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Background: Complement dysregulation has been documented in the molecular pathophysiology of COVID-19 and recently implicated in the relevant pediatric patient inflammatory responses. Aims: Based on our previous data in adults, we hypothesized that signatures of complement genetic variants would also be detectable in pediatric patients exhibiting COVID-19 signs and symptoms. Methods: We prospectively studied consecutive pediatric patients from our COVID-19 Units (November 2020-March 2021). COVID-19 was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Patient data were recorded by treating physicians that followed patients up to discharge. DNA was obtained from peripheral blood samples. Probes were designed using the Design studio (Illumina). Amplicons cover exons of complement-associated genes (C3, C5, CFB, CFD, CFH, CFHR1, CFI, CD46, CD55, MBL2, MASP1, MASP2, COLEC11, FCN1, FCN3 as well as ADAMTS13 and ΤHBD) spanning 15 bases into introns. We used 10ng of initial DNA material. Libraries were quantified using Qubit and sequenced on a MiniSeq System in a 2x150 bp run. Analysis was performed using the TruSeq Amplicon application (BaseSpace). Alignment was based on the banded Smith-Waterman algorithm in the targeted regions (specified in a manifest file). We performed variant calling with the Illumina-developed Somatic Variant Caller in germline mode and variant allele frequency higher than 20%. Both Ensembl and Refseq were used for annotation of the output files. A preliminary analysis (A) for the identification of variants of clinical significance was based on annotated ClinVar data, while a further and more selective analysis (B) was performed to identify missense complement coding variants that may biochemically contribute to the deregulation of innate responses during infection. This analysis was mainly based on the dbSNP and UniProt databases and available literature. Results: We studied 80 children and adolescents, 8 of whom developed inflammatory syndromes (MIS-C group). Among them, 41 were hospitalized and eventually all survived. In our preliminary analysis, patients exhibited heterogeneous variant profiles including pathogenic, benign, likely benign, and variants of unknown significance (median number of variants: 97, range: 61-103). We found a variant of ADAMTS13 (rs2301612, missense) in 39 patients. We also detected two missense risk factor variants, previously detected in complement-related diseases: rs2230199 in C3 (33 patients); and rs800292 in CFH (36 patients). Among them, 40 patients had a combination of these characterized variants. This combination was significantly associated with the presence of dyspnea (p=0.031) and cough (p=0.042). Furthermore, 27 patients had a pathogenic variant in MBL2 (rs1800450), and 7 a pathogenic deletion in FCN3 that have been previously associated with inflammatory syndromes.The results of our further analysis are summarized in Figure. We identified common variants, some well represented by relatively high frequencies (>70%) (rs11098044 in CFI, rs1061170 in CFH and rs12711521 in MASP2) and others less abundant, but varying considerably between the hospitalized group, the non-admitted group and the MIS-C individuals (rs2230199 in C3, rs1065489 in CFH, rs12614 and rs641153 in CFB, rs1800450 in MBL2, rs2273346 and rs72550870 in MASP2, rs72549154 in MASP3 and rs7567833 in COLEC11, all highlighted in Figure in red).). Structurally, the majority of these common variants of interest encode charge reversal mutations. These may influence protein-protein interactions in complex formations that are important for complement activation and/or regulation. Conclusion: In pediatric COVID-19 we have detected a novel set of complement missense coding variants some of which have been implicated earlier in inflammatory syndromes and endothelial stress responses. Certain combinations of mutations of alternative and/or lectin pathway components may increase the threshold dynamics of complement consumption and therefore alter COVID-19 phenotypes. Figure 1 Figure 1. Disclosures Gavriilaki: Alexion, Omeros, Sanofi Corporation: Consultancy; Gilead Corporation: Honoraria; Pfizer Corporation: Research Funding. Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials .

