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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.

Phylogenetic hypotheses for the largely South African genus Pelargonium L'Hér. (Geraniaceae) were derived based on DNA sequence data from nuclear, chloroplast and mitochondrial encoded regions. The datasets were unequally represented and comprised cpDNA trnL-F sequences for 152 taxa, nrDNA ITS sequences for 55 taxa, and mtDNA nad1 b/c exons for 51 taxa. Phylogenetic hypotheses derived from the separate three datasets were overall congruent. A single hypothesis synthesising the information in the three datasets was constructed following a total evidence approach and implementing dataset specific stepmatrices in order to correct for substitution biases. Pelargonium was found to consist of five main clades, some with contrasting evolutionary patterns with respect to biogeographic distributions, dispersal capacity, pollination biology and karyological diversification. The five main clades are structured in two (subgeneric) clades that correlate with chromosome size. One of these clades includes a "winter rainfall clade" containing more than 70% of all currently described Pelargonium species, and all restricted to the South African Cape winter rainfall region. Apart from (woody) shrubs and small herbaceous rosette subshrubs, this clade comprises a large "xerophytic" clade including geophytes, stem and leaf succulents, harbouring in total almost half of the genus. This clade is considered to be the result of in situ proliferation, possibly in response to late-Miocene and Pliocene aridification events. Nested within it is a radiation comprising c. 80 species from the geophytic Pelargonium section Hoarea, all characterised by the possession of (a series of) tunicate tubers.