Median MFI Research Articles

Treatment of De Novo DSA with IVIG Monotherapy After Lung Transplantation

<h3>Purpose</h3> The development of de novo donor-specific anti-HLA antibodies (DSA) is associated with worse outcomes in lung transplant recipients (LTxR). The purpose of this study is to evaluate IVIG monotherapy for de novo DSA in the absence of allograft dysfunction or clinical antibody-mediated rejection (AMR). <h3>Methods</h3> Adult LTxR between 2/2013 and 3/2021 treated with IVIG monotherapy for de novo DSA with no evidence of AMR were included. IVIG was initiated at 2g/kg followed by 1g/kg monthly for a minimum of 3 months or until clearance (up to 6 months). HLA Ab is routinely checked by Luminex SAB post-transplant at day 14, months 1, 3, 6, 9, 12, and 18, and monthly during IVIG treatment. Primary outcome was change in DSA after IVIG therapy. DSA clearance was defined as sum MFI less than 1000. <h3>Results</h3> 32 LTxR developed de novo DSA at a median of 33 days (18,143) post-transplant and were treated with IVIG. Mean follow-up time was 1330 days (861-1910). The median sum MFI for DSA at baseline was 4782 (2927,7490). DQ was the immunodominant DSA in 24 (75%) and only 2 (6%) patients had isolated class I DSA. MFI of sum DSA decreased by a median of 2993 (2051, 6358) after IVIG therapy; p < 0.0001. Eighteen (56%) cleared de novo DSA (Group 1) versus 14 (44%) did not clear (Group 2) at the end of IVIG therapy. There was no significant difference in age, gender, race, transplant indication or type, cPRA, HLA mismatch, or PGD. ACR and AMR tended to be more frequent in Group 2; ACR: 92.9% vs 61.1%, p=0.053; and AMR: 35.7% vs 5.6%, p=0.063. Overall survival for the entire cohort at 1, 3 and 5 years was 100%, 86%, 60%, respectively. There was no significant difference in chronic lung allograft dysfunction (CLAD) or survival between Groups (Table 1). <h3>Conclusion</h3> In LTxR, IVIG monotherapy is associated with significant reduction in de novo DSA. DSA clearance may result in less acute rejection. Randomized controlled trials are needed to determine the optimal strategy for managing de novo DSA.

Altered expressions of CXCR4 and CD26 on T-helper lymphocytes in hereditary hemorrhagic telangiectasia

BackgroundHereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease characterized by a deregulated neo-angiogenesis. Besides a mainly vascular phenotype (muco-cutaneous telangiectases, arteriovenous malformations), a specific risk of infection is suggested by case series of severe and atypical infections as well as by reports of decreased T and natural killer (NK) lymphocyte counts. As some evidence supports a dysregulation of the CXCR4/CXCL12 chemotactic axis of HHT endothelial cells, we hypothesized that a similar phenomenon could occur on lymphocytes.MethodsEighteen HHT patients with history of severe infection (HSI) were matched in age and sex with 18 HHT without HSI and 18 healthy control subjects (HC). We assessed the cell count and the surface expression of CXCR4 and CD26 (CXCL12 inactivating peptidase) of circulating T-helper and T-cytotoxic lymphocytes (including naive, memory and activated subsets) and NK cells.ResultsThe overall HHT group of 36 patients exhibited a reduction of circulating T-helper lymphocytes compared to HC (median: 517 vs. 1026 cells/mm3, p < 0.0001), correlated with age (r = − 0.46, p = 0.005), requirement of intravenous iron or blood transfusions (median: 291 vs. 627 cells/mm3, p = 0.03) and CXCR4 surface expression (r = 0.353, p = 0.0345). CXCR4 and CD26 membrane expression were both decreased on HHT T-helper lymphocytes (median MFI ratio: 4.49 vs. 5.74 for CXCR4 and 3.21 vs. 4.33 for CD26, p = 0.03 and 0.0018 respectively) with an unchanged CXCR4/CD26 ratio. The HHT group with HSI had a higher CXCR4/CD26 ratio on the total T-lymphocyte population, as well as on the T-helper population and its naive subset (median on naive T-helper cells: 2.34 vs. 1.32, p = 0.0002).ConclusionsOur findings support a dysregulation of the CXCL12/CXCR4 chemotaxis of T-helper lymphocytes in HHT patients, potentially linked to their T-helper lymphopenia and susceptibility to infection.

Potential Role of STAG1 Mutations in Genetic Predisposition to Childhood Hemato-Oncological Diseases