Background. Hematopoietic cell transplant (HCT) recipients who develop coronavirus disease 2019 (COVID-19), have dismal prognosis with approximately 20% mortality. Given the lack of a specific and effective therapy, the availability of various vaccination platforms against SARS-CοV-2 has generated optimism towards the development of a robust herd immunity. Notwithstanding the prioritization of HCT recipients to COVID-19 vaccination, limited information is available on whether and to what extent, they mount an immune response to SARS-CοV-2 vaccination as they were generally excluded from vaccination trials. Aim. To gain insights in the immune responses developed to SARS-CoV-2 vaccines under immunosuppression, we studied the humoral and cellular immune responses to SARS-CoV-2 vaccination in HCT recipients. Methods. We prospectively studied (April-July 2021), adult patients who had undergone HCT in our Unit and received two doses of a SARS-CoV-2 vaccine (as per international guidelines) after providing written informed consent. Responses were studied before each vaccination dose and 12-51 days later after the second dose. Neutralizing antibodies against SARS-CoV-2 (CoV-2-NAbs) were measured using an FDA approved methodology for diagnostic use (ELISA, cPass™ SARS-CoV-2 NAbs Detection Kit; GenScript, Piscataway, NJ, USA; cut-off value for a positive result set at ≥30%) and SARS-CoV-2 spike-specific T cells (spike-STs) by interferon-γ Elispot after pulsing peripheral blood mononuclear cells with spike pepmixes. Results. Humoral responses were studied on 65 patients, (50 allo-HCT/15 auto-HCT, Figure A). T cell responses were measured on 38/65 vaccinated patients (32 allo-HCT/6 auto-HCT) with a median of 3 (0.17-31) and 2 years (1.25-8) post allo- and auto-HCT respectively, and 19 healthy, unexposed vaccinees. One patient with prior COVID-19, was excluded from analysis. All patients were vaccinated with the Pfizer-BioNTech, except for 2 vaccinated with the AstraZeneca vaccine. Both CoV-2-NAbs and spike-STs were barely detectable before vaccination but could be detected in both allo- and auto-HCT patients after the first vaccination dose, reaching statistically significant increase after the second vaccination dose (p Conclusion . Herein, we report for first time humoral and T cell responses post SARS-CoV-2 vaccination in HCT recipients. Transplant recipients not under active and intense immunosuppression at the time of vaccination may benefit significantly from COVID-19 vaccination even though these responses are blunted compared to healthy individuals. However, for the severely immunocompromised patients it seems highly unlikely that they could be protected by vaccination and for this vulnerable population, different vaccination schemes or therapeutic platforms should be developed along with collateral measures including minimal exposure and immunization of caregivers and health care providers. Figure 1 Figure 1. Disclosures Gavriilaki: Alexion, Omeros, Sanofi Corporation: Consultancy; Pfizer Corporation: Research Funding; Gilead Corporation: Honoraria. Yannaki: SANDOZ: Speakers Bureau; Gilead: Speakers Bureau; Novartis: Speakers Bureau; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials .

Background: Several recent studies support the notion of excessive complement activation in patients with severe coronavirus disease-19 (COVID-19), with beneficial results of complement inhibition in case series. In this context, severe COVID-19 shares common characteristics with complement-mediated thrombotic microangiopathy (TMA). TMA is commonly characterized by genetic susceptibility, and presents with thrombocytopenia, anemia, increased lactate dehydrogenase (LDH), and organ damage (renal, neurological, cardiac). Aims: We hypothesized that genetic susceptibility would be also evident in patients with severe COVID-19 and would be associated with disease severity. Methods: We prospectively studied consecutive adult patients hospitalized with COVID-19 in our referral centers (April-May 2020). Diagnosis was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). COVID-19 severity was assessed based on World Health Organization's (WHO) criteria into moderate/severe, and critical disease. Additional data on patients' history and course were recorded by treating physicians that followed patients up to discharge or death. Patients' DNA was obtained from peripheral blood samples. Probes were designed using the Design studio (Illumina). Amplicons cover exonic regions of TMA-associated genes (Complement factor H/CFH, CFH-related, CFI, CFB, CFD, C3, CD55, C5, MCP, thombomodulin/THBD, ADAMTS13) spanning 15 bases into the intronic regions. We used 10ng of initial DNA material. Libraries were quantified using Qubit and sequenced on a MiniSeq System in a 2x150 bp run. Analysis was performed using the TruSeq Amplicon application (BaseSpace). Alignment was based on the banded Smith-Waterman algorithm in the targeted regions (specified in a manifest file). We performed variant calling with the Illumina-developed Somatic Variant Caller in germline mode and variant allele frequency higher than 20%. Both Ensembl and Refseq were used for annotation of the output files. Variants clinical significance was based on ClinVar and the current version of the Complement Database, as we have previously described. Results: We studied 60 patients, 40 with moderate/severe disease hospitalized in COVID-19 general ward (GW) and 20 with critical disease hospitalized in intensive care units (ICU). Among them, 11 patients succumbed due to COVID-19 disease. Patients laboratory characteristics are shown in Figure. In genetic analysis, patients presented heterogeneous variant profiles including pathogenic, benign, likely benign, and variants of unknown significance (median number of variants: 62, range: 51-89). Search in the Complement Database revealed seven patients, each carrying one pathogenic or likely pathogenic variant in C3, CD46, DGKE, and CFH. Based on ClinVar, we found a pathogenic variant of ADAMTS13 (rs2301612, missense) in 28 patients. We also detected two missense risk factor variants, previously detected in complement-related diseases: rs2230199 in C3 (13 patients); and rs800292 in CFH (26 patients). Among them, 22 patients had a combination of these characterized variants. This combination was significantly associated with critical disease that required intensive care (p=0.037), as well as low lymphocyte counts (p=0.021) and high neutrophil-to-lymphocyte ratio (p=0.050). In the multivariate model, critical disease was an independent predictor of double heterozygocity in these variants. Furthermore, one patient had a rare germline missense variant in CFI (rs112534524), previously detected in complement-related diseases. This patient suffered from critical disease but survived after long-term ICU hospitalization. Interestingly, five patients showed a likely protective missense variant in CFB (rs641153). Conclusion: We have detected for the first time rare and pathogenic TMA-associated variants in patients with severe COVID-19. Our findings of variants in complement-regulatory genes and ADAMTS13 suggest genetic susceptibility and define proof-of-concept for proper selection of patients that would benefit from complement inhibition. Table Disclosures Gavriilaki: Omeros Pharmaceuticals: Consultancy.

