain de l'accouchement.

Les caracte ´ristiques maternelles e ´tudie ´es e ´taient l'a ˆge maternel, la parite ´, le tabagisme, l'origine ge ´ographique, la cate ´gorie socioprofessionnelle, l'indice de masse corporelle, et les ante ´ce ´dents d'hypertension arte ´rielle et de diabe `te. Les caracte ´ristiques obste ´tricales e ´tudie ´es e ´taient le type de procre ´ation et les ante ´ce ´dents de pathologie hypertensive gravidique, d'accouchement pre ´mature ´, de cerclage, de fausse couche tardive ainsi que le nombre d'accouchement voie basse et de ce ´sarienne ante ´rieur.

La distinction a e ´te ´faite entre les ante ´ce ´dents de pre ´maturite índuite et spontane ´e apre `s relecture de chaque dossier. Les ante ´ce ´dents d'accouchement pre ´mature ´ont e ´te ´classe ´s en fonction de leur nombre, de 0 a `! 3. Par ailleurs, les caracte ´ristiques de l'accouchement, notamment le mode d'entre ´e en travail, le taux de pre ´maturite ´induite versus spontane ´e, la voie d'accouchement et le taux d'he ´morragie du post-partum, ont e ´te ´analyse ´es.

Le crite `re de jugement principal e ´tait le taux d'accouchement pre ´mature ´avant 34 SA. Les crite `res de jugement secondaires e ´taient le taux d'accouchement pre ´mature ´avant 32 SA, avant 28 SA et avant 24 SA, ainsi que les issues ne ´onatales.

Des analyses univarie ´es ont e ´te ´re ´alise ´es pour l'ensemble des caracte ´ristiques maternelles, obste ´tricales et ne ´onatales avec un test du Chi 2 ou de Fischer. Des analyses multivarie ´es a `l'aide de mode `les de re ´gression logistiques ont e ´te ´re ´alise ´es en prenant en compte comme facteur de confusion le nombre d'ante ´ce ´dents de fausse couche tardive et d'accouchement pre ´mature ´. L'ensemble des analyses a e ´te ´effectue ´au moyen du logiciel STATA 13.

Re ´sultats

Sur les 1972 grossesses ge ´mellaires ayant accouche ´dans le service entre 2002 et 2017, 113 (5,7 %) pre ´sentaient au moins un ante ´ce ´dent de fausse couche tardive ou d'accouchement pre

´mature ´avant 34 SA. Apre `s exclusion de 44 (38,9 %) patientes pre ´sentant uniquement des ante ´ce ´dents de pre ´maturite ´induite, 69 (61,1 %) patientes ont e ´te ´incluses. Le groupe « cerclage pre ´ventif » e ´tait constitue ´de 20 femmes (29,0 %) et le groupe « absence de cerclage pre ´ventif » de 49 femmes (71,0 %) (Fig. 1).

Les deux groupes e ´taient comparables concernant les caracte ´ristiques maternelles (Tableau 1), hormis l'origine ge ´ographique.

Concernant les ante ´ce ´dents obste ´tricaux, les femmes du groupe « cerclage pre ´ventif » pre ´sentaient significativement plus d'ante

´ce ´dents de fausses couches tardives (80,0 % versus 53,1 %, p = 0,038). On notait de fac ¸on significative plus d'ante ´ce ´dents de cerclage dans le groupe « cerclage pre ´ventif » (20,0 % versus 2,0 %, p = 0,03) (Tableau 1).

Le terme me ´dian du cerclage pre ´ventif e ´tait de 15 SA et 4 jours. Le terme me ´dian du de ´cerclage e ´tait de 35 SA et 3 jours. Quatre (8,2 %) patientes du groupe « absence de cerclage pre ´ventif » ont e ´te ćercle ´es a `chaud du fait de modifications cervicales. Concernant les caracte ´ristiques de la grossesse, il n'y avait pas de diffe ´rence significative entre les deux groupes sur le plan de la chorionicite ´avec une majorite ´de grossesse bichoriale biamniotique. Le taux d'hospitalisation pour menace d'accouchement pre ´mature ´ne diffe ´rait pas entre les 2 groupes. Les femmes du groupe « cerclage pre ´ventif » pre ´sentaient plus de rupture pre ´mature ´e des membranes (25,0 % vs 8,2 %, p = 0,06). Il n'existait pas de diffe ´rence significative sur le recours a `la tocolyse, ou a `la corticothe ´rapie maturative (Tableau 2).