Background. Genetic predisposition has been recently assessed in about 5-10% of pediatric tumors, including leukemias. Among genetic syndromes associated with an increased risk of developing leukemia, a possible link has been proposed for Cohesinopathies, like Cornelia de Lange Syndrome (CdLS), caused by mutations in Cohesin family genes. Somatic mutation in these genes, known for their fundamental role in cell cycle and DNA repair mechanisms, are described in myeloid malignancies (10-20% of AML, 50% of DS-AMKL, 5-15% of MDS and 10% of MPN) and solid tumours. We previously described the first germline variant of NIPBL in a CdL case with acute lymphoblastic leukemia (ALL). The identification of germline variants can provide new insights in the pathogenesis of hemato-oncological diseases.Objectives. This study aims to define the association of germline variants of the Cohesin family genes with predisposition to pediatric hemato-oncological diseases, including ALL. Functional assays have been performed on a STAG1 unique variant.Methods. We set up an NGS capture-base DNA panel, including 40 genes associated with 8 pathways associated to ALL predisposition, including Cohesin genes. Patients' bone marrow (BM) diagnostic samples have been analyzed together with a germline tissue (BM at remission or buccal brush). Bioinformatic analysis has been carried out by Sophia DDM software (Variant Fraction >5%; Coverage at least 500X; Variant Allele Frequency 1%) and variants were annotated as certainly pathogenic, potentially pathogenic or VUS.A Lymphoblastoid Cell Line (LCL) was obtained from PB of a MDS patient, then used for functional studies.Genomic stability and X-ray induced DNA-damage repair were evaluated in the STAG1-mutated LCL by Sister Chromatids Exchange (SCE) assay, cell cycle analysis (Propidium Iodide) and pH2AX staining.Results. Overall, 117 consecutive pediatric ALL patients and sporadic cases with familial recurrency of cancer or other hematological disorders have been analyzed through the NGS panel. A total of 225 mutations were identified in ALL, distributed as it follows: 77 variants in Transcription factors, 68 in Signaling-associated genes, 56 variants in RAS pathway, 44 in chromatin modifier genes, 38 variants in B-cell differentiation genes, 30 in DNA repair genes and 16 variants in genes associated to predisposition. Overall, 12/117 unique variants (5%) were found in Cohesins family genes; 11/12 were germline, while a single somatic variant was found in STAG2 in the same position as a germline variant. In details, 3 mutations were found in BRD2 gene while 2 variants were identified in STAG1, STAG2 and NIPBL genes, respectively. Only 1 variant was detected in SMC1A, SMC3 and BRD2. No mutations were detected in HDAC8 and RAD21 genes.Interestingly, the Arg1167Gln variant in STAG1 is located in a highly conserved region involved in solid tumors. A STAG1 variant (Arg1187Gln) was identified in a pediatric MDS patient, which was then further characterized in the LCL. First, we evaluated the effects on chromosomal stability, and we observed a higher number of abnormal chromatids exchanges in mutated LCL (STAG1 patient mean = 4.8 exchanges/metaphase compared to four control LCLs mean = 3.05; p <0.0001), as shown in the attached figure. Differences in cell cycle phases between STAG1-mutated LCL and controls, even after X-ray irradiation (3Gy - 6Gy), are not significant. Mutated cells showed a significantly lower capability to repair DNA after an ionizing radiation. γH2AX phosphorylation status of STAG1 mutated LCL appears higher also in basal condition (T0: 1.7X, p <0.01; T24: 2.2X, p <0.0001; T48: 2.4X, p <0.0001; MFI STAG1 over MFI control LCLs) and it remains at higher levels at 48 hours after the irradiation (T48: 3.7X, p<0.001 [3Gy]; 4.1X, p<0.0001 [6Gy]; MFI STAG1 over MFI control LCLs). These data are confirmed by the ratio of the STAG1-mutated LCL median MFI over controls (T48/T0: 3.42 vs 0.92, p <0.0001 [3Gy]; T48/T0: 2.33 vs 0.64, p <0.01 [6Gy]).Conclusion. In pediatric hematological diseases, NGS screening showed several variants among Cohesin genes, with either a potentially pathogenic or unknown significance.Our study confirms a considerable effect of a STAG1 germline variant on genetic instability that leads to oncogenesis, opening new scenarios for Cohesins' contribution to genetic predisposition to leukemias. [Display omitted] DisclosuresBiondi: Colmmune: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Bluebird: Other: Advisory Board; Novartis: Honoraria.

Midostaurin Reduces Bite® Mediated Cytotoxicity Against Acute Myeloid Leukemia

T-cell recruiting antibody constructs represent a promising approach to recruit T-cells to tumor cells independent of TCR-specificity. So far, CD33 has been the most commonly targeted antigen in AML. However, its ubiquitous expression pattern within the healthy myeloid hematopoietic system remains a major challenge. To reduce on-target off-leukemia toxicity, alternative AML-associated targets with a more restricted expression pattern are sought after. We evaluated CD135 (FLT3 ; FSM-like tyrosine kinase 3) as a target antigen for T-cell recruiting antibody based immunotherapy in AML. In 159/192 of AML patient samples (83%; MFI ratio of >1.5) a significant expression level was detected by flow cytometry with no differences in expression level at time of initial diagnosis versus relapse (Initial diagnosis: n=161, median MFI ratio 3.0 vs relapse: n=18, median MFI ratio 3.5). Expression level could neither be correlated to FLT3 mutational status (median MFI ratio FLT3-ITD vs FLT3 wild type (WT): 3.7 vs 4.0, n=52 and 173 respectively) nor to FLT3 allelic ratio (median MFI ratio allelic ratio high vs low: 4.1 vs 4.5, n=13 and 19 respectively). Importantly, CD34+/CD38- leukemia initiating cells (LICs) also expressed CD135 at a similar level compared to AML bulk cells (Median MFI ratio LIC: 2.4 vs bulk: 2.6, p=0.1, n=81). Bone marrow progenitor cells (CD45DIM/SSCLOW) cells and CD34+/CD38- expressing hematopoietic precursors from healthy bone marrow expressed CD135 to a significantly lower level compared to AML bulk cells and LICs, respectively (median MFI Ratio: AML bulk vs healthy progenitor cells: 3.2 vs 2.1, p<0.0001; LICs vs HSCs: 3.1 vs 0.7, p=0.0003). Next, we evaluated a CD135xCD3 BiTE® antibody construct (FLT3 BiTE®) for its ability to mediate cytotoxicity against AML cell lines and primary AML cells. Target antigen expression level was relevant for FLT3 BiTE® mediated cytotoxicity as demonstrated by preferential lysis of CD135bright (MM6, MFI ratio 15) vs CD135dim (OCI-AML3, MFI ratio 1.5) AML cell lines. Cytotoxicity against primary AML cells was tested in our long-term culture system using either allogeneic or autologous T cells (Krupka et al, Blood 2014). In the allogeneic set-up, with a fixed E:T ratio of 1:5, the FLT3 BiTE® antibody construct mediated efficient elimination of primary AML cells within 9 days of culture (median % specific lysis 75.9, n=6). This was accompanied by a strong T-cell proliferation (fold change CD2+ T cells: 7.9, n=6). In the autologous system, the E:T ratio was not fixed and influenced by the number of residual T-cells in the respective AML sample. Despite low E:T ratios (1:12-1:74) the FLT3 BiTE® induced high cytotoxicity and T-cell proliferation against primary AML cells within 14 days of culture (median specific lysis: 34.4%; fold change CD2 T cells: 6.6, n=7). We further investigated the influence of Midostaurin on FLT3 BiTE® mediated cytotoxicity against AML cells. Midostaurin (1 µM) drastically decreased FLT3 BiTE® mediated cytotoxicity in 7/8 patient samples (median % cytotoxicity FLT3 BiTE® vs +Midostaurin: 59.0 vs 39.9, p=0.02). This corresponded to a significant decrease in BiTE®- as well as CD3/CD28-mediated T-cell proliferation (median % CD3/CD28 mediated T-cell proliferation FLT3 BiTE® vs +Midostaurin: 56.4 vs 1.46, n=5). It has been reported that Midostaurin inhibits Akt phosphorylation (Kawai, J Bio Sci 2015), a major factor in T-cell proliferation, which might explain the decrease in T-cell proliferation and cytotoxicity. In summary, CD135 was shown to be expressed in the majority of AML patients on AML bulk cells as well as LICs with a restricted expression pattern in the healthy hematopoietic compartment. The FLT3 BiTE® antibody construct mediated strong cytotoxicity against primary AML cells. This correlated to profound T-cell activation and proliferation. The combinatorial approach with Midostaurin abrogated BiTE® mediated cytotoxicity due to a decrease in T-cell proliferation. As T-cell recruiting antibody construct mediated cytotoxicity relies on T-cell function, our data do not support the combinatorial approach with the FLT3 -TKI Midostaurin for targeted therapy of AML. DisclosuresLindl:AMGEN Inc: Research Funding. Krupka:Amgen, Inc: Research Funding; Amgen Research (Munich): Research Funding. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership. Kischel:AMGEN: Employment. Kufer:AMGEN Research Munich: Employment. Subklewe:Amgen, Inc.: Research Funding; Amgen Research (Munich): Research Funding.