Introduction: Reactivation of Epstein-Barr virus (EBV) is common in allogeneic hematopoietic cell transplantation (HCT). EBV infection leads to post-transplantation lymphoproliferative disease (PTLD), a life-threatening complication in this setting. Frequent molecular monitoring of viral load, especially in high risk patients and pre-emptive use of Rituximab has improved the outcome of EBV infection. However, the expansion of alternative transplants, leads to higher incidence and effective measures are warranted. Methods: We have retrospectively studied the clinical characteristics of EBV reactivation in consecutive patients that underwent HCT between 2007-2019, when pre-emptive administration of Rituximab was a standard of care in our Unit and possible correlations were sought. EBV reactivation was considered when viral load >8500 viral genomic copies (VGC)/ml in whole blood was documented during regular molecular monitoring with RQ-PCR. Patients received treatment with Rituximab, at a scheme according to physician's decision. We considered undetectable levels as resolution of infection. Patients with PTLD proven by lymph node biopsy were treated as previously described by our group. Results: Among 546 HCT recipients, EBV reactivation was detected in 100 patients, that suffered from hematologic malignancy (98) or aplastic anemia (2) and received grafts from matched sibling (23), unrelated (70) or haploidentical donors (12). Haploidentical donors were significantly higher in patients with EBV reactivation compared to our transplant population (12% versus 6%, p Five patients (two with haploidentical and three with unrelated donors) presented PTLD at 41 days post transplantation. ROC curve analysis identified a cut off of 67150 VGC/ml that predicts PLTD with 80% sensitivity and specificity (green line in Figure). Relapse free survival (RFS), overall survival (OS) and treatment-related mortality (TRM) in the entire cohort were similar regardless the EBV viral load or PTLD [4-year RFS 32.2%, 4-year OS 48.1% in a median follow up 29 months (4-216)]. ATG and chronic GVHD were independently associated with OS in the multivariate analysis (p Conclusion: Our study indicates that regular monitoring and use of preemptive therapy is an effective strategy for prevention of EBV related complications. RFS and OS were not associated to severity of EBV reactivation. A useful cut off of EBV load for PTLD prevention was identified (67150 VGC/ml, specificity and sensitivity: 80%). However, expanding use of alternative transplants warrants a more effective treatment strategy. In this setting, use of specific antiviral cytotoxic cell lines could enhance viral specific cell mediated immunity and provide a better outcome in immunocompromised HCT recipients. Disclosures Gavriilaki: Omeros Pharmaceuticals: Consultancy.