Concernant le crite `re de jugement principal, il n'existait pas de diffe ´rence significative entre les deux groupes (45,0 % versus 44,9 %, p = 0,99, OR brut : 1,00 [0,35-2,83]). Ces re ´sultats e ´taient inchange ´s apre `s ajustement sur le nombre d'ante ´ce ´dents de fausse couche tardive et d'accouchement pre ´mature ´(OR ajuste ´: 1,06 [0,33-3,44]).

Il n'existait pas de diffe ´rence significative concernant le caracte `re induit ou spontane ´de la pre ´maturite ´avant 34 SA dans les deux groupes (100 % de pre ´maturite ´spontane ´e dans le groupe « cerclage pre ´ventif » versus 81,8 % dans le groupe « absence de cerclage » p = 0,17). La re ´partition des termes d'accouchement ne diffe ´rait pas entre les deux groupes (Tableau 3).

Il n'existait pas non plus de diffe ´rence significative quant au de ´roulement du travail ou quant aux complications du postpartum (Tableau 4).

Il n'existait pas de diffe ´rence significative entre les deux groupes concernant les issues ne ´onatales, notamment pour le pH, l'APGAR < 4 a `5 minutes, la re ´animation ne ´onatale (Tableau 5).

Discussion

Dans cette e ´tude de cohorte re ´trospective de grossesses ge ´mellaires, il n'est pas mis en e ´vidence de diffe ´rence significative sur le taux de pre ´maturite ´avant 34 SA en cas de cerclage pre ´ventif indique ´sur des ante ´ce ´dents obste ´tricaux comparativement a l'absence de cerclage. Ce re ´sultat doit e ˆtre nuance ´par le fait que les femmes du groupe cerclage avaient des ante ´ce ´dents obste ´tricaux plus se ´ve `res que les femmes n'ayant pas eu de cerclage.

Le seul article publie ´concernant le cerclage pre ´ventif pour ante ´ce ´dents obste ´tricaux en cas de grossesses ge ´mellaires date de 1993. Il s'agissait d'une e ´tude en sous-groupe de 28 grossesses ge ´mellaires, dont [START_REF] Rafael | Cervical stitch (cerclage) for preventing preterm birth in multiple pregnancy. Cochrane Pregnancy and Childbirth Group[END_REF]

) Pe ´rine ´e complet 1 (6,2) 0 (0,0) Endome´trite du post-partum 0 (0,0) 0 (0,0) LA : liquide amniotique ; ARCF : anomalie du rythme cardiaque foetal. n'a pas permis de distinguer les ante ´ce ´dents obste ´tricaux des ante ´ce ´dents gyne ´cologiques [START_REF] Party On Cervical Cerclage | Final report of the Medical Research Council/Royal College of Obstetricians and Gynaecologists Multicentre Randomised Trial of Cervical Cerclage[END_REF].

Ces re ´sultats avaient e ´te ´repris dans une me ´ta-analyse de 2014 soulignant le manque de puissance compte tenu du faible nombre de patientes pre ´sentant des grossesses ge ´mellaires [START_REF] Rafael | Cervical stitch (cerclage) for preventing preterm birth in multiple pregnancy. Cochrane Pregnancy and Childbirth Group[END_REF].

En 2018, Han et al. ont publie ´une e ´tude de cohorte re ´trospective analysant l'efficacite ´du cerclage des grossesses ge ´mellaires, comparativement a `l'absence de cerclage, en cas d'ante ´ce ´dents obste ´tricaux, de col e ´chographiquement court ou de dilatation cervicale ! 1 cm. Parmi les 96 patientes cercle ´es, seulement 6 pre ´sentaient uniquement des ante ´ce ´dents obste

´tricaux. L'efficacite ´du cerclage indique ´uniquement sur ante

´ce ´dents obste ´tricaux n'a donc pas pu e ˆtre e ´tudie ´e [START_REF] Han | The impact of cerclage in twin pregnancies on preterm birth rate before 32 weeks[END_REF].