The impact of donor-specific anti-human leukocyte antigen antibodies in salvage haploidentical hematopoietic cell transplantation with posttransplant cyclophosphamide in patients with nonmalignant disorders.

The presence of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) has been recognized as a major risk factor for graft failure (GF) after haploidentical hematopoietic cell transplantation with posttransplant cyclophosphamide (haplo-PTCy). However, the role of DSAs in salvage haplo-PTCy for rescuing patients with nonmalignant disorders (NMDs) has not yet been reported. The present study retrospectively analyzed 22 patients with NMDs who underwent salvage haplo-PTCy from January 2008 to December 2017. The median age at the time of the rescue haplo-PTCy was 9 years (range, 1-26 years). Median time from the first transplant to second haplo-PTCy was 56 days (range, 37-591 days). Among all patients, six (27.3%) had DSAs, with a median DSA strength (mean fluorescence intensity [MFI]) of 5201 (range, 1412-11,543) in the first DSA testing. In addition, the median DSA MFI was 2672 (range, 832-10,498) before the bone marrow infusion. Overall, GF occurred in 5 (25%) of the 20 assessable patients. Three of four (75%) patients with DSAs experienced GF versus 2 of 16 (12.5%) DSA-negative patients (P=0.032). The median DSA MFI for patients with GF was 6437 (range, 1412-10,498) versus 1845 (range, 832-2672) for those who engrafted or had earlydeath (P=0.030). One-year event-free survival was significantly lower in DSA-positive patients than in those without DSAs (16.7% vs. 62.5%, P=0.002). DSA-negative patients had an acceptable 1-year survival of 62.5%. In conclusion, this study suggests that DSAs may be associated with deleterious outcomes after salvage haplo-PTCy in patients with NMDs.

P0739 / #1900: CD64, CD11A, AND CD18 LEUKOCYTES EXPRESSION IN A CASE SERIES OF CHILDREN WITH SARS-COV-2 MULTISYSTEM INFLAMMATORY SYNDROME AND KAWASAKI DISEASE: SIMILAR BUT NOT THE SAME

Aims & Objectives: A new paediatric inflammatory syndrome named as “pediatric multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS)” has been described. It has been compared to Kawasaki Disease. The aim of this report is adding to the PIMS-TS clinical and analytical description the application of immunophenotyping by flow cytometry (FC). We describe CD64, CD18, and CD11a leukocytes expression in three children with SARS-CoV2 infection and compare it with three cases of Kawasaki Disease. Methods: Three children were studied after informed consent obtained from their parents or legal guardians. Their blood samples were collected in sterile EDTA at room temperature or refrigerated at 4°C and analyzed within 24 hours. Expressions were measured in monocytes, neutrophils, and lymphocytes. At least 10 000 events were recorded for each sample. The intensity of CD64, CD18, and CD11a surface expression were measured as mean fluorescence intensity in arbitrary units (MFI). They were compared with three previous cases of KD. Results: The median CD64, CD18 and CD11a MFI expression in PIMS-TS versus KD cases are described in Figure 1. The CD64 and the CD11a expression on neutrophils and monocytes are higher. The CD11a in CD8 Lymphocytes is higher too.Conclusions: We compare for the first time the immunophenotype of children with PIMS-TS infection versus KD. We observed significant but higher upregulation of CD64, CD18, and CD11a expression. This response appears to be similar but different than in KD. Prospective studies with a higher number of cases should be conducted to confirm this observation.