Introduction: Endothelial dysfunction is a common denominator of graft-versus-host disease (GVHD) and transplant-associated thrombotic microangiopathy (TA-TMA). The latter is also characterized by excessive complement activation. Recent studies have introduced the Endothelial Activation and Stress Index (EASIX) as a potential predictor of survival in patients with GVHD. We hypothesized that EASIX would predict complement activation and survival in patients with GVHD and TA-TMA. Methods: We enrolled consecutive adult TA-TMA (International Working Group/IWG criteria), acute and/or chronic GVHD and control allogeneic hematopoietic cell transplantation (HCT) recipients in a 1:1:1 ratio (January 2015-December 2018). Plasma was collected and stored immediately at -80oC at the first day of confirmed TA-TMA or GVHD diagnosis and at a similar post-transplant period in control recipients. EASIX [lactate dehydrogenase (U/L) × creatinine (mg/dL)/thrombocytes (10⁹ cells per L)] was calculated at day 0, 30, 100 and last follow-up. Complement activation was detected measuring soluble C5b-9/membrane attack complex (ELISA, Quidel). Results: We studied 20 TA-TMA, 20 GVHD and 20 control patients (Table 1). TA-TMA developed at a median of 125 post-transplant day (range 9-2931); whereas the first day of confirmed GVHD diagnosis was at a median of 78 post-transplant day (range 16-145). EASIX at day 100 and last follow-up differed significantly among groups (p=0.014 and p=0.001, Table 1), although there was no significant difference between TA-TMA and GVHD patients. In contrast, soluble C5b-9 was significantly higher in TA-TMA compared to GVHD (p=0.008) and control patients (p Furthermore, EASIX at day 0 and last follow-up was significantly associated with overall survival (p=0.013 and p=0.046). Among other pre-transplant factors studied (age, disease type and phase at transplant, donor), EASIX at day 0 was an independent predictor of overall survival (beta=2.627, p=0.029). Conclusion: Our study shows for the first time that EASIX predicts complement activation and overall survival in patients with endothelial dysfunction syndromes and control HCT recipients. Our findings suggest that EASIX may be a useful dynamic marker reflecting the course of endothelial dysfunction in these patients. Disclosures No relevant conflicts of interest to declare.

Introduction: ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats) activity remains a key tool in differential diagnosis of thrombotic microangiopathies (TMAs). However, ADAMTS13 testing is not readily available in many hospitals. Recently, PLASMIC and PLASIC scores have been developed to facilitate rapid recognition of TTP. We aimed to evaluate their usefulness compared to ADAMTS13 testing in a real-world cohort of TMA patients. Methods: We enrolled consecutive patients with samples referred to our Center for ADAMTS13 measurement due to TMA over the last 2 years. Samples were collected either at first diagnosis or relapse before initiation of treatment. ADAMTS13 activity was measured with a commercially available and validated ELISA kit (Technozym, Diapharma). Clinical data were retrospectively collected from referring centers. Management was based on treating physicians' decisions. TTP was defined as severe ADAMTS13 deficiency (activity≤10%); while secondary TMAs were diagnosed in patients with cancer, connective tissue disorders or hematopoietic cell transplantation recipients (transplant-associated TMA). Atypical hemolytic uremic syndrome (aHUS) remained a diagnosis of exclusion in patients with ADAMTS13>10%. PLASMIC was calculated based on seven variables: platelets, hemolysis, cancer, transplant, MCV, INR, creatinine; while MCV was not included in PLASIC, as previously described. ROC curve analysis was performed to determine the sensitivity and specificity of scores. Multivariate binary or logistic regression models were performed when appropriate. Results: We studied 50 TMA patients. Combined clinical and laboratory data conferred the following TMA classification: TTP in 36 patients (72%), transplant-associated TMA in 7 (14%), other secondary TMA in 5 and aHUS in 2. PLASMIC score was intermediate in 2 and high in another 4 patients without TTP. The PLASIC score was high in 5 patients without TTP, leading to less false positive results compared to PLASMIC (p Plasma exchange was commenced in the majority of patients (42/50, 84%). Among TTP patients, the majority (77%) received rituximab as salvage or prophylactic treatment. Rituximab administration was associated with platelet (p=0.003) and ADAMTS13 (p=0.015) levels at diagnosis. The complement inhibitor eculizumab was administered in 3 patients with TA-TMA, who achieved TMA resolution. With a median follow-up of 2.9 years (range 0.3-26.3), overall survival was significantly lower in patients with secondary TMAs (p Conclusion: PLASMIC and PLASIC scores are excellent tools in TMA patients without secondary causes. While PLASMIC and PLASIC scores conferred similar outcomes, the PLASIC score requires six instead of seven variables, is classified as low/high omitting the intermediate category and leads to less false positive results. Further validation of the PLASIC score might confirm its clinical value. In addition, the role of ADAMTS13 levels in guiding rituximab administration needs to be further investigated. When an underlying etiology is detected, ADAMTS13 testing is necessary to exclude TTP and facilitate further therapeutic decisions. Figure 1 Disclosures Panayiotidis: Bayer: Other: Support of clinical trial.