Ainsi, il n'existe, a `notre connaissance, aucune publication traitant du cerclage pre ´ventif sur ante ´ce ´dent obste ´trical seul en cas de grossesse ge ´mellaire, ne rendant pas possible la comparaison de nos re ´sultats avec la litte ´rature. En cas de grossesse ge ´mellaire, dans les autres indications possibles, l'efficacite ´du cerclage est discute ´e. En effet, il n'a pas e ´te ´montre ´de be ´ne ´fice en l'absence d'ante ´ce ´dent [START_REF] Seki | Prophylactic cervical cerclage for the prevention of early premature delivery in nulliparous women with twin pregnancies[END_REF][START_REF] Eskandar | Cervical cerclage for prevention of preterm birth in women with twin pregnancy[END_REF], ou en cas de cerclage e ´choguide ´ [START_REF] Rafael | Cervical stitch (cerclage) for preventing preterm birth in multiple pregnancy. Cochrane Pregnancy and Childbirth Group[END_REF][START_REF] Berghella | Cerclage for short cervix on ultrasonography: meta-analysis of trials using individual patient-level data[END_REF].

Concernant l'efficacite ´du cerclage a `chaud des grossesses ge ´mellaires, les donne ´es disponibles montraient une diminution significative du taux d'accouchement pre ´mature ´, quel que soit le terme, sans re ´duction de la mortalite ´pe ´rinatale. Ne ´anmoins, seules les femmes cercle ´es avaient rec ¸u des traitements concomitants par indome ´tacine ou par antibiotique, associe ´s a `une prolongation de la grossesse, responsable d'un biais majeur et a prendre en compte dans l'interpre ´tation des re ´sultats [START_REF] Roman | Cerclage in twin pregnancy with dilated cervix between 16 to 24 weeks of gestation: retrospective cohort study[END_REF]. Notre e ´tude pre ´sente plusieurs forces. Le nombre relativement important de patientes incluses pour la proble ´matique traite ´e, le caracte `re unicentrique ayant permis une homoge ´ne ´isation des pratiques et des prises en charge des femmes. On peut e ´galement souligner que toutes les donne ´es concernant les patientes ont e ´te ŕecueillies de manie `re prospective, permettant d'assurer une bonne validite ´des donne ´es.

De me ˆme, la distinction entre la pre ´maturite ´induite et spontane ´e, que cela soit au niveau des ante ´ce ´dents obste ´tricaux ou au cours de la grossesse e ´tudie ´e, a pu e ˆtre faite, ce qui n'ai quasiment jamais pre ´cise ´lors des e ´tudes des moyens de pre ´vention de la pre ´maturite ´en cas d'ante ´ce ´dents obste ´tricaux.

La principale limite de cette e ´tude est la pre ´sence d'un biais d'indication du cerclage, partiellement pris en compte par l'ajustement. L'indication du cerclage a sans doute e ´te ´porte ´e pour des ante ´ce ´dents plus graves : plus d'ante ´ce ´dents de cerclage a chaud lors des pre ´ce ´dentes grossesses, plus d'ante ´ce ´dents de fausses couches tardives. Par ailleurs, certaines patientes n'ont pas e ´te ´cercle ´es alors me ˆme qu'elles auraient e ´te ´candidates a `un cerclage du fait d'une prise en charge tardive dans notre centre. Ainsi l'absence de be ´ne ´fice du cerclage peut e ˆtre explique ´e par un niveau de risque initial plus e ´leve ´dans le groupe cerclage.

Une autre limite est que, malgre ´le nombre important de patientes incluses pour la proble ´matique traite ´e, cette e ´tude manque de puissance du fait des faibles effectifs de chaque groupe. Le design re ´trospectif et le fait que les populations ne soient pas comparables limitent l'interpre ´tation des re ´sultats.

Bien que cela soit probablement tre `s difficile a `mettre en oeuvre en pratique, un essai contro ˆle ´randomise ´prospectif serait inte ´ressant a `re ´aliser au sein d'une population plus large, afin de statuer sur l'inte ´re ˆt du cerclage dans la pre ´vention de la pre ´maturite ´en cas de grossesse ge ´mellaire avec ante ´ce ´dent obste ´trical.

Conclusion

Cette e ´tude met en e ´vidence, en cas de grossesse ge ´mellaire avec ante ´ce ´dents obste ´tricaux de fausse couche tardive ou de pre ´maturite ´, un taux d'accouchement pre ´mature ´similaire entre le groupe des femmes cercle ´es ayant plus d'ante ´ce ´dents de FCT et les femmes du groupe non cercle ´es. En attendant d'autres e ´tudes de me ´thodologie plus robuste sur le sujet, l'indication du cerclage dans ces situations doit e ˆtre e ´tudie ´e au cas par cas.

De ´claration de liens d'inte ´re ˆts

Les auteurs de ´clarent ne pas avoir de liens d'inte ´re ˆts.

Re ´fe ´rences

Fig. 1 .

 1 Fig. 1. Population d'e ´tude.