Stand-Alone Middle Meningeal Artery Embolization for Treatment of Giant Chronic Subdural Hematoma in Elderly Frail Patients: A Case Series

ABSTRACT BACKGROUND Previous case series have demonstrated safety and efficacy of middle meningeal artery (MMA) embolization as both adjuvant and stand-alone treatment of chronic subdural hematoma (cSDH). The safety and efficacy of stand-alone MMA embolization for treatment of giant (&gt;150 ml) cSDH in specifically elderly, frail patients is not well studied. OBJECTIVE To evaluate the safety and efficacy of stand-alone MMA embolization for treatment of giant cSDH in elderly, frail patients in whom traditional surgical intervention was deemed high risk. METHODS We reviewed the records of consecutive patients referred for MMA embolization and identified patients ≥ 75 yr with modified Frailty Index (mFI) ≥ 2 who underwent stand-alone MMA embolization for cSDH treatment with at least one follow-up imaging study. RESULTS A total of 42 patients were referred for MMA embolization with 5 patients meeting inclusion age and frailty criteria. The average age was 82.2 ± 6.8 yr. The median mFI was 3.0 (interquartile range (IQR) 2-4). Four patients were either on aspirin or thrombocytopenic. The average initial cSDH volume was 187.8 ± 31 ml with average initial midline shift of 8.0 ± 2.1 mm. The average length of stay was 4 ± 1.1 d. The average cSDH volume decrease on follow-up imaging was 68.5% ± 11.9%. Follow-up average midline shift decreased by 70% to 2.4 ± 1.4 mm. There were no peri-procedural or in-hospital complications. CONCLUSION In frail, elderly patients with giant cSDH, stand-alone MMA embolization was effective in reducing cSDH volume and improving presenting symptoms without complications.

The Accumulating Deficits Model for Postoperative Mortality and Readmissions: Comparison of Four Methods Over Multiple Calendar Year Cohorts.

To assess 4 measures of the accumulating deficits model of frailty for postoperative mortality and readmissions including their stability over time. Frailty has been assessed by multiple methods. It is unclear whether variation in how frailty is measured is important and would be stable over time. Rockwood's 57-item frailty index was mapped onto 14,568 ICD9 diagnosis codes from Healthcare Cost and Utilization Project State Inpatient Database for the state of Florida (HCUP-SID-FL) for calendar years 2011 to 2015, inclusive, with 962 ICD9 codes matching onto 42 items. This became the modified frailty index (mFI) used. Three measures of the mFI were differentiated: the number of admission diagnoses, number of chronic conditions upon admission, and number of increased deficits accumulated during the admission. The Charlson Co-Morbidity Index was a fourth measure of frailty. The mFI of patients who survived or died and were readmitted or not were compared. Across all years, 4,796,006 patient observations were compared to the number of diagnoses matched on the 42 items of the mFI. The median mFI scores for each method was statistically significantly higher for patients who died compared those that survived and for patients readmitted compared to patients not readmitted for all years. There was little-to-no variation in the year to year median mFI scores. The 4 methods of calculating frailty performed similarly and were stable. The actual method of determining the accumulated deficits may not be as important as enumerating their number.

PD-L1/PD-1 Pattern of Distribution within Bone Marrow Microenvironment Cells in Patients with Smoldering Myeloma and Active Multiple Myeloma

Despite the promising results of immune-checkpoint blockade in the treatment of many tumors, the use anti-PDL-1/PD-1 antibodies in multiple myeloma (MM) still remains debated and under observation for the high toxicity in combination with immunomodulatory drugs or for the lack of a clear evaluation of the PD-L1/PD-1 distribution in MM patients. Literature data on PD-L1/PD-1 expression by CD138+ and bone marrow (BM) cells in MM patients are discordant and none of them compared patients with active and smoldering myeloma (SMM). This suggests the need to better define PD-L1/PD-1 distribution in the BM immune-microenvironment in MM patients, to identify those that could benefit from PD-L1/PD-1 blockade. In this study, we isolated mononuclear cells from BM aspirates in a cohort of patients with monoclonal gammopathies (total number= 107) including 39 patients with SMM and 78 with active MM, including both newly diagnosed (MMD) and relapsed MM (MMR). We compared the expression profile of PD-L1/PD-1 axis on CD138+ cells, CD14+ monocytes and T cells (both CD4+ and CD8+), by flow-cytometry. Results were correlated with clinical parameters, as International Staging system (ISS), cytogenetic risk, bone disease. BM sera were also collected to measure the levels of different soluble factors known to regulate PD-L1 expression or to exert pro/anti-tumor activity in MM (IL-6, IL-10, IL-27, IFN-γ). Results from ELISA assay were examined in relation with flow-cytometry data. We found that neither PD-L1 expression on CD138+ cells nor PD-1 on CD4+/CD8+ cells significantly differ between SMM and MM patients; although, CD14+PD-L1+% increases with disease progression (SMM vs MMD vs MMR: median: 39.14 vs 59.49 vs 47.66, p=0.14) without reaching a statistical significance. Analysis on the total cohort revealed that PD-L1 is expressed at higher levels on CD14+CD16+ non-classical monocytes compared with classical monocytes (17.41 vs 23.09, p&amp;lt;0.0001), independently from the stage of disease. Of note, relapsed MM patients, which are currently the main candidates for anti-PD-L1/PD-1 treatment, showed an inverted CD4+/CD8+ ratio as compared with SMM (0.75 vs 1.22, p=0.024) and MMD (0.75 vs 1.50, p=0.001), along with high levels of pro-tumoral IL-6 and a positive correlation between CD14+PD-L1+% and CD8+PD-1+% cells (p=0.013), suggesting a highly compromised immune-microenvironment with a low amount of effector cells. Among the cytokines tested on the total cohort, the anti-tumoral IL-27 BM serum levels inversely correlated with PD-L1 MFI only on CD14+ cells (p=0.025), with CD8+PD-1+% (p=0.013) and with the immunesuppressive cytokine IL-10 serum levels (p=0.035), independently from the stage of disease. Noteworthy, we did not find any correlation between PD-L1/PD-1 profile and BM levels of either IFN-γ, known to in vitro up-regulate PD-L1 expression on MM cell lines, or IL-6 levels, the main MM pro-survival factor. Focusing on patients with active MM, those with ISS=II and III showed increased PD-L1 expression on CD14+ cells (ISS II+III vs I, median MFI 20.35vs14.59, p=0.005) and higher CD8+PD-1+% (II+III vs I, 4.35vs2.58, p=0.022) compared with ISS=I patients. Analysis on PD-L1/PD-1 expression in relation with the cytogenetic features of our cohort of patients revealed that CD138+ cells from hyperdiploid patients express higher levels of PD-L1 compared with not-hyperdiploid ones (25.66 vs 13.96, p=0.001), suggesting that the expression of this immune checkpoint does not contribute as a high risk factor in MM disease. Finally, we investigated PD-L1/PD-1 distribution, in relation with the presence of bone lesions, and we found that not-osteolytic patients have higher CD8+PD-1+% compared with osteolytic ones (p=0.071). In conclusion, our data show a similar PD-L1/PD-1 expression pattern between SMM and MM patients, and higher PD-L1 intensity on the non-classical monocytes CD14+CD16+ compared with classical CD14+CD16- cells. On the other hand, an inverted CD4+/CD8+ ratio characterizes relapsed MM patients, together with high amount of MM pro-survival IL-6 and low anti- tumor IL-27 BM serum levels. Overall these data suggest that, despite the similarity in PD-L1/PD-1 expression, SMM and early MM patients rather than relapsed MM, could represent the potential subset which could better benefit of anti PD-L1/PD-1 therapy, in light of their less compromised immune-microenvironment. Disclosures Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Participation in congresses, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: Participation in congresses; GSK: Other: Clinical study sponsorship, Research Funding; Janssen Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Other: Clinical study sponsorship; participation in congresses, Research Funding; Millennium Pharmaceutical: Other: Clinical study sponsorship, Research Funding.