Introduction: Transplant-associated thrombotic microangiopathy (TA-TMA) is a potentially life-threatening complication of allogeneic hematopoietic cell transplantation (HCT). In light of encouraging results by complement inhibition treatment, a few studies and case series have reported complement-related genetic variants in patients with TA-TMA. However, several issues remain undisclosed, regarding both the incidence of such variants and the clinical importance of pre-transplant genetic profiling. Within this context, we hypothesized that pre-transplant genetic susceptibility is evident in adult TA-TMA patients but not in their donors or control transplanted patients and also investigated the association of genetic variants with response to treatment and survival. Methods: To this end, we enrolled consecutive adult allogeneic HCT recipients diagnosed with TA-TMA according to the International Working Group criteria between 2014-2017. Patients were managed based on institutional policy with conventional treatment including withdrawal of calcineurin or mTOR inhibitors, steroid administration and/or plasma infusion/plasma exchange. To test our hypothesis, we also studied donors of the enrolled patients with available samples and age- and sex-matched control HCT recipients. Genomic DNA was extracted from pre-transplant peripheral blood samples of TA-TMA patients, donors and controls. Probes were designed using the Design studio (Illumina). The amplicons cover the exonic regions of complement regulatory genes (complement factor H/CFH, CFH-related, CFI, CFB, CFD, C3, CD55, C5, CD46, thombomodulin/THBD) and TMA-associated ADAMTS13, spanning 15 bases into the intronic regions. After quality assessment, each sample was treated independently in order to produce the appropriate mapping of the reads against the human reference genome hg19. Variant calling was performed using the Genome Annotation Toolkit and variant annotation using annovar together with supplemental databases. Variants with minor allele frequency lower than 1% were considered rare. Results: We studied 30 patients that presented TA-TMA at median + 73 (9-540) days with full hematopoietic constitution, 18 donors of our patients and 30 controls, without significant differences in transplant characteristics (Table). Donors of patients presented significantly lower number of detected (p=0.039) and rare (p=0.049) variants per sample, as well as variants in exonic, splicing or UTR regions (p=0.025) compared to TA-TMA patients. In control patients, we also observed a significantly lower number of rare variants in ADAMTS13 (p=0.002), CD46 (p=0.001), CFH (p=0.010), CFI (p=0.031) and CFB (p=0.016) compared to TA-TMA patients (Graph). Variants previously reported to be pathogenic in TMAs were found in ADAMTS13 and CFB. While heterozygous pathogenic mutations were present in both TA-TMA and control samples, homozygous pathogenic mutations were evident only in 4 TA-TMA patients (p=0.038). Regarding clinical outcomes, 21 of 30 TA-TMA patients (70%) were refractory to conventional treatment. Refractory patients presented a significantly increased incidence of variants in exonic, splicing or UTR regions (p=0.045) compared to responders. 12 patients received eculizumab based on institutional policy. Despite initial laboratory response to eculizumab treatment, only 4 of 12 survived. 19 out of 30 patients succumbed to treatment-related mortality, which was also associated with significantly increased number of variants in exonic, splicing or UTR regions (p=0.012). Conclusions: Increased incidence of pathogenic, rare and exonic variants in TA-TMA patients supports a relevant role for genetic susceptibility that is not evident in control HCT recipients or patients' donors. This, combined with the finding that exonic variants were associated with refractoriness to treatment and increased mortality, indicates that pre-transplant genetic profiling may be useful to intensify monitoring and early intervention in high-risk patients. In this complex setting, the functional and clinical role of genetic variants needs to be further investigated in prospective studies. Disclosures Gavriilaki: European Hematology Association: Research Funding. Stamatopoulos:Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

The development of CD3+/CD8+/CD57+ cytotoxic cell expansions after allogeneic hematopoietic cell transplantation (allo-HCT) driven by antigenic stimulation, viral or associated with chronic graft-versus-host disease (cGvHD), has been suggested as related with favorable outcome. Rituximab, an anti-CD20 humanized monoclonal antibody, has been linked to the development of oligo- or even monoclonal expansions of CD3+/CD8+/CD57+ T-large granular lymphocytes (T-LGLs) that can manifest with neutropenia of delayed origin in relation to Rituximab administration. We have recently reported remarkable skewing of the T-cell receptor (TR) gene repertoire in two allo-HCT transplanted patients with delayed neutropenia associated with T-LGL expansions developing in a context of GvHD and Rituximab administration for EBV reactivation. Prompted by these preliminary findings, we here extend our immunogenetic studies of the TR repertoire in patients receiving