CT-371: Impact of Donor-Specific Anti-HLA Antibody on Platelet Transfusion Refractoriness in Haplo-Identical Hematopoietic Stem Cell Transplant Patients: A Single-Center Experience

Context Outcomes of haplo-identical hematopoietic stem cell transplantation (haploSCT) have recently improved due to better control of allo-reactive reactions related to the major HLA mismatch between the recipient and the donor. Presence of pretransplant donor-specific anti-HLA antibodies (DSA) is associated with higher risk of graft failure. However, little is known regarding the association between the presence of these antibodies and platelet transfusion refractoriness (PTR) post-haploSCT that could predict poor transplant outcomes. Objective The aim of this study is to evaluate the association between DSA status of the recipient and the corrected platelet count increment (CCI) from day 0-100 post-haploSCT as an objective measure of PTR. Design This is a retrospective study that comprises chart review of patients who underwent haplo-SCT between June 2015 and January 2020 at the American University of Beirut medical center in Lebanon. Main outcome measures Pretransplant panel reactive antibodies (PRA) and DSA status at the time of transplant were collected, and the 24-hour CCI was calculated for every platelet donor transfusion. Results We identified 69 patients with a median age of 36 years (range: 16-77). 44 patients (64%) were males and 52 patients (75%) had myelodysplastic syndrome or acute leukemia. Median time from diagnosis to transplant was 13.8 months (range: 2.5-91.7), and 43 patients (62%) were in complete remission at transplant. Of all patients, only 6 patients (8.7%) were DSA+ at time of transplant, 14 (20.3%) were PRA+/DSA-, and the remaining 49 (71%) were PRA-. All 6 patients with DSAs underwent desensitization with a median MFI decrease of 375 (range: -541-1603). One of these 6 did not reach platelet nor leukocyte engraftment. Patients with persistent DSAs had lower mean CCI (9.27, range: 4.58-12.37) compared to those without DSAs (mean CCI: 11.93, range: 1.34-29.71; p=0.017). Statistical significance was maintained upon comparing PRA+DSA+ patients to PRA+DSA- and to PRA- patients with mean CCI of 14.04 (range: 1.39-29.71; p=0.005) and 11.38 (range: 1.34-28.00, p=0.024), respectively. Conclusions Presence of anti-HLA DSAs in haploSCT recipients, even at MFI lower than 1500, was associated with increased PTR. Larger number of patients is needed to evaluate its impact on survival outcomes.

P1604SIGNIFICANT DETECTION OF UNEXPLAINED HLA ANTIBODIES WITH SINGLE-ANTIGEN BEADS IN PATIENTS ON THE WAITING-LIST WITHOUT PREVIOUS SENSITIZING EVENTS

Abstract Background and Aims The introduction of more sensitive and specific tools as solid phase immunoasssays has improved the ability to detect HLA antibodies. The techniques based on solid phase immunoassays in Luminex system can be performed either as screening assays (with pooled antigen panels that use bead populations coated in affinity-purified class I or class II HLA molecules) or to assess the specificity with single-antigen bead (SAB) assays (single cloned allelic HLA antigen).The sensitivity of each assay Is different, and their results may be discordant. Our objective was to evaluate the potential discrepancies between tests in a cohort of patients on our waiting list with no previous sensitizing events, for their potential influence in the access to transplantation. Method Observational study of 184 patients included in the kidney-transplant waiting list on March 31st, 2019. All possible sensitizing events (pregnancy, transfusion and previous transplants) were registered. HLA antibodies where analyzed both by screening and SAB tests. Results We limited the observations to males (64.7% of the cohort), as most women (95.4%) had a known sensitizing event, not being possible to discard possible pregnancies in the remaining. Of men, 46.2% had a sensitizing event and 63.8% did not. All 63 unsensitized patients showed a negative screening test, and 49 (77,78%) had at least one SAB done being positive in 73.4% (36/49). The positive SAB yielded a cPRA &amp;gt;10% in 30 cases (between 21-85%), mostly (27/30) due to HLA class II antibodies (88.8% with at least one DQ antibody) with a median MFI of 1571 (range:752-17650). A thorough review of specificities showed that 81% DQB1* and 89.6% DQA1* antibodies did not show consistency in all beads included for the same allele. Conclusion Screening and SAB assays show a significant discordancy in unsensitized men included in the waiting list for kidney transplantation. Automated detection of antibodies by SAB without a proper specialized interpretation may wrongly limit access to transplantation.

Circulating Neutrophils Do Not Predict Subclinical Coronary Artery Disease in Women with Former Preeclampsia.

Introduction: Preeclampsia (PE) represents a hypertensive pregnancy disorder that is associated with increased cardiovascular disease (CVD) risk. This increased risk has been attributed to accelerated atherosclerosis, with inflammation being a major contributor. Neutrophils play an important role in the onset and progression of atherosclerosis and have been associated with vascular damage in the placenta as well as the chronic inflammatory state in women with PE. We therefore investigated whether circulating neutrophil numbers or reactivity were associated with the presence and severity of subclinical atherosclerosis in women with a history of PE. Methods: Women aged 45–60 years with a 10 to 20 years earlier history of early onset preeclampsia (delivery <34 weeks of gestation) (n = 90), but without symptomatic CVD burden were screened for the presence of subclinical coronary artery disease (CAD) using both contrast-enhanced and non-contrast coronary CT angiography. Subclinical CAD was defined as a coronary artery calcium (CAC) score ≥100 Agatston Units and/or ≥50% coronary luminal stenosis. We assessed whether the numbers and activity of circulating neutrophils were associated with the presence of subclinical CAD and as secondary outcome measurements, with the presence of any calcium (CAC score > 0 AU) or stenosis, categorized as absent (0%), minimal to mild (>0 and <50%), and moderate to severe (≥50%) narrowing of the coronary artery. Blood was drawn just before CT and neutrophil numbers were assessed by flow cytometry. In addition, the presence of the chemokine receptors CXCR2 and CXCR4, which are known to be instrumental in neutrophil recruitment, and neutrophil activity upon stimulation with the bacterial peptide N-Formylmethionyl-leucyl-phenylalanine (fMLF) was assessed by flow cytometry. Results: Of the participating women, with an average age of 49 years, 13% (12 out of 90) presented with subclinical signs of CAD (CAC score ≥100 AU and/or ≥50% luminal stenosis), and 37% (33 out of 90) had a positive CAC score (>0). Total white blood cell count and neutrophil counts were not associated with the presence of subclinical CAD or with a positive CAC score. When assessing the presence of the chemokine receptors CXCR4 and CXCR2, we observed a slight decrease of neutrophil CXCR2 expression in women with CAC (median MFI 22.0 [interquartile range (IQR) 20.2–23.8]) compared to women without CAC (23.8 [IQR 21.6–25.6], p = 0.02). We observed no differences regarding neutrophil CXCR4 expression. In addition, expression of the early activity marker CD35 was slightly lower on neutrophils of women with subclinical CAD (median MFI 1.6 [IQR 1.5–1.9] compared to 1.9 [IQR 1.7–2.1] in women without CAD, p = 0.02). However, for all findings, statistical significance disappeared after adjustment for multiple testing. Conclusion: Our findings indicate that neutrophil counts and (re)activity are not directly associated with silent CAD disease burden and as such are not suitable as biomarkers to predict the presence of subclinical CAD in a high-risk population of women with a history of preeclampsia.

Patients with BRCA mutated ovarian cancer may have fewer circulating MDSC and more peripheral CD8+ T cells compared with women with BRCA wild-type disease during the early disease course.

Immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are associated with immunologic tolerance and poor prognosis in ovarian cancer (OvCa). We hypothesized that women with germline BRCA1 and BRCA2 mutation-associated (gBRCAm) OvCa would have fewer circulating immunosuppressive immune cells compared to those with BRCA wild-type (BRCAwt) disease during their early disease course (<5 years post-diagnosis) where gBRCAm is a favorable prognostic factor. We collected and viably froze peripheral blood mononuclear cells (PBMCs) from patients with recurrent OvCa olaparib clinical trials (NCT01445418/NCT01237067). Immune subset analyses were performed using flow cytometry for Tregs, exhausted CD8+ T cells, monocytes and MDSCs. Functional marker expression, including cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3) and programmed cell death protein 1 (PD-1) was evaluated. Data were analyzed using FlowJo. Pretreatment PBMCs were collected from 41 patients (16 gBRCAm/25 BRCAwt). The percentage of MDSCs among viable CD45+ PBMC was lower in gBRCAm OvCa compared with BRCAwt OvCa (median 0.565 vs. 0.93%, P=0.0086) but this difference was not seen in those women >5 years post-diagnosis. CD8+ T cells among viable CD45+ PBMCs and CTLA-4+/CD8+ T cells were higher in gBRCAm carriers than patients with BRCAwt, in particular for those <5 years post-diagnosis (median 20.4 vs. 9.78%, P=0.031 and median MFI 0.19 vs. 0.22, P=0.0074, respectively). TIM-3 expression on Tregs was associated with poor progression-free survival, independent of gBRCAm status (P<0.001). Our pilot data suggested that patients with gBRCAm OvCa may have fewer circulating MDSCs but higher CD8+ T cells in PBMCs during their early disease course. This may contribute to the observed survival benefit for these women in their first post-diagnosis decade.