Rituximab post allo-HCT. The study group was comprised of 9 patients (including the two previously reported) aged 14-50 years (median 41) who were subjected to myeloablative allo-HCT (4 from matched related, 3 from matched unrelated donors), haplo-identical transplantation (1) or Reduced Intensity Conditioning-allo-HCT from sibling donor (1), all for hematological malignancies. All patients received Rituximab consecutively between 2010-2013 either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. In all patients TR gene repertoire analysis was performed at least one year after the transplantation (range 12-72 months), when immune reconstitution normally would have been achieved, and 5-24 months after the first treatment with Rituximab. Each patient received a mean of 7 cycles of Rituximab (range, 1-14). TRBV-TRBD-TRDJ gene rearrangements were PCR-amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data was interpreted using the IMGT/V-QUEST tool. A total of 164 sequences were analyzed (9-25/case, median=18) revealing 106 productive TRBV-TRBD-TRBJ rearrangements. Among the 29 TRBV functional genes identified only three accounted for 48% of cases: (i) TRBV27*01 (25%), (ii) TRBV6-5*01 (13%), (iii) TRBV6-2*01 (10%). Of note, TRBV27*01 has been reported as the most frequent TRBV gene in Rituximab-related late-onset neutropenia in CLL. All cases were found to carry clusters of identical (>=2) rearrangements corresponding to clonotypes. In the majority of cases (5/9), 2-4 (median 3) immunodominant clonotypes accounted for over 30% of the analyzed sequences (frequency of immunodominant clonotype/case 13-40%). Lymphocyte subpopulation analysis by flow cytometry in 6 patients revealed T-LGL expansion. Samples from additional time points (spanning a period of 10 years), pre- and post- Rituximab, were studied in one patient. Analysis of 71 sequences demonstrated progressive expansion of a certain clonotype overtime, associated with the emergence of steroid-refractory autoimmune hemolytic anemia in a context of CD3+CD8+CD57+ lymphoproliferation. This particular clonotype dominated the repertoire by far, thus establishing a diagnosis of T-LGL leukemia. which, remarkably, proved to be of donor origin (97% and 30% donor chimerism in T lymphocytes and total hematopoeisis, respectively). No association of oligoclonality to stronger GvL effect could be found among the rest of the patients. However, a strong correlation with cGvHD (100% vs 25% among polyclonal cases) was identified. Late-onset neutropenia was documented in 4/9 patients, regardless of the composition of the repertoire i.e whether it was polyclonal or oligo(mono)clonal. In conclusion, we report frequent development of oligoclonal cytotoxic T-cell populations after Rituximab treatment post allo-HCT likely of multifactorial evidence. Direct evidence of the anti-leukemic effect of this phenomenon could not be provided, however, the observed association of oligoclonality with GvHD and the development of a possible “T-LGL leukemia vs leukemia” effect in one patient is noteworthy and merits further investigation. Finally, the observed skewing of the TR gene repertoire strongly implicates antigen selection in the development of cytotoxic T-cell expansions after allo-HCT. Disclosures No relevant conflicts of interest to declare.

The JAK2 V617F mutation is of high diagnostic value in the evaluation of myeloproliferative neoplasms (MPN) as it helps to document clonality; in addition, it may also predict for response to hydroxyurea treatment. According to recent studies, the presence of bone marrow (BM) fibrosis at diagnosis may be associated with the clinical evolution of MPNs, in particular development of secondary acute myeloid leukemia (AML) or transformation to myelofibrosis (MF), however the underlying mechanisms remain unknown. In this study we characterized in detail subgroups of patients with Polycythemia Vera (PV) and Essential Thrombocythemia (ET) carrying the JAK2 V617F mutation (M-JAK2) or displaying BM fibrosis at diagnosis with the ultimate aim of identifying potential associations and/or overlapping phenotypes. The present single-institution patient cohort included 118 cases diagnosed according to WHO 2008 criteria. Patient characteristics were as follows: (i) Diagnosis: PV/ET, 37/82; (ii) Gender: male/female, 58/60; (iii) median age at diagnosis: 59.8 years (range, 25-90). M-JAK2 was detected in 86/118 (72.9%) cases [PV: 32/37 (86.5%) - ΕΤ: 54/82 (65%)]. BM fibrosis was observed in 28/112 (25%) cases [PV: 10/34 (29.4%), ΕΤ: 18/78 (23%)], grade I in 24/28 (85%) cases and grade II in 4 cases (all with ΕΤ). Thirteen patients without BM fibrosis at diagnosis underwent a second BM biopsy at a median time of 4.7 years (range, 1-10): BM fibrosis was observed in 5/13 (38.4%), 4 carrying M-JAK2, of whom only one had received anagrelide before the second BM biopsy. With a median follow up of 6 years (range 1-10), one of these five patients developed AML. There was no statistically significant association between M-JAK2 and BM fibrosis at diagnosis, neither in the entire cohort, nor in each MPN (ET or PV) separately. In PV: (i) M-JAK2 was significantly (p Disclosures: No relevant conflicts of interest to declare.