The Fully Human Anti-CD47 Antibody SRF231 Has Dual-Mechanism Antitumor Activity Against Chronic Lymphocytic Leukemia (CLL) Cells and Increases the Activity of Both Rituximab and Venetoclax

The Fully Human Anti-CD47 Antibody SRF231 Has Dual-Mechanism Antitumor Activity Against Chronic Lymphocytic Leukemia (CLL) Cells and Increases the Activity of Both Rituximab and Venetoclax

Correlation between Pre-Transplant Bead-Based Screening of Anti-HLA Antibodies and Single Antigen Arrays

Introduction Generalization of solid-phase bead-based assays has dramatically modified the definition of HLA sensitization in patients waiting for an organ transplant. Because of their lower cost, Luminex screening tests are often the first line and the main follow-up technique, single antigen (SA) approach being performed only when screening is considered positive. As no data are available, we aimed at exploring the correlations between these two techniques, the predictive value and the optimal thresholds of screening tests. Methods We studied 4430 recipients of their first organ-transplant, with at least one pre-transplantation screening and one SA test, both from the manufacturer One Lambda. We reanalyzed all raw MFI data for these two techniques. For screening tests, we considered the bead with the highest signal-to-noise ratio calculated according to the manufacturer’s recommendations. Results Transplants were mainly kidney (64%), liver (13%), heart (12%) and lung (10%). Mean recipient age was 48.8±18.8 years. During pre-transplant follow-up, median [IQR] of peak screening ratios was 3.8[2.1-9.7] for class 1 and 2.1[1.3-4.4] for class 2. Patients with a history of pregnancy or transfusion had significantly higher screening ratios than patients without: for class 1, 7.6 [3.5-32.8] versus 3.9 [2.4-7.2] and for class 2, 3.2 [1.7-9.6] versus 2.3[1.5-4.1], (Wilcoxon test, p<1.10-11). Figure 1 shows frequency of sensitization according to two frequently used thresholds for screening and SA tests.For class 1 and 2, there was a linear relation between the screening ratio and the SA bead with maximal MFI, but it was significant only for ratios above the median value. Principal component analysis revealed that screening ratio mostly correlated with the sum of SA beads MFI, maximal SA bead MFI and the number of positive SA beads (Figure 2). We performed ROC (Receiver Operating Characteristic) analysis to find the optimal screening threshold predictive of a positive SA test (at least one positive bead with a MFI > 1000). For class 1 and 2, AUC were 0.719 and 0.642 and optimal ratio thresholds were 3.3 and 2.1.The median MFI of SA profiles corresponding to ratios above these thresholds were 3635 versus 1291 for class 1 and 2088 versus 1204 for class 2 (Wilcoxon test compared to sera below the threshold, p< 2.2e-16). However, using these thresholds, 25% of sera considered as negative for screening had a class 1 maximal MFI > 2419, and a class 2 maximal MFI > 2063. Conclusion In a low immunological risk population of patients waiting for a transplant, definition of HLA sensitization is highly dependent on the solid phase assay technique and the threshold chosen. The correlation between Luminex screening and SA is significant only for screening ratios above the median value. The predictive value of screening tests regarding results of SA is low to moderate and optimal thresholds are different for class 1 and 2. This study was realized with the financial support of Assistance Publique Hôpitaux de Paris, Sandoz and CSL Behring France.

Decreased CD22 expression and intracellular signaling aberrations in B cells of patients with systemic sclerosis.

The objective of the study was to explorethe phenotype and intracellular signaling events of B cells in patients with systemic sclerosis (SSc). Peripheral blood B cell surface markers CD19 and CD22 were evaluated by flow cytometry in 23 patients with SSc and seven healthy individuals. Levels of intracellular kinases Lyn, Syk and P-Y 348 Syk along with phosphatase SHP-1 were examined with Western immunoblotting in selected patients. P-Y 822 CD22 was subsequently evaluated flow cytometrically in antigen receptor stimulated B cells. A statistically significant decrease in CD22 B cell surface expression was found in the diffuse subset of patients (median CD22 MFI ± SD was 5.90 ± 2.35 vs 10.20 ± 1.88 for patients vs healthy controls respectively; p = 0.021), while no statistically significant change was found regarding CD19. CD22 underexpression was more pronounced when interstitial lung disease (ILD) was present (median CD22 MFI ± SD was 5.90 ± 2.25 vs 10.20 ± 1.88 for patients with ILD vs healthy controls respectively; p = 0.011). CD22 phosphorylation following B cell receptor (BCR) stimulation was also found to be impaired in patients with diffuse SSc (median change in MFI ± SD was 0.28 ± 0.09 vs 0.38 ± 0.08 for patients vs healthy controls respectively; p = 0.034). Low CD22 expression was arithmetically correlated with kinase Lyn underexpression (Pearson coefficient 0.926; p = ns) in B cells from a small sample of patients. These results suggest that CD22 underexpression and impaired phosphorylation along with implications for Lyn kinase aberrations could contribute to the activated B cell phenotype in SSc.

Frailty Predicts Morbidity and Mortality after Colectomy for Clostridium difficile Colitis

Frailty has been noted as a powerful predictive preoperative tool for 30-day postoperative complications. We sought to evaluate the association between frailty and postoperative outcomes after colectomy for Clostridium difficile colitis. The National Surgical Quality and Improvement Program cross-institutional database was used for this study. Data from 470 patients with a diagnosis of C. difficile colitis were used in the study. Modified frailty index (mFI) is a previously described and validated 11-variable frailty measure used with the National Surgical Quality and Improvement Program to assess frailty. Outcome measures included serious morbidity, overall morbidity, and Clavien IV (requiring ICU) and Clavien V (mortality) complications. The median age was 70 years and body mass index was 26.9 kg/m2. 55.6 per cent of patients were females. 98.5 per cent of patients were assigned American Society of Anesthesiologists Class III or higher. The median mFI was 0.27 (0-0.63). Because mFI increased from 0 (non-frail) to 0.55 and above, the overall morbidity increased from 53.3 per cent to 84.4 per cent and serious morbidity increased from 43.3 per cent to 78.1 per cent. The Clavien IV complication rate increased from 30.0 per cent to 75.0 per cent. The mortality rate increased from 6.7 per cent to 56.2 per cent. On a multivariate analysis, mFI was an independent predictor of overall morbidity (AOR: 13.0; P < 0.05), mortality (AOR: 8.8; P = 0.018), cardiopulmonary complications (AOR: 6.8; P = 0.026), and prolonged length of hospital stay (AOR: 6.6; P = 0.045). Frailty is associated with increased risk of complications in C. difficile colitis patients undergoing colectomy. mFI is an easy-to-use tool and can play an important role in the risk stratification of these patients who generally have significant morbidity and mortality to begin with.