Abstract 3862 Subgroups of CLL cases defined on the basis of the immunogenetic features of their B cell receptors (BcRs), especially those cases belonging to subsets with stereotyped BcRs, exhibit differential responses to immune stimulation through either the BcR or the Toll-Like Receptors (TLRs). This indicates distinct, subset-biased modalities of BcR collaboration with specific TLRs that may extend to the control of cell proliferation or apoptosis, B cell anergy and/or TLR tolerance. In both normal and CLL B cells, the final signaling outcome can be controlled by microRNAs (miRNAs) targeting critical signaling molecules downstream of the BcR and/or the TLRs. With this in mind, here we investigated the impact of immune signaling on miRNA expression and function. We used two paradigmatic stereotyped subsets with distinct molecular and clinicobiological figures: (1) subset #1: unmutated (U) IGHV1/5/7-IGKV1(D)-39 IgM BcRs, aggressive disease, the largest U-CLL subset; and, (2) subset #4: mutated (M) IGHV4–34/IGKV2–30 IgG BcRs, indolent disease, the largest M-CLL subset. We have recently shown that these two subsets show differential functional outcomes after TLR stimulation with subset #4 selectively responsive to TLR1/2 but not to TLR7 stimulation, thus contrasting subset #1 which shows the opposite profile. Based on the above, we cultured negatively selected CD19+ B cells from subset #1 and subset #4 cases under the following conditions: (1) non stimulated; (2) stimulated through the BcR with anti-IgM and anti-IgG, respectively; (3) stimulated through TLR7 (Imiquimod) for subset #1, or through TLR1/2 (Pam3CSK4) for subset #4; and, (4) BcR/TLR co-stimulated. Using RQ-PCR Arrays, we measured the expression levels of a series of 33 miRNAs relevant for CLL biology with potential targets in BCR and/or TLR signaling pathways. In subset #1, none of the TLR ligands affected miRNA expression. On the contrary, TLR1/2 stimulation in subset #4 significantly (p Disclosures: No relevant conflicts of interest to declare.

Abstract 44 The role of antigen in the development of CLL is underscored by the biased immunoglobulin (IG) gene repertoire expressed by the malignant clones, the prognostic implications of B-cell receptor (BcR) structure and the identification of subsets of patients with quasi-identical, stereotyped BcRs. The structural homology of BcRs implies a role for a limited set of antigens or structurally related epitopes in leukemogenesis. Antigen recognition and host defense rely on multiple mechanisms acting synergistically in order to mount an effective immune response. These include specific stimulation through the BcR and non-specific stimulation through innate immune receptors, of which the major class is the Toll-like receptor (TLR) family. The available data on TLR expression in CLL are limited and derive from small series of patients. In the present study we performed a systematic gene expression profiling of the TLR signaling pathway in a large series of 191 patients with CLL. The analysis extended from all TLRs known to be functional in normal B cells to adaptors, effectors, inhibitors and members of the NFKB, JNK/p38, NF/IL6, and IRF signaling pathways downstream of TLR signaling. In addition, differences in gene expression profiles were sought for among subgroups of cases defined by BcR molecular features, such as the repertoire and mutational status of the IG heavy variable (IGHV) genes or the expression of stereotyped BcRs. CD19+ B lymphocytes were negatively selected from peripheral blood samples. The gene expression profile of the TLR signalling pathway was determined by the RT2Profiler™ PCR Array kit (PAHS-018A array, SABiosciences). At cohort level, among the receptors, high expression was recorded for TLR7 and CD180, intermediate for TLR1, TLR6 and TLR10 and low expression for TLR2 and TLR9. TLR4 and TLR8 were characterized by low to undetectable expression, with significant variations between patients, while TLR3 and TLR5 were not expressed in any case. The vast majority of the adaptors (e.g. MyD88, TICAM1, TRAF6), the effectors (UBE2N) and the members of the NFKB, JNK/p38, NF/IL6, and IRF signaling pathways downstream of TLR signaling were intermediately to highly expressed, while the inhibitors of TLR activity (TOLLIP, SIGIRR/TIR8) were generally low to undetectable, suggesting that the TLRs signalling pathway is active in CLL. Further comparison in subgroups of cases carrying mutated vs. unmutated IGHV sequences (124 and 67 cases, respectively) revealed upregulation of CD80, CD86, IL6, IFNG and TLR4 and downregulation of TLR8 and NFKBIL1 in the mutated subgroup. Significant differences in gene expression profiles were also found in subgroups of cases with stereotyped receptors. Comparison of subset #4 (mutated IGHV4-34/1GKV2-30 BcR, 10 cases) vs. subset #1 (unmutated IGHV1/5/7-IGKV1(D)-39 BcR, 10 cases) vs. subset #8 (unmutated IGHV4-39/IGKV1(D)-39 BcR, 4 cases) revealed: (i) upregulation of TLR7 and NFKB1A and downregulation of CD86 and TLR4 in #1 vs #4 cases; (ii) upregulation of MAP4K4 and TLR4 and downregulation of NFKB1A and CD180 in #8 vs #1 cases; and, (iii) upregulation of LY96 and downregulation of RIPK2 and CD86 in #8 vs #4 cases. In conclusion, the TLR gene expression profile of CLL is consistent with derivation from antigen-experienced B cells. Significant variations were identified in different subgroups of cases defined by BcR molecular features, indicating distinctive activation patterns of the TLR signaling pathway, especially among cases assigned to subsets with stereotyped BcRs, with potential implications about the nature of the antigenic stimulation. Disclosures: No relevant conflicts of interest to declare.

Abstract 1297 Relapse remains the most common cause of treatment failure in patients with hematological malignancies undergoing allogeneic hematopoietic cell transplantation (allo-HCT). Prediction of relapse by detection of minimal residual disease (MRD) is not always feasible, especially in AML and MDS, because of lack of clonal markers suitable for MRD detection by flow cytometry or PCR-based approaches. For this reason the kinetics of hematopoietic chimerism between the donor and the recipient have been proposed as essential for early intervention; however, the precise predictive value of monitoring chimerism has remained unresolved. In the present study, we retrospectively explored whether monitoring chimerism of unfractionated bone marrow (UBM) samples can be predictive of impending relapse in the setting of allo-HCT. The study group included 68 patients (37 males, 31 females) with a median age of 38 years (range, 8–63) who underwent allo-HCT for (i) AML, n=65 and, (ii) MDS, n=3 and were followed-up for a median time of 31 months (range 1–98). All patients were not evaluable for MRD by flow cytometry or molecular techniques due to lack of an informative clonal marker. Sixty-two and 6 patients received myeloablative (MA) or reduced intensity conditioning (RIC), respectively. The donor was a matched sibling in 47/68 patients, matched unrelated donor in 18/68, while 3 patients underwent haploidentical allo-HCT from a mismatched relative. The chimerism status was assessed by genotyping short tandem repeat polymorphisms (STRs) of the VWA, FES, THO1, SE33 and F13A1 genes; in the case of male recipient/female donor pairs, the chimerism was assessed by determination of allelic variants of the AMEL genes. The monitoring was based on STR/AMEL genotyping at days +14, +30, +60, +90 post-transplantation and thereafter every 3 months for up to 2 years. Following the current practice, patients were stratified as having complete chimerism (CC), decreasing recipient mixed chimerism (DMC), increasing recipient mixed chimerism (IMC), increasing and decreasing mixed chimerism (MC) and stable mixed chimerism (SMC) at regular time intervals (0-3 months, 3–6 months, 6–9 months, 9–12 months, 12–18 months, 18–24 months). Patients with a Disclosures: No relevant conflicts of interest to declare.

Abstract 2240 Poster Board II-217 Molecular monitoring of the CMV viral load in the blood after allogeneic hematopoietic cell transplantation (allo-HCT) by quantitative real-time polymerase chain reaction (RQ-PCR) assays is considered as the most important measure for CMV disease prevention and may prompt the initiation of preemptive therapy. Molecular assays have a high negative predictive value, yet their precise role in the establishment of CMV infection in biological specimens other than plasma has not been defined conclusively. Furthermore, several technical aspects remain unresolved, in particular the use of cut-offs for positivity, given that, generally, viral loads (viral genome copies, VGC) less than 0,5-2,5log10 cannot provide linearity in the results. We retrospectively evaluated the clinical significance of positive RQ-PCR tests for CMV DNA in biological fluids other than plasma from 73 patients after allo-HCT, with a special emphasis on samples with a low viral load ( Disclosures: No relevant conflicts of interest to declare.