Using anti-CD38 immunotherapy to enhance anti-tumor T-cell immunity in chronic lymphocytic leukemia (CLL)

Abstract Increased CD38 expression on chronic lymphocytic leukemia (CLL) cells correlates with a negative prognosis, however, its utility as a therapeutic target in this malignancy remains unknown. Our investigations show that the anti-CD38 therapeutic mAb, daratumumab (Dara) induces direct apoptosis of CLL cells and promotes their immune-mediated clearance (through ADCC). Notably, CD38 expression is quite high on a subset of CLL-cells (CD19+/CD38hi/CD24+/IL-10+; B-regulatory-like phenotype), which promote Treg expansion while prohibiting CD8+ cytotoxic T-cells (cTLs). Indeed, immunosuppression is a hallmark of CLL and patients often present with increased number of Tregs and Th2 cells but decreased effector T cells and cTLs. It has recently been reported that CD38hi Tregs are significantly immunosuppressive and when examining this population in peripheral blood mononuclear cells (PBMCs) from CLL patients (n=13), we noted that &amp;gt;50% of CD4+/CD25+/FoxP3+ cells were CD38hi (median MFI ~600). We hypothesized that Dara would kill CD38hi CLL cells à decreased CD38hi Treg formation/direct killing à expansion of cTLs. Indeed, ex-vivo treatment of CLL-patient derived PBMCs showed that Dara was highly lethal to both CD38hi “Breg-like” CLL cells and Tregs; associated with decrease in IL-10, increase in IFN-γ, effector T cells, Th17 subsets and proliferation of cTLs. Altogether this pointed towards recovery of anti-tumor immunity which was further evidenced by enhanced tumor-specific cytolytic activity of cTLs. Our pre-clinical data demonstrates that targeting CD38 in CLL has an anti-tumor effect via 1.) lethal effect on CLL-Bregs and Tregs and 2.) tumor-specific expansion of cTLs, which we will test clinically in an upcoming trial.

Frailty Predicts Morbidity after Colectomy for Ulcerative Colitis

The rate of ulcerative colitis (UC), an inflammatory bowel disease, has been on the rise in the United States for the last several decades. Colectomy can be performed when other treatment options cannot provide a reasonable quality of life to patients with UC. Frailty has been shown to be a strong tool for evaluating preoperative risk factors for poor postoperative outcomes. The National Surgical Quality and Improvement Program cross-institutional database was used for this study. Data from 943 patients who underwent colectomy for UC between 2005 and 2012 were evaluated. Modified frailty index (mFI) is a previously described and validated 11-variable frailty measure used in the National Surgical Quality and Improvement Program to assess frailty. Outcome measures included serious morbidity; overall morbidity; cardiopulmonary, septic, and wound complications; and Clavien class IV (requiring ICU) and V (mortality) complications. Median age was 46 years and median body mass index was 25.5 Kg/m2. In all, 54.3 per cent of patients were male and 39.38 per cent of patients were American Society of Anesthesiologists Class lll or higher. The median mFI was 0 (0-0.54). As the mFI increased from 0 (nonfrail) to 0.18 and above, the overall morbidity increased from 25.40 to 52.1 per cent (P < 0.05), serious morbidity increased from 14.9 to 42.1 per cent (P < 0.05), septic complications increased from 9.87 to 21.49 per cent (P < 0.05), cardiopulmonary complications increased from 2.98 to 23.14 per cent (P < 0.05), Clavien class IV complications increased from 3.5 to 26.5 per cent (P < 0.05), and Clavien V complications increased from 0.16 to 6.61 per cent (P < 0.05). On multivariate analysis, mFI was an independent predictor of septic complications [Adjusted Odds Ratio (AOR): 31.26; P = 0.006], cardiopulmonary complications (AOR: 216.3; P ≤ 0.001), serious morbidity(AOR: 66.8; P ≤ 0.001), overall morbidity (AOR: 25.5; P ≤ 0.001), Clavien class IV (AOR: 204.9; P ≤ 0.001) complications, and return to the operating room (AOR: 14.29; P = 0.048). Frailty is associated with an increase in morbidity and mortality after colectomy in patients with UC. mFI is an easy-to-use tool and can play an important role in the risk stratification of these patients.

Long-term Outcomes of Kidney Transplantation in Patients With High Levels of Preformed DSA: The Necker High-Risk Transplant Program.

There is an increasing number of anti-HLA sensitized and highly sensitized renal transplant candidates on waiting lists, and the presence of donor-specific alloantibodies (DSAs) at the time of transplantation leads to acute and chronic antibody-mediated rejection (AMR). Acceptable short-term outcomes have been described, notably because of desensitization protocols, but mid- and long-term data are still required. Our high immunologic risk program included 95 patients with high peak or day 0 DSA levels (mean fluorescence intensity [MFI] > 3000) with a complement-dependent cytotoxicity-negative crossmatch, who received a posttransplant desensitization protocol starting at day 0 with high-dose intravenous immunoglobulin, plasma exchanges, and eventually rituximab. Their characteristics were compared with a control group including 39 patients with a lower immunologic risk (MFI between 500 and 3000 at day 0) who received the same posttransplant desensitization. The median MFI of the immunodominant class I or II DSA in the peak or day 0 serum was 9421 (interquartile range, 4959-12 610). An AMR occurred during the first posttransplant year in 31 patients (32.6%), and at one year, the rate of chronic AMR was 39.5%. The 1-, 3-, 5- and 7-year death-censored allograft survival rates were 98%, 91%, 86%, and 78%, respectively, with concomitant recipient survival rates of 97%, 93%, 85%, and 79%, respectively. These results suggest that DSA-sensitized patients with high MFI levels can receive transplantation across the HLA-barrier, with the use of an intensified posttransplant immunosuppressive therapy starting at day 0 combined with close clinical, immunologic, and histologic monitoring.